Podcasts about tnfr1

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.01.551503v1?rss=1 Authors: Swanson, K. A., Nguyen, K. L., Gupta, S., Ricard, J., Bethea, J. R. Abstract: Upregulation of soluble tumor necrosis factor (sTNF) cytokine signaling through TNF receptor 1 (TNFR1) and subsequent neuronal hyperexcitability are observed in both animal models and human chronic neuropathic pain (CNP) (Clark et al., 2013; Empl et al., 2001; Ji et al., 2018; Lindenlaub and Sommer, 2003). To test the hypothesis that supraspinal circuitry is critical to pain chronification, we studied the intersect between supraspinal TNFR1 mediated neuronal signaling and sex specificity by selectively removing TNFR1 in Nex+ neurons in adult mice (NexCreERT2::TNFR1f/f). We determined that following chronic constriction injury (CCI), pain resolves in males; however, female acute pain transitions to chronic. Subsequently, we investigated two downstream pathways, p38MAPK and NF-{kappa}B, important in TNFR1 signaling and injury response. We detected p38MAPK and NF- {kappa}B activation in male cortical tissue; however, p38MAPK phosphorylation was reduced in NexCreERT2::TNFR1f/f males. We observed similar behavioral results following CCI in NexCreERT2::p38MAPKf/f mice. Previously, we established estrogen's ability to modulate sTNF/TNFR1 signaling in CNP, which may contribute to female prevalence of CNP (Bouhassira et al., 2008; Claiborne et al., 2006; de Mos et al., 2007; Del Rivero et al., 2019; Li et al., 2009). To explore the intersection between estrogen and inflammation in CNP we used a combination therapy of an estrogen receptor {beta} (ER {beta}) inhibitor with a sTNF/TNFR1 or general p38MAPK inhibitor. We determined both combination therapies lend "male-like" therapeutic relief to females following CCI. These data suggest that TNFR1/p38MAPK signaling in Nex+ neurons in CNP is male-specific and lack of therapeutic efficacy following sTNF inhibition in females is due to ER {beta} interference. These studies highlight sex-specific differences in pathways important to pain chronification and elucidate potential therapeutic strategies that would be effective in both sexes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.08.23.505002v1?rss=1 Authors: Sharma, S., Borski, C., Hanson, J., Garcia, M. A., Link, C. D., Hoeffer, C. A., Chatterjee, A., Nagpal, P. Abstract: Acute activation of innate immune response in the brain, or neuroinflammation, protects this vital organ from a range of external pathogens and promotes healing after traumatic brain injury. However, chronic neuroinflammation leads to the activation of immune cells like microglia and astrocytes causes damage to the nervous tissue, and is causally linked to a range of neurodegenerative diseases such as Alzheimers diseases (AD), Multiple Sclerosis (MS), Parkinsons diseases (PD), and many others. While neuroinflammation is a key target for a range of neuropathological diseases, there is a lack of effective countermeasures to tackle it, and existing experimental therapies require fairly invasive intracerebral and intrathecal delivery due to difficulty associated with the therapeutic crossover between the blood-brain barrier (BBB), making such treatments impractical to treat neuroinflammation long-term. Here, we present the development of an optimal neurotherapeutic using our NanoligomerTM discovery engine, by screening downregulation of several proinflammatory cytokines (e.g., Interleukin-1{beta} or IL-1{beta}, tumor necrosis factor-alpha or TNF-, TNF receptor 1 or TNF-R1, Interleukin 6 or IL-6), inflammasomes (e.g., NLRP1), key transcription factors (e.g., nuclear factor kappa-B or NF-{kappa}{beta}) and their combinations, as upstream regulators and canonical pathway targets, to identify and validate the best-in-class treatment. Using our high-throughput drug discovery, target validation, and lead molecule identification using a bioinformatics and AI-based ranking method to design sequence-specific peptide molecules to up-or down-regulate gene expression of the targeted gene at will, we used our discovery engine to perturb and identify most effective upstream regulators and canonical pathways for therapeutic intervention to reverse neuroinflammation. The lead neurotherapeutic was a combination of NanoligomersTM targeted to NF-{kappa}{beta} (SB.201.17D.8_ NF-{kappa}{beta}1) and TNFR1 (SB.201.18D.6_TNFR1), which were identified using in vitro cell-based screening in donor-derived human astrocytes, and further validated in vivo using a mouse model of lipopolysaccharide (LPS)-induced neuroinflammation. The combination treatment SB_NI_111 was delivered without any special formulation using a simple intraperitoneal injection of low-dose (5mg/kg) and was found to significantly suppress the expression of LPS-induced neuroinflammation in mouse hippocampus. These results point to the broader applicability of this approach towards the development of therapies for chronic neuroinflammation-linked neurodegenerative diseases, sleep countermeasures, and others, and the potential for further investigation of the lead neurotherapeutic molecule as reversible gene therapy. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

