Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

National Institute of Allergy and Infectious Diseases, NIH

NGF increases expression and content of substance P in developing and mature spinal sensory neurons. The role this neurotrophin plays in peptide release, however, is less clear. Accordingly, we examined substance P release from cultures of mature rat sensory neurons, which do not require NGF for survival. Neurons grown without NGF have a low but detectable basal release, which increases with depolarization by KCI (50 mM) but never achieves statistical significance. In contrast, basal release is 3 times higher from neurons that have been cultured in the presence of NGF, and KCl depolarization triples the amount of SP released. Stimulation with capsaicin (10(-7) M) yields similar results. Residual peptide remaining after capsaicin stimulation is refractory to release for up to 24 h. Bradykinin does not induce SP secretion from mature neurons nor does it potentiate the action of capsaicin. GDNF, which also increases SP content, mimics NGF. Addition of NGF to the bath during release does not directly induce SP secretion, nor does it alter the effects of KCI, capsaicin, or bradykinin. It appears therefore that NGF increases SP release indirectly by increasing intracellular stores.

Nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) regulate substance P release in adult spinal sensory neurons.

NGF augmente l&#39;expression et le contenu des neurones sensoriels spinaux de la substance P dans le d&eacute;veloppement et mature. Le r&ocirc;le de cette neurotrophine dans lib&eacute;ration peptidique, cependant, est moins clair. En cons&eacute;quence, nous avons examin&eacute; la lib&eacute;ration de substance P de cultures de neurones sensoriels rat mature, qui ne n&eacute;cessitent pas de NGF pour la survie. Neurones cultiv&eacute;s sans NGF ont une faible mais d&eacute;tectable lib&eacute;ration basale, qui augmente avec la d&eacute;polarisation par KCI (50 mM) mais jamais atteint une signification statistique. En revanche, sorti basale est 3 fois plus de neurones qui ont &eacute;t&eacute; cultiv&eacute;es en pr&eacute;sence de NGF et d&eacute;polarisation de KCl triple la quantit&eacute; de SP. Stimulation avec capsa&iuml;cine (10(-7) M) donne des r&eacute;sultats similaires. Peptide r&eacute;siduel restant apr&egrave;s que la stimulation de la capsa&iuml;cine est r&eacute;fractaire &agrave; lib&eacute;rer jusqu&#39;&agrave; 24 h. la bradykinine n&#39;induit pas de s&eacute;cr&eacute;tion de SP de neurones matures ni il potentialise l&#39;action de la capsa&iuml;cine. GDNF, qui augmente &eacute;galement le contenu de la SP, imite NGF. Ajout de NGF au bain au cours de la lib&eacute;ration n&#39;induit pas directement la s&eacute;cr&eacute;tion de SP, ni Il alt&egrave;re les effets de KCI, capsa&iuml;cine ou la bradykinine. Il semble donc que NGF augmente la lib&eacute;ration de SP indirectement en augmentant les r&eacute;serves intracellulaires.

Nerve growth factor (NGF) et le facteur neurotrophique d&eacute;riv&eacute; de cellules gliales (GDNF) r&eacute;gulent la lib&eacute;ration de substance P dans les neurones sensoriels spinales adultes.

NGF aumenta l&#39;espressione e il contenuto dei neuroni sensoriali spinali sostanza P in via di sviluppo e maturo. Il ruolo che svolge questo neurotrophin nel rilascio del peptide, tuttavia, &egrave; meno chiaro. Di conseguenza, abbiamo esaminato il rilascio di sostanza P dalle colture di neuroni sensoriali ratto maturo, che non necessitano di NGF per la sopravvivenza. I neuroni coltivati senza NGF hanno un rilascio basale basso ma rilevabile, che aumenta con la depolarizzazione di KCI (50 mM) ma non raggiunge la significativit&agrave; statistica. Al contrario, rilasciato basale release &egrave; 3 volte pi&ugrave; elevato di neuroni che sono stati coltivati in presenza di NGF e depolarizzazione KCl triplica la quantit&agrave; di SP. Stimolazione con capsaicina (10(-7) M) produce risultati simili. Peptide residuo rimanente dopo stimolazione di capsaicina &egrave; refrattaria a rilasciare per fino a 24 h. bradichinina non indurre secrezione SP da neuroni maturi e non fa potenziare l&#39;azione della capsaicina. GDNF, che aumenta anche il contenuto di SP, imita NGF. Aggiunta di NGF al bagno durante il rilascio non induce direttamente la secrezione SP, n&eacute; fa alterare gli effetti della bradichinina, capsaicina o KCI. Sembra quindi che NGF aumenta rilascio SP indirettamente aumentando i depositi intracellulari.

Nerve growth factor (NGF) e fattore neurotrophic riga-derivato di cellule gliali (GDNF) regolano il rilascio di sostanza P nel adulti spinali neuroni sensoriali.

