Expression of 6 Biomarkers in Liver Grafts After Pediatric Liver Transplantation : Correlations with Histology, Biochemistry, and Outcome

Background: Subclinical graft inflammation and fibrosis after pediatric liver transplantation (LT) are common. Biomarkers are needed that precede and are associated with these changes and graft outcome. Material/Methods: We evaluated immunohistochemical expression of 6 biomarkers [alpha-smooth muscle actin (alpha-SMA), collagen I, decorin, vimentin, P-selectin glycoprotein ligand-1 (PSGL-1), and CD34] in biopsies taken intraoperatively at LT (baseline) (n=29) and at 11.3 years after LT (first follow-up) (n=51). Liver biochemistry and graft histology were assessed at the first follow-up and at final assessment (19.6 years after LT) (n=48). Second follow-up biopsies for histology were available from 24 patients. The immunostainings were correlated with liver histology, biochemistry, and outcome at these time-points. Results: Baseline levels of the biomarkers were unrelated to presence of fibrosis at follow-up. Increased a-SMA, collagen I levels, decorin, and vimentin were associated with simultaneous fibrosis at the first follow-up (p=0.001-0.027). Increased SMA, collagen I, decorin, vimentin, PSGL-1, and CD34 expression at first follow-up were associated with simultaneous portal inflammation (p=0.001-0.025). alpha-SMA, decorin, and vimentin expression were increased in patients without fibrosis at the first follow-up but who developed fibrosis in second follow-up (p=0.014 p=0.024 and p=0.024). Significant fibrosis (F2) and markedly increased alpha-SMA, collagen I, decorin, and vimentin levels at first follow-up were associated with suboptimal liver status at the final assessment (p=0.002-0.042). Conclusions: The expression of the biomarkers at LT was unrelated to later development of graft fibrosis. alpha-SMA, decorin, and vimentin were associated with later graft fibrosis and suboptimal liver status.Peer reviewe

Compressive stress-mediated p38 activation required for ER alpha plus phenotype in breast cancer

Breast cancer is now globally the most frequent cancer and leading cause of women's death. Two thirds of breast cancers express the luminal estrogen receptor-positive (ER alpha + ) phenotype that is initially responsive to antihormonal therapies, but drug resistance emerges. A major barrier to the understanding of the ER alpha-pathway biology and therapeutic discoveries is the restricted repertoire of luminal ER alpha + breast cancer models. The ER alpha + phenotype is not stable in cultured cells for reasons not fully understood. We examine 400 patient-derived breast epithelial and breast cancer explant cultures (PDECs) grown in various three-dimensional matrix scaffolds, finding that ER alpha is primarily regulated by the matrix stiffness. Matrix stiffness upregulates the ER alpha signaling via stress-mediated p38 activation and H3K27me3-mediated epigenetic regulation. The finding that the matrix stiffness is a central cue to the ER alpha phenotype reveals a mechanobiological component in breast tissue hormonal signaling and enables the development of novel therapeutic interventions. Subject terms: ER-positive (ER + ), breast cancer, ex vivo model, preclinical model, PDEC, stiffness, p38 SAPK. Reliable luminal estrogen receptor (ER alpha+) breast cancer models are limited. Here, the authors use patient derived breast epithelial and breast cancer explant cultures grown in several extracellular matrix scaffolds and show that ER alpha expression is regulated by matrix stiffness via stress-mediated p38 activation and H3K27me3-mediated epigenetic regulation.Peer reviewe

Compressive stress-mediated p38 activation required for ERα + phenotype in breast cancer

