Mechanisms of adaptation and reconstruction in the hypoperfused brain

Ischemic damage to the brain poses a long-term disability or even death. Endogenous recovery cannot fully restore functionality by itself, leading to deficits in cognition and motor function. This thesis investigates microenvironmental changes mediated by the molecules interleukin 6 (IL-6) and SorCS2 in murine models of ischemia. Our first study focuses on the effect of the inflammatory cytokine IL-6 in carotid stenosis, evaluating changes in the brain parenchyma and other associated factors. IL-6 plays an ambivalent role in the hypoperfused brain: it is associated with poor outcome such as deterioration in motor function, but it is also essential for recovery processes. In this study, mice underwent unilateral carotid artery occlusion, and IL-6 overexpression in astrocytes was induced on day 2 after surgery. Motor skills and health status were monitored for 21 days using behavioral tests, and changes in cerebral connectivity and brain remodeling were examined using diffusion tensor imaging. Data analysis showed that IL-6 overexpression has no adverse effect on the overall condition and fosters connectivity changes in the brain. In total, 10 out of 14 tracts were increased, mainly in interhemispheric networks. Proteome analysis of the ipsilesional striatal fiber tracts and the contralesional motor cortex showed alterations in expression levels of plasticity-associated proteins. Caprin-1 was more abundantly expressed on the ipsilesional side, and the GABA-transporter Gat1 was downregulated on the contralesional side, reducing inhibitory GABA signaling. Both are targets for further studies and potential drug targets for translatory research. The second part of the thesis investigates the effects of a SorCS2 knockout in stroke models. This Vps10p sorting receptor is a regulator of vesicular trafficking and secretion, possibly involved in endogenous IL-6 regulation. The study showed that contrary to prior knowledge, not only neurons but also astrocytes express SorCS2 after stroke, and TGF β likely mediates induction. IL-6 was not confirmed to be regulated in in vivo assays. Ablation of SorCS2 correlated with endostatin expression, mediating effects on regenerative processes such as angiogenesis. Our third study focuses on improving animal research. Data from standardized behavioral tests were collected in one curated database. This allows meta-analysis of data and can reduce animal suffering and the number of experimental animals due to higher experimental standards and prediction of human endpoints. It also generated a new starting point for innovative research concepts based on existing animal data. In summary, the thesis contributes insights into post-ischemic recovery processes and found several potential targets for further research on therapeutic interventions for brain ischemia. It also contributes to the 3R approach of reducing animal suffering in experimental research.Eine ischämische Schädigung des Gehirns kann zu einer langfristigen Behinderung bis hin zum Tod führen. Die körpereigene Regeneration kann verlorene Funktionalität nicht vollständig wiederherstellen, was zu kognitiven und motorischen Defiziten führt. Diese Arbeit erforscht die durch die Moleküle IL-6 und SorCS2 in Ischämiemodellen bei Mäusen vermittelten, lokalen Veränderungen des Mikromileus. Die erste Studie untersucht die Wirkung des inflammatorischen Zytokins Interleukin 6 (IL-6) bei einer Karotisstenose auf Veränderungen des Hirnparenchyms. IL-6 spielt im hypoperfundierten Gehirn eine ambivalente Rolle, da es einerseits mit einem schlechte-ren Verlauf korreliert, andererseits aber auch für den Erholungsprozess wichtig ist. In dieser Studie wurde bei Mäusen ein einseitiger Verschluss der Arteria carotis communis vorgenommen und zwei Tage später eine Überexpression von IL-6 in Astrozyten indu-ziert. Die motorischen Fähigkeiten und der Gesundheitszustand wurden 21 Tage lang mittels Verhaltensversuchen beobachtet und Veränderungen in der zerebralen Konnek-tivität sowie der Umbau des Gehirns untersucht. Die Überexpression von IL-6 hatte keine nachteiligen Auswirkungen auf den Gesundheitszustand und begünstigte eine Zunahme der Konnektivität im Gehirn bei 10 von 14 veränderten Verbindungen, vor allem zwischen den Hemisphären. Die Proteomanalyse der ipsilesionalen striatalen Faserbahnen und des kontraläsionalen motorischen Kortex zeigte Veränderungen in den Expressionsni-veaus von Plastizitäts-assoziierten Proteinen wie Caprin-1 und dem GABA-Transporter Gat1. Beide Moleküle sind Kandidaten für weitere Untersuchungen und potenzielle Ziel-strukturen für translationale Forschung. Im zweiten Teil der Arbeit wird die Auswirkungen eines SorCS2-Knockouts in Schlagan-fallmodellen untersucht. Die Hypothese war, dass SorCS2 an der endogenen IL-6-Regu-lation beteiligt ist. Die Studie zeigte, dass im Gegensatz zu bisherigen Erkenntnissen nach einem Schlaganfall nicht nur Neuronen, sondern auch Astrozyten TGF-β-vermittelt SorCS2 exprimieren. Es wurde nicht bestätigt, dass IL-6 durch SorCS2 reguliert wird. Ein SorCS2-Knockout korrelierte hingegen mit der Endostatin-Expression. Diese hat Auswir-kungen auf regenerative Prozesse wie Angiogenese. Drittens konnte durch Agglomeration von Verhaltensdaten eine kuratierte Datenbank er-stellt werden, um eine Meta-Analyse der Daten zu ermöglichen. Somit kann die Zahl der Versuchstiere aufgrund höherer experimenteller Standards und das Tierleid durch Vor-hersage humaner Endpunkte reduziert werden. Zusammenfassend gewährt diese Arbeit Einblicke in postischämische Erholungspro-zesse und zeigt potenzielle Zielstrukturen für die weitere Erforschung therapeutischer In-terventionen bei Ischämien auf, während sie gleichzeitig einen Beitrag zum 3R-Ansatz zur Verringerung des Tierleidens in der experimentellen Forschung leistet