CoQ10 supplementation associated with improved trauma patient outcomes Urmia University of Medical Sciences (Iran) July 23 2021.    Findings from a trial reported on July 12, 2021 in the Journal of Nutritional Science revealed benefits for hospitalized traumapatients who were given supplements that contained coenzyme Q10.  The trial enrolled 40 men and women with traumatic injury and low plasma levels of CoQ10. Participants received a placebo or 400 milligrams CoQ10 daily for seven days. Blood samples collected at the beginning and end of the trial were analyzed for interleukin 6 (IL-6), which may be elevated during inflammation, and the oxidative stress markers malondialdehyde (MDA) and thiobarbituric acid reactive substances (TBARS). Body composition was also assessed at these time points, as well secondary outcomes that included Sequential Organ Failure Assessment (SOFA) and the Glasgow Coma Scale (GCS).  While interleukin-6 levels at the beginning of the study were similar between the CoQ10 and placebo groups at an average of 175.05 pg/mL and 177.82 pg/mL, they were reduced by 76.99 pg/mL in the CoQ10 group and 17.35 pg/mL in the placebo group. MDA values averaged 232.37 picograms per milliliter (pg/mL) and 239.96 pg/mL and were lowered by 88.84 pg/ml among participants who received CoQ10 and by 26.23 pg/mL among those who received a placebo. In comparison with the placebo group, fat free mass, skeletal muscle mass and body cell mass increased among those who received CoQ10. GCS and SOFA scores, and duration of hospital stay, ICU stay and ventilator use also improved among treated patients.  “To date, no randomized clinical trial study has been conducted to evaluate the effect of CoQ10 supplementation in traumatic mechanical ventilated patients and we hypothesized that CoQ10 administration in these patients could have beneficial effects on biochemical and clinical factors,” the authors wrote. “We have shown that CoQ10 could improve some of the clinical and anthropometric parameters in patients with a traumatic injury.”     Nigella sativa (black seed) prevents covid-induced vascular damage, scientists conclude   Oriental Institute of Science and Technology (India), July 27, 2021 New research published in the journal Vascular Pharmacology shows that Nigella sativa, also known as black seed or black cumin, binds to ACE2 in the lungs, effectively stopping the Wuhan coronavirus (Covid-19) from inducing inflammation and vascular damage. Researchers out of India investigated the effects of nigellidine, an indazole alkaloid of black seed, using molecular docking for binding to different angiotensin-binding proteins, as well as the Chinese Virus spike glycoprotein. They found that nigellidine “strongly binds” to the Chinese Virus spike protein at what is known as the hinge region or active site opening, which may in turn hamper its binding to the nCoV2-ACE2 surface. “Nigellidine effectively binds in the Angiotensin-II binding site / entry pocket,” the study explains. “Nigellidine showed strong binding to mono / multi-meric ACE1.” This process of ACE blocking could, the study goes on to suggest, restore angiotensin levels and restrict vasoturbulence in Chinese Virus patients, while the receptor blocking could help to stop resulting inflammation and vascular impairment. “Nigellidine may slow down the vaso-fluctuations due to Angiotensin deregulations in Covid patients,” the paper further explains. “Angiotensin II-ACE2 binding (ACE-value -294.81) is more favorable than nigellidine-ACE2. Conversely, nigellidine-ACE1 binding-energy / Ki is lower than nigellidine-ACE2 values indicating a balanced-state between constriction-dilatation.” Nigellidine also binds to the viral spike proteins, which when taken by Chinese Virus patients, and especially those who fall in the elderly category, could greatly reduce their risk of suffering complications or death. Nigellidine impairs SARS-CoV-2 infection, “cytokine storm” through numerous mechanisms In a related study that was published last year in the journal Europe PMC, researchers learned that nigellidine inhibits the Chinese Virus infection in several other ways. It was discovered early on in the “pandemic” that many of those who tested “positive” for the virus were suffering associated “cytokine storms,” in which their immune systems were over-responding and causing more damage, or even death. Nigellidine was then studied and discovered to possess certain properties that inhibit cytokine storms, as well as impede the SARS CoV-2 virus from causing infection. It is also hepato- and reno-protective, meaning it protects against liver damage. Beyond this, nigellidine was determined to possess unique immunomodulatory and anti-inflammatory characteristics, as well as antioxidant potential strong enough to inhibit important proteins associated with the Chinese Virus. In their quest to uncover possible “drug” candidates to protect patients against hyper-inflammation and other associated problems, the researchers learned that nigellidine – and more than likely other black seed constituents – helps tremendously with preventing negative side effects. Along with nigellicine, nigellidine is found in the seed coat of Nigella sativa. Both of these constituents in their sulfated forms are extremely bioavailable, and along with thymoquinone and dithymoquinone, two other black seed components, they show strong antioxidant, antibacterial, anti-hypertensive, anti-inflammatory and immunomodulatory effects. Black seed extracts have been shown in other experiments to decrease oxidative stress, effectively lowering the risk of inflammation-related diseases. We now know that this includes the Wuhan coronavirus (Covid-19). Black seed is also recognized as a metabolic protector, helping to improve lipid and blood sugar levels. “Most importantly, in SARS CoV-2 infection ACE-2 mediated impairment of aldosterone system may be repaired by,” the study further explains, providing relevant information to the current “pandemic.” “Vasorelaxant and anti-hypertensive function of [black seed] helps in the modulation of renin angiotensin system (RAS) or the diuretic activity, which is one of the major targets of COVID. It might have great protective role during post infective secondary disorder of the peripheral vasculature namely cardiac and renal systems. In most of the instances patients die due to this organ dysfunction/failure in COVID-19 infection.” By quelling