Breast cancer is now globally the most frequent cancer and leading cause of women's death. Two thirds of breast cancers express the luminal estrogen receptor-positive (ER alpha + ) phenotype that is initially responsive to antihormonal therapies, but drug resistance emerges. A major barrier to the understanding of the ER alpha-pathway biology and therapeutic discoveries is the restricted repertoire of luminal ER alpha + breast cancer models. The ER alpha + phenotype is not stable in cultured cells for reasons not fully understood. We examine 400 patient-derived breast epithelial and breast cancer explant cultures (PDECs) grown in various three-dimensional matrix scaffolds, finding that ER alpha is primarily regulated by the matrix stiffness. Matrix stiffness upregulates the ER alpha signaling via stress-mediated p38 activation and H3K27me3-mediated epigenetic regulation. The finding that the matrix stiffness is a central cue to the ER alpha phenotype reveals a mechanobiological component in breast tissue hormonal signaling and enables the development of novel therapeutic interventions. Subject terms: ER-positive (ER + ), breast cancer, ex vivo model, preclinical model, PDEC, stiffness, p38 SAPK.Reliable luminal estrogen receptor (ER alpha+) breast cancer models are limited. Here, the authors use patient derived breast epithelial and breast cancer explant cultures grown in several extracellular matrix scaffolds and show that ER alpha expression is regulated by matrix stiffness via stress-mediated p38 activation and H3K27me3-mediated epigenetic regulation.</p