Macrophage Inhibitory Cytokine 1 (MIC-1/GDF15) Decreases Food Intake, Body Weight and Improves Glucose Tolerance in Mice on Normal & Obesogenic Diets

Food intake and body weight are controlled by a variety of central and peripheral factors, but the exact mechanisms behind these processes are still not fully understood. Here we show that that macrophage inhibitory cytokine-1 (MIC-1/GDF15), known to have anorexigenic effects particularly in cancer, provides protection against the development of obesity. Both under a normal chow diet and an obesogenic diet, the transgenic overexpression of MIC-1/GDF15 in mice leads to decreased body weight and fat mass. This lean phenotype was associated with decreased spontaneous but not fasting-induced food intake, on a background of unaltered energy expenditure and reduced physical activity. Importantly, the overexpression of MIC-1/GDF15 improved glucose tolerance, both under normal and high fat-fed conditions. Altogether, this work shows that the molecule MIC-1/GDF15 might be beneficial for the treatment of obesity as well as perturbations in glucose homeostasis

Macrophage inhibitory cytokine-1 (MIC-1/GDF15) slows cancer development but increases metastases in TRAMP prostate cancer prone mice

Macrophage inhibitory cytokine-1 (MIC-1/GDF15), a divergent member of the TGF-β superfamily, is over-expressed by many common cancers including those of the prostate (PCa) and its expression is linked to cancer outcome. We have evaluated the effect of MIC-1/GDF15 overexpression on PCa development and spread in the TRAMP transgenic model of spontaneous prostate cancer. TRAMP mice were crossed with MIC-1/GDF15 overexpressing mice (MIC-1fms) to produce syngeneic TRAMPfmsmic-1 mice. Survival rate, prostate tumor size, histopathological grades and extent of distant organ metastases were compared. Metastasis of TC1-T5, an androgen independent TRAMP cell line that lacks MIC-1/GDF15 expression, was compared by injecting intravenously into MIC-1fms and syngeneic C57BL/6 mice. Whilst TRAMPfmsmic-1 survived on average 7.4 weeks longer, had significantly smaller genitourinary (GU) tumors and lower PCa histopathological grades than TRAMP mice, more of these mice developed distant organ metastases. Additionally, a higher number of TC1-T5 lung tumor colonies were observed in MIC-1fms mice than syngeneic WT C57BL/6 mice. Our studies strongly suggest that MIC-1/GDF15 has complex actions on tumor behavior: it limits local tumor growth but may with advancing disease, promote metastases. As MIC-1/GDF15 is induced by all cancer treatments and metastasis is the major cause of cancer treatment failure and cancer deaths, these results, if applicable to humans, may have a direct impact on patient care