inflammation, black seed could save lives from covid Laboratory studies have found that intake of Nigella sativa significantly improves the parameters for hyperglycemia and diabetes control, as well as glycated hemoglobin and insulin resistance. Based on this, experts believe that nigellidine specifically could play an important role in fighting the Chinese Virus by “docking” to the proteins and inflammatory molecules that can cause a cytokine storm – mainly TNF-? receptors such as TNFR1, TNFR2 and IL1R. “In the experimental rat model the source of this drug Nigella sativa; black cumin seed extracts were tested for its role on antioxidant, hepatic and renal status,” the paper states. “This work will help in the urgent therapeutic intervention against COVID-19 global pandemic.” “In the current study, we have decisively shown by molecular modeling that nigellidine can bind in the active sites of several important proteins of SARS CoV 2, several host receptors specific for SARS CoV-2 induced inflammatory markers IL1, IL6, TNF-?. Moreover, the extract from black cumin seed has been shown in experimental rat to be highly antioxidative, hepato- and reno-protective. Further studies are necessary to verify the potential effects of nigellidine in in vivo laboratory experimental animal model.”   Vitamin D supplementation improves recovery time of children with pneumonia at pediatric hospital Cairo University (Egypt), July 20, 2021 According to news reporting originating from Cairo, Egypt, by NewsRx correspondents, research stated, “Despite the well-recognized effect of vitamin D in metabolism and homeostasis, there is now growing interest in its probable association with pneumonia. This study aims to supply vitamin D3 (Cholecalciferol) (100,000 IU) to pneumonic children to minimize the duration of illness and improve their outcome.” Our news editors obtained a quote from the research from Cairo University, “A double-blinded, randomized, placebo-controlled trial was conducted in a Pediatric Cairo University affiliated hospital. An intervention arm (93 children) and a control arm (98 children), who had pneumonia with an insufficient or deficient level of vitamin D and whose parental permission was obtained, were enrolled in the trial. All children were treated with antibiotics according to WHO guidelines. Children were given a single injection of 1 mL of 100,000 IU of vitamin D3 or placebo. Clinical data were recorded every eight hours for all children. Outcomes were assessed 7 days after vitamin D injection. The primary outcome variable was the change in serum level of 25(OH)D, while the secondary outcomes were the medical state of the assigned cases (improvement or death) and duration between enrollment and hospital discharge for improved cases. In the supplementation group, the percentage of patients who suffered either deficient (38.7%) or insufficient levels (61.3%) of 25 (OH)D at day one had significantly decreased in the seventh day to (11.8%) and (52.7%), respectively. Kaplan--Meier plots highlighted that the median time to recover of the placebo group was significantly longer than that of the supplementation group (Log Rank P value < .001). VDD was detected in pediatric critical care children.” According to the news editors, the research concluded: “In pneumonic children with high VDD, it is illustrated that Vitamin D supplementation is accompanied by lowered mortality risk and pSOFA scores, reduced time to recover, and improved PaO2/FiO(2).”   Physical activity could combat fatigue, cognitive decline in cancer survivors University of Illinois, July 26, 2021 A new study indicates that cancer patients and survivors have a ready weapon against fatigue and "chemo brain": a brisk walk. Researchers at the University of Illinois, along with collaborators at Digital Artefacts in Iowa City, Iowa, and Northeastern University in Boston, looked at the association between physical activity, fatigue and performance on cognitive tasks in nearly 300 breast cancer survivors. "The data suggest that being more physically active could reduce two of the more commonly reported symptoms in breast cancer survivors: fatigue and cognitive impairment," said study leader Edward McAuley, a professor of kinesiology and community health at Illinois. "Most people think, 'If I exercise, I'll become tired.' In our study, exercise actually was associated with reduced fatigue, which in turn was associated with better cognitive function." Cognitive impairment, such as memory problems or shortened attention spans, is a common complaint among cancer patients and survivors, and is thought to be similar to decline due to aging. Past Illinois research has explored the effect of physical fitness on age-related cognitive decline, so the researchers wondered whether cancer survivors would respond similarly to exercise. "Other studies of cancer survivors have relied on small samples of cancer survivors, and used self-reporting measures of physical activity and cognitive function, which can be very biased," said postdoctoral researcher Diane Ehlers, the first author of the study, which is published in the journal Breast Cancer Research and Treatment. "What makes our study novel is that we had objective measures for both physical activity and cognitive performance, and a nationwide sample of breast cancer survivors." The researchers worked with Digital Artefacts -- developer of the commercial neuroscience app BrainBaseline - to create an iPad app tailored to this study. The app included questionnaires and activities designed to measure attention, memory and multitasking skills. The researchers also sent each participant an accelerometer to track daily physical activity. "We found that higher levels of daily moderate-to-vigorous physical activity were associated with better performance on the cognitive