Kolesteroliaineenvaihdunnan häiriöt keskushermoston perinnöllisissä sairauksissa

The central nervous system (CNS) is the most cholesterol-rich organ in the body. Cholesterol is essential to CNS functions such as synaptogenesis and formation of myelin. Significant differences exist in cholesterol metabolism between the CNS and the peripheral organs. However, the regulation of cholesterol metabolism in the CNS is poorly understood compared to our knowledge of the regulation of cholesterol homeostasis in organs reached by cholesterol-carrying lipoprotein particles in the circulation. Defects in CNS cholesterol homeostasis have been linked to a variety of neurodegenerative diseases, including common diseases with complex pathogenetic mechanisms such as Alzheimer s disease. In spite of intense effort, the mechanisms which link disturbed cholesterol homeostasis to these diseases remain elusive. We used three inherited recessive neurodegenerative disorders as models in the studies included in this thesis: Niemann-Pick type C (NPC), infantile neuronal ceroid lipofuscinosis and cathepsin D deficiency. Of these three, NPC has previously been linked to disturbed intracellular cholesterol metabolism. Elucidating the mechanisms with which disturbances of cholesterol homeostasis link to neurodegeneration in recessive inherited disorders with known genetic lesions should shed light on how cholesterol is handled in the healthy CNS and help to understand how these and more complex diseases develop. In the first study we analyzed the synthesis of sterols and the assembly and secretion of lipoprotein particles in Npc1 deficient primary astrocytes. We found that both wild type and Npc1 deficient astrocytes retain significant amounts of desmosterol and other cholesterol precursor sterols as membrane constituents. No difference was observed in the synthesis of sterols and the secretion of newly synthesized sterols between Npc1 wild type, heterozygote or knockout astrocytes. We found that the incorporation of newly synthesized sterols into secreted lipoprotein particles was not inhibited by Npc1 mutation, and the lipoprotein particles were similar to those excreted by wild type astrocytes in shape and size. The bulk of cholesterol was found to be secreted independently of secreted NPC2. These observations demonstrate the ability of Npc1 deficient astrocytes to handle de novo sterols, and highlight the unique sterol composition in the developing brain. Infantile neuronal ceroid lipofuscinosis is caused by the deficiency of a functional Ppt1 enzyme in the cells. In the second study, global gene expression studies of approximately 14000 mouse genes showed significant changes in the expression of 135 genes in Ppt1 deficient neurons compared to wild type. Several genes encoding for enzymes of the mevalonate pathway of cholesterol biosynthesis showed increased expression. As predicted by the expression data, sterol biosynthesis was found to be upregulated in the knockout neurons. These data link Ppt1 deficiency to disturbed cholesterol metabolism in CNS neurons. In the third study we investigated the effect of cathepsin D deficiency on the structure of myelin and lipid homeostasis in the brain. Our proteomics data, immunohistochemistry and western blotting data showed altered levels of the myelin protein components myelin basic protein, proteolipid protein and 2 , 3 -cyclic nucleotide 3 phosphodiesterase in the brains of cathepsin D deficient mice. Electron microscopy revealed altered myelin structure in cathepsin D deficient brains. Additionally, plasmalogen-derived alkenyl chains and 20- and 24-carbon saturated and monounsaturated fatty acids typical for glycosphingolipids were found to be significantly reduced, but polyunsaturated species were significantly increased in the knockout brains, pointing to a decrease in white matter. The levels of ApoE and ABCA1 proteins linked to cholesterol efflux in the CNS were found to be altered in the brains of cathepsin D deficient mice, along with an accumulation of cholesteryl esters and a decrease in triglycerols. Together these data demonstrate altered myelin architecture in cathepsin D deficient mice and link cathepsin D deficiency to aberrant cholesterol metabolism and trafficking. Basic research into rare monogenic diseases sheds light on the underlying biological processes which are perturbed in these conditions and contributes to our understanding of the physiological function of healthy cells. Eventually, understanding gained from the study of disease models may contribute towards establishing treatment for these disorders and further our understanding of the pathogenesis of other, more complex and common diseases.Keskushermosto, eli aivot ja selkäydin, sisältää suhteellisesti enemmän kolesterolia kuin mikään muu elin. Keskeiset keskushermoston toiminnot, kuten viestejä solusta toiseen välittävien synapsien ja hermosäikeitä eristävien myeliinituppien muodostuminen on riippuvaista kolesterolin saatavuudesta. Kolesteroliaineenvaihdunnan säätely keskushermostossa on erilaista kuin muissa elimissä, sillä veri-aivoeste estää verenkierron mukana kulkevien kolesterolia sisältävien lipoproteiinikappaleiden pääsyn keskushermostoon. Tästä syystä keskushermoston solut valmistavat tarvitsemansa kolesterolin itse. Mikäli kolesterolin muodostuminen keskushermostossa on estynyt, on seurauksena vakavia kehityshäiriöitä. Tässä väitöskirjassa on tutkittu kolmea väistyvästi periytyvän neurologisen sairauden poistogeenista hiirimallia. Sairaudet, joiden malleja on käytetty, ovat Niemann-Pick tyyppi C-tauti (NPC), infantiili neuronaalinen seroidi lipofuskinoosi (INCL) ja cathepsin D-entsyymin puutos. Näistä NPC on aikaisemmin liitetty kolesteroliaineenvaihdunnan häiriöhin, sillä se johtaa massiiviseen kolesterolin kertymään soluissa. Ensimmäisessä osatyössä tutkittiin kolesterolin ja sen esiasteiden sekä lipoproteiinikappaleiden muodostumista ja erittymistä Npc1-geenipuutteisissa keskushermoston tukisoluissa, astrosyyteissä. Sekä terveet että poistogeeniset solut pystyvät muodostamaan kolesterolia. Sekä terveiden että poistogeenisten astrosyyttien solukalvoista löydettiin kolesterolin lisäksi suhteellisesti suuria määriä kolesterolin esiastetta desmosterolia. Desmosterolia on useimmissa solutyypeissä hyvin vähän. Lipoproteiinikappaleiden muodostuminen ja sterolien eritys ei ollut estynyt poistogeenisissä soluissa. Nämä havainnot osoittavat, että Npc1-puutteiset astrosyytit pystyvät muodostamaan ja erittämään steroleja. INCL aiheutuu Ppt1-entsyymin toiminnan puutoksesta soluissa. Toisessa osatyössä tutkittiin 14000 geenin ilmentymistä terveissä ja Ppt1-puutteisissa hiiren hermosoluissa. Useiden kolesterolin muodostumiseen osallistuvia entsyymejä koodittavien geenien ilmentyminen oli lisääntynyt Ppt1-puutteisissa soluissa ja vastaavasti kolesterolin muodostumien poistogeenisissä soluissa oli koholla verrattuna terveisiin soluihin. Nämä löydökset liittävät Ppt1-puutoksen kolesteroliaineenvaihdunnan häiriöön keskushermoston hermosoluissa. Kolmannessa osatyössä tutkittiin cathepsin D-entsyymin puutoksen vaikutusta myeliinin rakenteeseen ja rasva-aineenvaihduntaan aivoissa. Keskeisten myeliinin rakenneproteiinien määrät olivat muuttuneet cathepsin D-puutteisissa aivoissa ja myeliinin rakenne oli poikkeava. Poistogeenisissä aivoissa todettiin poikkeavat määrät myeliinille tyypillisiä rasva-aineita ja monityydyttymättömiä rasvahappoja viitaten valkean aineen eli myelinisoidun aivokudoksen tuhoon. Kolesterolin eritykseen liittyvien proteiinien määrät olivat muuttuneet poistogeenisissä aivoissa, joissa myös todettiin viisi kertaa enemmän kolesterolin varastomuotoa kolesteroliestereitä ja varastorasvojen, triglyseridien, määrän lasku verrattuna terveeseen kudokseen. Yhdessä nämä havainnot osoittavat, että cathepsin D-puutteisilla hiirillä on poikkeava myeliinin rakenne ja muutoksia aivojen kolesteroliaineenvaihdunnassa. Keskushermoston kolesteroliaineenvaihdunnan häiriöiden on arveltu myötävaikuttavan myös useiden keskushermostoa rappeuttavien sairauksien syntyyn. Näihin kuuluu yleisiä mutta tautimekanismiltaan monimutkaisia sairauksia, esimerkiksi Alzheimerin tauti. Aihetta on tutkittu runsaasti, mutta on edelleen epäselvää, millä mekanismeilla kolesteroliaineenvaihdunnan häiriöt vaikuttavat näiden sairauksien syntyyn ja etenemiseen. Kolesteroliaineenvaihdunnan ja keskushermoston rappeutumisen välistä yhteyttä voidaan valottaa käyttämällä tautimalleja, joissa on jonkin tunnetun yksittäisen geenin puutos. Näiden mallien avulla voidaan selvittää kolesteroliaineenvaihdunnan säätelyä terveessä keskushermostossa ja lisätä ymmärrystä niiden sairauksien kehittymisestä, joiden tautimekanismi on monimutkaisempi