Artritis reumatoide en afrocolombianos de Quibdó

Objectives: Little data is available on the prevalence and incidence of rheumatoid arthritis (RA) or the genetic and environmental factors that influence RA risk and severity in non-Caucasian populations. The prevalence of RA in Caucasians and some Native American populations is 1% or more; in contrast, low prevalences of RA have been reported in some African populations. We determined the hospital incidence (HI) and period prevalence (PP) of RA in African Colombians in Quibdo, Colombia, by using data collected at the Hospital San Francisco de Asis, a primary-to-tertiary care center. Genetic and immunologic studies of factors that influence RA risk and severity, such as HLA genes, immunoglobulin-A (IgA) rheumatoid factor (RF), and antikeratin antibodies (AKA) were performed. African Colombians with RA also were compared with Mestizo RA patients from Medellín, Colombia. Methods: To determine the HI, all the outpatient charts for 1995 were reviewed (n = 3,044). PP during 1996 (Jan-Dec) was assessed by stratified sampling of all African Colombians aged 18 or more having arthralgia. Participants completed a survey and a pretested standard questionnaire, had hands and feet X-rays, and provided a blood sample. Total and IgA RF were measured by turbidimetry and ELISA, respectively; AKA were assessed by indirect immunofluorescence on rat esophagus. HLA-DRB1 and DQB1 alleles were determined by polymerase chain reaction technique with primers of specific sequence and by reverse dot blot. Results: The HI was 0.65 cases per 1,000 person years. There were 321 individuals with arthralgia (0.3%; 95% CI, 0.28-0.3), 18 of whom fulfilled the American College of Rheumatology criteria for RA (PP in the general population, 0.01%; 95% CI, 0.008-0.02). Lower erosion scores were seen in African Colombian patients compared to Mestizos (n = 56), although duration of disease was similar in each group. No association between any HLA allele and RA risk or RA severity or between autoantibodies and RA severity was observed in African Colombians. Comparisons showed no significant differences between African Colombians and Mestizo patients in the presence of RF (total and IgA), AKA, age at onset, extra-articular manifestations, formal education level, and history of malaria. Conclusions: These results suggest that RA in African Colombian patients from Quibdo is rare, may be less severe in terms of radiographic damage than in Colombian Mestizo patients, and lacks association to HLA-DRB1 and DQB1 alleles. Additionally, RF (total and IgA) and AKA are not markers of progression and activity of the disease in this population

Serum macrophage inhibitory cytokine 1 in rheumatoid arthritis: a potential marker of erosive joint destruction

OBJECTIVE: The transforming growth factor beta superfamily member macrophage inhibitory cytokine 1 (MIC-1) is expressed upon macrophage activation, regulated by the p53 pathway, and linked to clinical events in atherosclerosis and cancer. Since rheumatoid arthritis (RA) shares similar etiopathologic mechanisms with the above diseases, we sought to determine the clinical utility of determining MIC-1 serum levels and MIC-1 genotype in the management of RA. METHODS: Ninety-one RA patients were recruited. Serum was collected from 83 of these patients and synovial biopsy samples were collected from the remaining 8 patients. Of the 83 patients from whom serum was collected, 61 were treated on an outpatient basis (defined as having nonsevere disease), and 22 patients went on to undergo hemopoietic stem cell transplantation (HSCT) (defined as having severe disease). RESULTS: Serum levels of MIC-1 were higher in RA patients and reflected disease severity independently of classic disease markers. MIC-1 was detected in rheumatoid synovial specimens, and allelic variation of MIC-1 was associated with earlier erosive disease and severe treatment-resistant chronic RA. Additionally, algorithms including serum and/or allelic variation in MIC-1 predicted response to HSCT, the presence of severe disease, and joint erosions. CONCLUSION: Determination of serum levels of MIC-1 and MIC-1 genotype may be clinically useful in the management of RA as well as in selection of patients for HSCT, since they predict disease course and response to therapy. The data indicate a potential role for MIC-1 in RA pathogenesis. These results warrant larger prospective studies to fully delineate and confirm a role for MIC-1 genotyping and serum estimation in patient selection for HSCT and in the management of R