tasks measuring attention, memory and multitasking," Ehlers said. "What was notable was that physical activity's effect on cognitive performance was mediated by fatigue. This provides evidence that physical activity interventions targeting fatigue in cancer patients and survivors might provide promising models for improving cognitive function as well." Next, the researchers plan to conduct further studies to establish causation and further explore the pathways of how physical exercise improves cognitive performance. They are working with Digital Artefacts to conduct an iPhone-based study and focusing on diverse populations of breast cancer survivors. "The message for cancer patients and survivors is, get active!" Ehlers said. "Even if it's 10-minute bouts of brisk walking. It's not a magical cure-all, but we've seen many benefits of physical activity for cancer patients and survivors."   Cannabidiol promotes oral ulcer healing by inactivating CMPK2-mediated NLRP3 inflammasome Sichuan University (China), July 26, 2021 Xingying Qi, West China Hospital of Stomatology, Sichuan University, Chengdu, China, presented the oral session "Cannabidiol Promotes Oral Ulcer Healing by Inactivating CMPK2-Mediated NLRP3 Inflammasome" at the virtual 99th General Session & Exhibition of the International Association for Dental Research (IADR), held in conjunction with the 50th Annual Meeting of the American Association for Dental Research (AADR) and the 45th Annual Meeting of the Canadian Association for Dental Research (CADR), on July 21-24, 2021. The oral ulcer is a common oral inflammatory lesion with severe pain but little effective treatment is currently available. Cannabidiol (CBD) is recently emerging as a therapeutic agent for inflammatory diseases. However, the underlying mechanisms are not fully elucidated. Qi and colleagues sought to investigate whether and how CBD could play a therapeutic role in the oral ulcer. Oral ulcer models were performed in the tongue of C57BL/6 mice by acid etching or mechanical trauma, followed by CBD local administration. Samples were harvested for macroscopic and histological evaluation. CBD oral spray on acid- or trauma-induced oral ulcers on mice tongues inhibited inflammation, relieved pain and accelerated lesions closure in a dose-dependent manner. The results show that CBD accelerates oral ulcer healing by inhibiting CMPK2-mediated NLRP3 inflammasome activation and pyroptosis, which is mediated mostly by PPARγ in nucleus and partially by CB1 in plasma membrane. This data may shed light on the development of new therapeutic strategies for oral ulcers.   Algal solution: Could Spirulina modify the microbiome to protect against age-related damage? Louvain Drug Research Institute (Belgium), July 25 2021 Spirulina might help protect against age-related liver inflammation by modifying pathways in the microbiome, say researchers. Consumption of spirulina could help protect against hepatic inflammation in the elderly, according to the new animal research published in Nutrients. Belgian researchers carried out tests on mice, which suggest that the algae Spirulina has an impact on the gut microbiota, which in turn activates the immune system in the gut and improves inflammation in the liver that is associated with ageing. Led by senior author Professor Nathalie Delzenne from the Louvain Drug Research Institute in Belgium, the team said oral feeding of Spirulina was found to modulates several immunological functions involving, among others, the TLR4 pathway in old mice. “The fact that its oral consumption can influence both gut immunity and systemic sites, such as the liver, suggests that its immune action is not confined to the gut immune system,” wrote the team – who said the findings open the way to new therapeutic tools “in the management of immune alterations in aging, based on gut microbe-host interactions.” Furthermore, they suggested that improvement of the homeostasis in the gut ecosystem ‘could be essential' during the aging process, “and, in this perspective, dietary manipulation of the gut microbiota of the elderly with Spirulina, may represent a tool for preserving a healthy gastrointestinal microbial community in addition to its beneficial effects on immune function.” Study details Delzenne and colleagues noted that while the possible cardiovascular and immune support benefits of Spirulina have been fairly widely reported, the new study brings a fresh approach by testing whether the effects could be related to a modulation of gut micrbiota. In the trial, young mice aged three months were fed a standard diet, while older mice aged 24 months were fed a standard diet either with or without 5% Spirulina for six weeks. Upton supplementation with Spirulina, the team reported several changes to gut microbiota composition, including an increase in Roseburia and Lactobacillus populations. “Interestingly, parameters related to the innate immunity are upregulated in the small intestine of Spirulina-treated mice,” said the team. “Furthermore, the supplementation with Spirulina reduces several hepatic inflammatory and oxidative stress markers that are upregulated in old mice versus young mice.” Expression of several genetic and biochemical markers of inflammation and immunity were altered by supplementation with Spirulina, said the team. In particular, the transcription factor Foxp3 – involved in the differentiation of T cells into regulatory T cells (Tregs) – and MCP1 were increased due to Spirulina supplementation in old mice. Old mice that consumed Spirulina also showed activation of several immune parameters including Foxp3 in the ileum – suggesting an improvement of the gut immune function upon Spirulina treatment in this segment, said the Belgian researchers. Furthermore, Spirulina supplementation upregulated both TLR2 and TLR4 