MLN64 Is Involved in Actin-mediated Dynamics of Late Endocytic Organelles

MLN64 is a late endosomal cholesterol-binding membrane protein of an unknown function. Here, we show that MLN64 depletion results in the dispersion of late endocytic organelles to the cell periphery similarly as upon pharmacological actin disruption. The dispersed organelles in MLN64 knockdown cells exhibited decreased association with actin and the Arp2/3 complex subunit p34-Arc. MLN64 depletion was accompanied by impaired fusion of late endocytic organelles and delayed cargo degradation. MLN64 overexpression increased the number of actin and p34-Arc-positive patches on late endosomes, enhanced the fusion of late endocytic organelles in an actin-dependent manner, and stimulated the deposition of sterol in late endosomes harboring the protein. Overexpression of wild-type MLN64 was capable of rescuing the endosome dispersion in MLN64-depleted cells, whereas mutants of MLN64 defective in cholesterol binding were not, suggesting a functional connection between MLN64-mediated sterol transfer and actin-dependent late endosome dynamics. We propose that local sterol enrichment by MLN64 in the late endosomal membranes facilitates their association with actin, thereby governing actin-dependent fusion and degradative activity of late endocytic organelles

Respiratory complex I regulates dendritic cell maturation in explant model of human tumor immune microenvironment.

BACKGROUND: Combining cytotoxic chemotherapy or novel anticancer drugs with T-cell modulators holds great promise in treating advanced cancers. However, the response varies depending on the tumor immune microenvironment (TIME). Therefore, there is a clear need for pharmacologically tractable models of the TIME to dissect its influence on mono- and combination treatment response at the individual level. METHODS: Here we establish a patient-derived explant culture (PDEC) model of breast cancer, which retains the immune contexture of the primary tumor, recapitulating cytokine profiles and CD8+T cell cytotoxic activity. RESULTS: We explored the immunomodulatory action of a synthetic lethal BCL2 inhibitor venetoclax+metformin drug combination ex vivo, discovering metformin cannot overcome the lymphocyte-depleting action of venetoclax. Instead, metformin promotes dendritic cell maturation through inhibition of mitochondrial complex I, increasing their capacity to co-stimulate CD4+T cells and thus facilitating antitumor immunity. CONCLUSIONS: Our results establish PDECs as a feasible model to identify immunomodulatory functions of anticancer drugs in the context of patient-specific TIME