Generation and characterization of mice with null mutation of the chloride intracellular channel 1 gene

Summary: CLIC1 belongs to a family of highly conserved and widely expressed intracellular chloride ion channel proteins existing in both soluble and membrane integrated forms. To study the physiological and biological role of CLIC1 in vivo, we undertook conditional gene targeting to engineer Clic1 gene knock-out mice. This represents creation of the first gene knock-out of a vertebrate CLIC protein family member. We first generated a Clic1 Knock-in (Clic1FN) allele, followed by Clic1 knockout (Clic1-/-) mice by crossing Clic1FN allele with TNAPcre mice, resulting in germline gene deletion through Cre-mediated recombination. Mice heterozygous or homozygous for these alleles are viable and fertile and appear normal. However, Clic1-/- mice show a mild platelet dysfunction characterized by prolonged bleeding times and decreased platelet activation in response to adenosine diphosphate stimulation linked to P2Y12 receptor signaling

The propeptide mediates formation of stromal stores of PROMIC-1: Role in determining prostate cancer outcome

The extracellular matrix (ECM) is a reservoir of cellular binding proteins and growth factors that are critical for normal cell behavior, and aberrations in the ECM invariably accompany malignancies such as prostate cancer. Carcinomas commonly overexpress macrophage inhibitory cytokine 1 (MIC-1), a proapoptotic and antitumorigenic transforming growth factor-β superfamily cytokine. Here we show that MIC-1 is often secreted in an unprocessed propeptide containing form. It is variably processed intracellularly, with unprocessed forms being secreted from several tumor lines, including prostate carcinoma lines, PC-3 and LNCaP. Once secreted, only unprocessed proMIC-1 binds ECM, demonstrating for the first time the occurrence of extracellular stores of MIC-1. The propeptide mediates this association via its COOH-terminal 89 amino acids. Xenograft models bearing tumors secreting various engineered forms of MIC-1 show that the propeptide regulates the balance between ECM stores and circulating serum levels of mature MIC-1 in vivo. The absence of propeptide results in ∼ 20-fold increase in serum MIC-1 levels. The significance of stromal MIC-1 stores was evaluated in prostate cancer tissue cores, which show major variation in stromal levels of MIC-1. Stromal MIC-1 levels are linked to prostate cancer outcome following radical prostatectomy, with decreasing stromal levels providing an important independent predictor of disease relapse. In low-grade localized prostate cancer (Gleason sum score ≤6), the level of MIC-1 stromal stores was the best predictor of future relapse when compared with all other clinicopathologic variables. The secretion and ECM association of unprocessed proMIC-1 is likely to play a central role in modulating local bioavailability of MIC-1 which can affect patient outcome in prostate cancer and other epithelial tumors

Generation and characterization of mice with null mutation of the chloride intracellular channel 1 gene

CLIC1 belongs to a family of highly conserved and widely expressed intracellular chloride ion channel proteins existing in both soluble and membrane integrated forms. To study the physiological and biological role of CLIC1 in vivo, we undertook conditional gene targeting to engineer Clic1 gene knock-out mice. This represents creation of the first gene knock-out of a vertebrate CLIC protein family member. We first generated a Clic1 Knock-in (Clic1FN) allele, followed by Clic1 knock-out (Clic1−/−) mice by crossing Clic1FN allele with TNAP-cre mice, resulting in germline gene deletion through Cre-mediated recombination. Mice heterozygous or homozygous for these alleles are viable and fertile and appear normal. However, Clic1−/− mice show a mild platelet dysfunction characterized by prolonged bleeding times and decreased platelet activation in response to adenosine diphosphate stimulation linked to P2Y₁₂ receptor signaling.10 page(s