expression in the ileum of aged mice. “In accordance with these results, a solution of Spirulina (5%) exhibited a TLR4 agonist activity similar to the one reached in old-SP mice, suggesting a direct effect of the Spirulina, itself, on the TLR4 pathway,” they added. Microbiome mechanisms While the positive effect of Spirulina on the microbiome and liver inflammation is clear, the team noted that the mechanism by which the algae could change the composition of the intestinal microbiota remains unanswered. One possible mechanism could be the presence of antimicrobial substances produced by Spirulina, they said. “On the other hand, antimicrobial peptides (AMPs) could be mediators of the nutritional modulation of the gut microbiota.” “In the present study, RegIIIγ and Pla2g2 were increased by the supplementation with Spirulina, suggesting that the host contributes to the reduction and modification of the microbial community by modulating the production of specific AMPs,” they added.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.16.341644v1?rss=1 Authors: Lantoine, J., Proces, A., Villers, A., Halliez, S., Buee, L., Ris, L., Gabriele, S. Abstract: Traumatic brain injury (TBI) remains one of the leading causes of mortality and morbidity worldwide. Despite its high prevalence and extensive efforts to develop neuroprotective therapies, effective treatments for TBI are still limited. Among important neuronal damages, TBI induces structural and functional alterations of astrocytes, the most abundant cell type in the brain. Injured astrocytes respond in diverse mechanisms that result in reactive astrogliosis and are involved in the physiopathological mechanisms of TBI in an extensive and sophisticated manner. The establishment of effective neuroprotective treatments for TBI requires to better understand the complex biochemical interactions between activated astrocytes and neurons that contribute to the secondary injury. To address this challenge, we studied in vitro the role of mechanically injured astrocytes on the growth and synaptic connections of cortical neuronal networks of controlled architectures grown on well-defined protein micropatterns. Astrocytes were cultivated on elastic membranes and mechanically activated by stretching cycles. The culture media of healthy or activated astrocytes was then introduced on neuronal networks. We analyzed the neuronal viability, the neurite growth and the synaptic density of neuronal networks to understand the role of the inflammatory molecules secreted by mechanically activated astrocytes. Furthermore, we cultivated neuronal networks during 13 days with different doses of TNF- in order to decipher its individual contribution among the other cytokines. Here we show that the ratio of tubulin to synapsin area was significantly higher in neuronal networks treated with either 4 or 2 doses of TNF-, suggesting that TNF- can promote the tubulin polymerization process. Assuming that TNF- can bind to either TNFR1 or TNFR2 receptors, which lead respectively to the cell survival or the cell apoptosis, we studied the modulation of the both TNF- receptors in response to the medium of mechanically activated astrocytes and different doses of TNF-. Our findings indicate that the amount of both receptors increases with the maturation of the network. In addition, we observed a significant modulation of the amount of TNFR1 and TNFR2 in response to the media of injured astrocytes that leads to a large imbalance between both receptors, suggesting an important role for TNF-signaling in the physiopathological mechanisms of TBI. Copy rights belong to original authors. Visit the link for more info

Die von intrinsichen renalen Zellen und infiltrierenden Leukozyten exprimierten Zytokine sind zentrale Vermittler entzündlicher Nierenerkrankungen. Tumor Nekrose Faktor-α (TNF) ist ein solches proinflamatorisches Zytokin, das in der glomerulären Entzündungsreaktion involviert ist. Die funktionelle Rolle von TNF wurde in Tiermodellen der Glomerulonephritis belegt. Die biologischen Effekte von TNF werden durch die beiden funktionell eigenständigen TNF-Rezeptoren TNFR1 (CD120a) und TNFR2 (CD120b) vermittelt. Neuere Daten zeigen, dass in Modellen einer Immunkomplex-Glomerulonephritis wie der nephrotoxische Serumnephritis die beiden TNF-Rezeptoren in vivo unterschiedliche Funktionen bei der glomerulären Entzündung vermitteln können. Der vorliegenden Arbeit liegt die Hypothese zugrunde, dass Tnfr1 und Tnfr2 unterschiedliche inflammatorische TNF-Effekte in Glomeruli vermitteln. Daher war das Ziel dieser Arbeit, Expression und Funktion der beiden TNF-Rezeptoren in Maus-Glomeruli zu charakterisieren und die Tnfr-abhängig exprimierten Entzündungsmediatoren in Maus-Glomeruli zu identifizieren. Aufbauend auf den Ergebnissen dieser Arbeit könnten selektive, Tnfr-spezifische Therapien zur Hemmung der glomerulären Entzündungsreaktion entwickelt werden. Zudem wurde in dieser Arbeit die funktionelle Rolle der beiden TNF-Rezeptoren im MRL/lpr-Mausmodell der Lupusnephritis untersucht, um eine selektive Tnfr-Blockade als mögliche Therapiestrategie zu charakterisieren. Hierfür war eine Rückkreuzung von Tnfr1- und Tnfr2-defizienten C57BL/6J-Mäusen in den MRL/lpr-Hintergrund erforderlich. Um TNF-Rezeptor-1- und 2-vermittelte inflammatorische Signalwege in Glomeruli zu identifizieren wurde die Expression und die Funktion der beiden TNF-Rezeptoren in Mausnieren, in isolierten Glomeruli ex vivo und murinen glomerulären Endothel- und Mesangialzellen in vitro untersucht. In normaler Mausniere konnte eine Tnfr1- und Tnfr2-mRNA- und Protein-Expressionen präferentiell in Glomeruli im Vergleich zum Tubulointerstitium nachgewiesen werden. Die Expression von beiden TNF-Rezeptoren und die TNF-induzierte Induktion von Tnfr2-mRNA-Expression wurde auch in vitro sowohl in murinen glomerulären Endothel- als auch Mesangialzelllinien bestätigt. Die prominente glomeruläre TNF-Rezeptor-Expression korrelierte mit einer konstitutiven glomerulären mRNA-Expression von Adhäsionsmolekülen wie Icam-1, Vcam-1, E- und P-Selektin und Chemokinen wie Ccl2, Ccl3 und Ccl5. Eine intraperitoneale TNF-Injektion induzierte die Expression dieser Mediatoren präferentiell in Glomeruli. Diese in vivo TNF-Exposition führte zu einer raschen glomerulären Akkumulation von Leukozyten einschließlich Neutrophilen und mononukleären Phagozyten, die mittels einer kompartimentspezifischer Durchflußzytometrie analysiert wurden. Um Tnfr-abhängige inflammatorische Effekte in intrinsischen glomerulären Zellen unabhängig von infiltrierenden Leukozyten zu untersuchen, wurde eine Microarray-Gene-Expressionsanalyse an intakten Glomeruli durchgeführt, die aus Wildtyp und Tnfr-defizienten Mäusen isoliert und anschließend mit TNF ex vivo stimuliert wurden. Die meisten TNF-Effekte wurden ausschließlich durch Tnfr1 vermittelt, unter anderem die induzierte mRNA-Expression von Adhäsionsmolekülen, proinflammatorischen Chemokinen, Komplement-Faktoren und proapoptotischen Molekülen. Im Gegensatz dazu fanden wir nur vier Tnfr2-abhängig exprimierte Gene, einschließlich einer kleinen GTPase der Rab-Familie (Rab6b). Diese Ergebnisse wurden durch quantitative RT-PCR-Analysen von TNF-stimulierten Glomeruli und primären Mesangialzellen bestätigt. Weitere Untersuchungen zeigten allerdings auch einen Beitrag von Tnfr2 bei der gesteigerten glomerulären Expression von Adhäsionsmolekülen und Chemokine nach Stimulation mit niedrigen TNF-Konzentrationen auf. Im Gegensatz zur Wildtyp-Kontrolle fehlte in TNF-stimulierten Tnfr1-defizienten Glomeruli die Sekretion verschiedener proinflammatorischer Chemokine beinahe vollständig. Interessanterweise war die Proteinexpression auch in Tnfr2-defizienten Glomeruli signifikant herunterreguliert. Folglich sind die meisten inflammatorischen TNF-Effekte in Glomeruli via Tnfr1 durch die induzierte Expression von proinfammatorischen Mediatoren wie Adhäsionsmolekülen und Chemokinen vermittelt. Darüber hinaus dürfte Tnfr2 zu dieser inflammatorischen Antwort beitragen, wenn Glomeruli niedrigen TNF-Konzentrationen ausgesetzt sind. Ferner scheint Tnfr2 posttranskriptionell die Chemokinsekretion in Glomeruli nach einer TNF-Exposition zu beeinflussen, möglicherweise durch die Tnfr2-abhängig exprimierte Rab GTPase Rab6b, die am intrazellulären Transport und der Sekretion von inflammatorischen Molekulen beteiligt sein könnte. In Bezug auf Tnfr-spezifische, anti-inflammatorische Therapien weisen die hier präsentierten Ergebnisse somit darauf hin, dass eine selektive Tnfr1-Blockade eine glomeruläre, insbesondere durch Granulozyten und Makrophagen vermittelte Entzündung verbessern könnte, möglicherweise bei geringer Hemmung immunregulatorischer und antimikrobieller Funktionen von TNF, die redundant durch Tnfr2 vermittelt werden könnten. Dagegen erscheint aufgrund der erhobenen Daten im MRL/lpr-Mausmodell eine Blockade von TNF oder beider Rezeptoren bei der Lupusnephritis, in der glomeruläre Neutrophileninfiltrate keine entzündliche Rolle spielen, weniger erfolgversprechend. Gleichzeitig weisen die vorliegenden Ergebnisse auf eine immunsuppressive, die systemische Immunreaktivität beim SLE begrenzende Funktion von Tnfr2 hin.

Die vorliegende Arbeit untersuchte die Rolle der Perizyten bei steriler Inflammation. Bisher war in diesem Zusammenhang der Einfluss der Perizyten nicht bekannt, ebenso wenig ob und wie sie zu Entzündungsreaktionen beitragen. Weiterhin war der Einfluss der Perizyten auf die interstitielle Migration myeloider Zellen in vivo unerforscht. Hier konnte gezeigt werden, dass Perizyten durch eine Vielzahl von Rezeptoren wie TLR2, TLR4, TNFR1, FPR2 in der Lage sind inflammatorische Reize zu detektieren und daraufhin einen proinflammatorischen Phänotyp annehmen. Dieser ist durch die vermehrte Expression von NLRP3 sowie des Adhäsionsmoleküls ICAM-1 und die Sekretion von Chemokinen wie CXCL1, IL8 und CCL2 gekennzeichnet. Weiterhin wird das Chemokin-ähnliche Molekül MIF von aktivierten Perizyten sowohl sezerniert als auch an der Oberfläche präsentiert. Die ausgeschütteten Chemokine beeinflussen wiederum Monozyten und neutrophile Granulozyten durch ihre chemotaktische Wirkung. Auch konnte ein anti-apoptotischer sowie aktivierender Effekt der Perizyten auf neutrophile Granulozyten gezeigt werden, was die Überlebensdauer dieser Zellen im interstitiellen Gewebe signifikant verlängert. Anhand eines Mausmodells und der 2-Photonen Mikroskopie wurde gezeigt, dass Perizyten auch in vivo einen entscheidenden Beitrag zur Rekrutierung neutrophiler Granulozyten und Monozyten zur Inflammation leisten. Zum ersten Mal wurde die Interaktion myeloider Zellen mit Perizyten in vivo visualisiert und genauer charakterisiert. Diese Interaktion beeinflusst die interstitielle Migration neutrophiler Granulozyten und Monozyten abhängig davon, ob ein Stimulus für gerichtete oder ungerichtete Migration vorliegt. Es wurde deutlich, dass Perizyten sowohl einen chemotaktischen als auch einen haptotaktischen Reiz auf myeloide Leukozyten ausüben, was an einer Polarisierung der Zellen zu erkennen ist. Ebenso tragen sie durch die Interaktion zur Aktivierung der myeloiden Zellen in vivo bei. Diese Arbeit leistet demnach einen Beitrag zur genaueren Definition der Rolle von Perizyten bei steriler Inflammation. Hierfür wurden die zellulären und molekularen Mechanismen in vitro und die in vivo ablaufenden Prozesse bei der interstitiellen Migration myeloider Zellen genauer charakterisiert. Dabei konnten Perizyten als neuer Zelltyp identifiziert werden, der Gewebeschäden detektiert und aktiv zur akuten Entzündungsreaktion beiträgt indem er die Rekrutierung und Funktionalität myeloider Leukozyten unterstützt.

Introduction: Elevated serum levels of the proinflammatory cytokine tumor necrosis factor alpha (TNF alpha) correlate with an increased risk for atherothrombotic events and TNF alpha is known to induce prothrombotic molecules in endothelial cells. Based on the preexisting evidence for the impact of TNF alpha in the pathogenesis of autoimmune disorders and their known association with an acquired hypercoagulability, we investigated the effects of TNF alpha and the role of the TNF receptor subtypes TNFR1 and TNFR2 for arteriolar thrombosis in vivo. Methods: Arteriolar thrombosis and platelet-rolling in vivo were investigated in wildtype, TNFR1-/-, TNFR2-/- and TNFR1-/R2-/-C57BL/6 mice using intravital microscopy in the dorsal skinfold chamber microcirculation model. In vitro, expression of prothrombotic molecules was assessed in human endothelial cells by real-time PCR and flow cytometry. Results: In wildtype mice, stimulation with TNF alpha significantly accelerated thrombotic vessel occlusion in vivo upon ferric chloride injury. Arteriolar thrombosis was much more pronounced in TNFR1-/- animals, where TNF alpha additionally led to increased platelet-endothelium-interaction. TNF alpha dependent prothrombotic effects were not observed in TNFR2-/- and TNFR1-/R2- mice. In vitro, stimulation of human platelet rich plasma with TNF alpha did not influence aggregation properties. In human endothelial cells, TNF alpha induced superoxide production, p-selectin, tissue factor and PAI-1, and suppressed thrombomodulin, resulting in an accelerated endothelial dependent blood clotting in vitro. Additionally, TNF alpha caused the release of soluble mediators by endothelial cells which induced prothrombotic and suppressed anticoagulant genes comparable to direct TNF alpha effects. Conclusions: TNF alpha accelerates thrombus formation in an in vivo model of arteriolar thrombosis. Its prothrombotic effects in vivo require TNFR2 and are partly compensated by TNFR1. In vitro studies indicate endothelial mechanisms to be responsible for prothrombotic TNF alpha effects. Our results support a more selective therapeutic approach in anticytokine therapy favouring TNFR2 specific antagonists.

TRADD spielt als Adaptermolekül eine zentrale Rolle in der Signaltransduktion von LMP1 und TNF-Rezeptor 1. Während es allerdings durch den TNFR1 neben der Aktivierung verschiedener Signalwege auch zur Induktion von Apoptose und Nekrose kommt, handelt es sich bei LMP1 um ein Protein mit transformierendem Potential. Bei den jeweiligen TRADD-Bindestellen von LMP1 und TNFR1 handelt es sich um zwei strukturell vollkommen unterschiedliche Domänen. Und auch auf der Seite von TRADD wird die Bindung über zwei verschiedene Domänen vermittelt. Im Rahmen dieser Doktorarbeit sollte die Frage beantwortet werden, ob die TRADD-Bindestelle intrinsisch die biologischen Effekte der Signaltransduktion bestimmt oder ob diese durch den Rezeptorkontext festgelegt werden. Zur Beantwortung dieser Frage wurde in einem Domain Swapping Experiment die TRADD-Bindestelle des konstitutiv aktiven LMP1-TNFR1 sowie des TNFR1 gegen die putative TRADD-Bindestelle von LMP1 ausgetauscht. Es konnte erstmals gezeigt werden, dass die Aminosäuren 370-386 die vollständige TRADD-Bindestelle von LMP1 umfassen. Weiter konnte gezeigt werden, dass diese Aminosäuren im LMP1-TNFR1- sowie im TNFR1-Kontext ausreichend sind, um den NF-κB und den JNK1 Signalweg zu aktivieren. Die Aktivierung des JNK1 Signalweges durch LMP1-TNFR1-CTAR2 verläuft unabhängig von TRAF2 und abhängig von TRAF6 und auch die Aktivierung des NF-κB Signalweges durch dieses Rezeptorkonstrukt verläuft TRAF6-abhängig. Damit konnte gezeigt werden, dass die LMP1-spezifischen Charakteristika der Signaltransduktion durch die TRADD-Bindestelle festgelegt und mit ihr zusammen übertragen werden. Obwohl die Aminosäuren 370-386 von LMP1 funktionell sind, sind sie auch im LMP1-TNFR1 sowie im TNFR1 Kontext nicht in der Lage Apoptose zu induzieren. Damit konnte im Rahmen dieser Doktorarbeit gezeigt werden, dass die Aminosäuren 370-386 von LMP1 intrinsisch und unabhängig vom Rezeptorkontext den nicht-apoptotischen Phänotyp der Signaltransduktion festlegen. Außerdem wurde im Rahmen dieser Doktorarbeit die Beteiligung von TRAF7 an der Signaltransduktion von LMP1 untersucht. Dazu wurde traf7 aus einer cDNA kloniert. Zusätzlich wurden verschiedene Deletionsmutanten sowie Fusionen mit dem fluoreszierenden Protein mRFP hergestellt. Es konnte eine Threonin-Phosphorylierung von TRAF7(1-383) nachgewiesen werden. Mittels Fluoreszenzmikroskopie konnte eine Lokalisierung von TRAF7 in vesikulären Strukturen beobachtet werden. Eine Mutante, der der RING- sowie der Zink-Finger fehlen, zeigte hingegen eine gleichmäßige zytosolische Verteilung. Außerdem konnte in dieser Doktorarbeit mit Hilfe von spezifischer siRNA gezeigt werden, dass TRAF7 an der Aktivierung des JNK1 Signalweges durch LMP1 beteiligt ist.

The study is subdivided into two different parts: the first part deals with the development of a method to gain uterus milk in vivo during the preimplantation periode in cattle for the investigation of regulatory factors. The second part investigates different proteases in bovine follicles 20 hours after GnRH (Gonadotropin releasing hormone) injection (shortly bevor ovulation) for comparable as well as in the corpus luteum (CL) during oestrous cycle and induced luteolysis. In addition apoptotic as well as anti-apoptotic factors were evaluated in the CL during oestrous cycle and induced luteolysis. For the development of a method for gaining uterus milk in vivo during the first 24 days of gravidity in cattle, nine heifers were cycle synchronised using the Ovsynch method and artificially inseminated. Before flushing an epiduralanaesthesia was given and both uterus horns were flushed with 13ml 0.9% NaCl using a balloon embryo transfer catheter at day 5, 7, 12, 17 and 24 of gravidity. The catheter was placed 1cm cranial to the bifurcatio uteri in both horns. It was possible to retrive between 3ml and 13ml of the used flushing fluid. The uterus milk from the ipsilateral horn was inspected for an embryo and an EDTA-stabilisator was given to the uterus milk of both horns. An infection of the uterus occured in three heifers after the second and in five heifers after the third flushing. In one heifer no infection was found. Between day 17 and day 24 all heifers showed clear signs of oestrus. It was possible to detect progesterone, oestradiol-17-beta, PGF2alpha and VEGF via enzyme immunoassay (EIA) and radio immunoassay (RIA), respectively. Because of the occurred infection no statistic analysis was made. But it could be seen that the level of progesterone ranged between

In dieser Arbeit sollte untersucht werden, auf welchem Wege die beiden Zelltodrezeptoren TNFR1 und Fas Apoptose in den Zelllinien A9 und SV80 ausführen. Ausgangspunkt waren widersprüchliche Erkenntnisse über die Signalwege von TNFR1 und Fas. So war in der Literatur beschrieben, daß TNFR1 und Fas die gleichen intrazellulären Signalmoleküle benutzen um Apoptose auszulösen. Als diese Signalmoleküle wurden FADD, TRADD und FLICE identifiziert. Ergebnisse aus unserem Labor zeigten jedoch, daß in A9 Zellen der durch Fas oder TNFR1 ausgelöste Zelltod unterschiedliche Signalwege beschreiten musste, da sich der durch TNFR1 ausgelöste Zelltod bezüglich der Hemmbarkeit durch verschiedene Inhibitoren der Apoptose deutlich von dem durch Fas ausgelösten Zelltod unterschied. Es sollte untersucht werden, ob in SV80 und A9 Zelllinien FADD als gemeinsames Signalprotein beider Zelltodrezeptoren benutzt wird, oder bereits auf dieser Ebene ein Unterschied festzustellen ist. Dazu wurde FADD kloniert, und die Mutante DFADD von FADD hergestellt, die in der Lage ist, den bisher bekannten Signalweg von FADD in einer dominant negativen Weise zu hemmen. FADD und DFADD wurden in A9 und SV80 Zellen zur Expression gebracht. In SV80 Zellen führte Überexpression von FADD wie erwartet zu Zelltod, Expression von DFADD schützte nach Stimulation des TNFR1 oder Fas wie erwartet vor Zelltod. Expression von FADD in A9 Zellen führte zu Zelltod. Überraschenderweise führte jedoch auch Expression von DFADD in A9 zu Zelltod. Es war unerwartet, daß die Mutante, die den bisher bekannte Signalweg von FADD blockieren sollte, selbst Zelltod auslösen kann. Um die Art der Wirkung dieser Mutante DFADD weiter zu charakterisieren wurde versucht, den durch DFADD vermittelten Zelltod durch verschiedene Klassen von Hemmstoffe der Apoptose zu inhibieren. Zum einen Apoptoseinhibitoren, die über Hemmung der Caspasen wirken, zum anderen solche, die durch Hemmung der Atmungskette Apoptose unterbinden. Des weiteren wurde das Signalmolekül RAIDD und verschiedene dominant negative Mutanten kloniert, um zu untersuchen, ob DFADD in der Lage ist weitere Signalproteine zu aktivieren und so Zelltod auszulösen.