Complete nerve transection results in loss of sensation and paralysis of the involved extremity. Such injury drastically reduces content of the nociceptive peptides, substance P, and somatostatin in the dorsal horn of the spinal cord and dorsal root ganglia innervating the limb. Partial nerve injuries occur more commonly in clinical practice, however, and frequently result in the development of chronic neuropathic pain. To investigate mechanisms underlying this pathologic pain syndrome, rats were subjected to partial sciatic nerve transection. Withdrawal thresholds determined with Von Frey hairs dropped dramatically in the operated limb. On postoperative Day 4, thresholds had decreased from 15 g to less than 5 g on the operated side, whereas those on the contralateral (unoperated) side or those from sham-operated rats did not change. Sciatic hemisection had no effect on total content of either substance P or somatostatin in the dorsal spinal cord and lumbar dorsal root ganglia as measured by radioimmunoassay on postoperative Days 4, 7, or 14. However, when examined immunohistochemically, there was a marked redistribution of both peptides associated with partial transection. On the contralateral side or in sham-operated rats, both substance P and somatostatin were confined to the superficial laminae of the dorsal horn. By contrast, on the operated side, content of both peptides was reduced by more than half in the superficial laminae. There was a compensatory increase in content in the deeper laminae where nociceptive peptides are not usually found. Redistribution of substance P and somatostatin may be due to axonal sprouting, increased peptide expression by interneurons, or aberrant expression of nociceptive peptides by neurons normally mediating mechanical sensation. The presence of increased levels of nociceptive peptides in regions of the spinal cord that mediate innocuous sensation may underlie development of allodynia.

Partial sciatic nerve transection causes redistribution of pain-related peptides and lowers withdrawal threshold.

Transection nerveuse compl&egrave;te se traduit par la perte de sensation et de la paralysie du membre impliqu&eacute;. Telle blessure r&eacute;duit consid&eacute;rablement le contenu des peptides nociceptives, substance P et la somatostatine dans la corne dorsale des moelle &eacute;pini&egrave;re et des ganglions rachidiens innervant le limbe. L&eacute;sions des nerfs partielle surviennent plus fr&eacute;quemment dans la pratique clinique, cependant et fr&eacute;quemment d&eacute;boucher sur l&#39;&eacute;laboration de la douleur neuropathique chronique. Afin d&#39;&eacute;tudier les m&eacute;canismes qui sous-tendent ce syndrome de la douleur pathologique, a soumis des rats &agrave; la r&eacute;section partielle du nerf sciatique. Seuils de retrait d&eacute;termin&eacute;es avec des poils de Von Frey a chut&eacute; dramatiquement &agrave; la branche exploit&eacute;e. Sur 4 de jour postop&eacute;ratoire, seuils avaient diminu&eacute; de 15 g &agrave; moins de 5 g sur le c&ocirc;t&eacute; op&eacute;r&eacute;, tandis que ceux du c&ocirc;t&eacute; controlat&eacute;ral (op&eacute;r&eacute;s) ou ceux des rats op&eacute;r&eacute;s n&#39;a pas chang&eacute;. H&eacute;misection sciatique est sans effet sur la teneur totale de la substance P ou la somatostatine dans la moelle &eacute;pini&egrave;re dorsale et lombaire les ganglions rachidiens, telle que mesur&eacute;e par dosage radio-immunologique jours postop&eacute;ratoires, 4, 7 ou 14. Cependant, quand examen immunohistochimique, il y avait une redistribution nette des deux peptides associ&eacute;e &agrave; une r&eacute;section partielle. Du c&ocirc;t&eacute; controlat&eacute;ral ou chez les rats op&eacute;r&eacute;s, la substance P et la somatostatine &eacute;taient limit&eacute;s aux limbes superficielles de la corne dorsale. En revanche, du c&ocirc;t&eacute; op&eacute;r&eacute;, contenu des deux peptides a &eacute;t&eacute; r&eacute;duite de plus de la moiti&eacute; dans les limbes superficielles. Il y avait une augmentation compensatoire de contenu dans les limbes plus profondes o&ugrave; les peptides nociceptives sont trouvent pas habituellement. Redistribution de la substance P et la somatostatine peut &ecirc;tre due &agrave; la repousse axonale, expression accrue de peptide par les interneurones ou expression aberrante des peptides nociceptives de neurones normalement m&eacute;diation sensation m&eacute;canique. La pr&eacute;sence des niveaux accrus de peptides nociceptives dans les r&eacute;gions de la moelle &eacute;pini&egrave;re qui m&eacute;dient innofensive sensation pourrait expliquer le d&eacute;veloppement d&#39;allodynie.

R&eacute;section partielle du nerf sciatique provoque la redistribution des peptides associ&eacute;s &agrave; la douleur et abaisse le seuil de retrait.

Transezione completa del nervo si traduce in perdita di sensazione e paralisi dell&#39;arto coinvolto. Tali lesioni riducono drasticamente il contenuto dei peptidi nocicettivo, sostanza P e della somatostatina nel corno dorsale dei gangli del midollo spinale e spinali che innervano l&#39;arto. Lesioni nervose parziale si verificano pi&ugrave; comunemente nella pratica clinica, tuttavia e spesso il risultato nello sviluppo di dolore neuropatico cronico. Per indagare i meccanismi alla base di questa sindrome di dolore patologico, ratti sono stati sottoposti a transezione parziale nervo sciatico. Soglie di ritiro determinate con peli di Von Frey &egrave; sceso drasticamente l&#39;arto operato. 4 Giorno postoperatorio, soglie avevano diminuito da 15 g a meno di 5 g sul lato operato, mentre quelli sul lato controlaterale (non operato) o quelli da ratti sham-operated non cambia. Emisezione sciatico ha avuto alcun effetto sul contenuto totale della sostanza P o somatostatina nei gangli spinali lombari e dorso del midollo spinale come misurato da dosaggio radioimmunologico giorni postoperatori, 4, 7 o 14. Tuttavia, quando ha esaminato Immunoistochimicamente, c&#39;era una marcata ridistribuzione di entrambi peptidi associati transezione parziale. Sul lato controlaterale o nei ratti sham-operated, sostanza P e la somatostatina furono confinati alle lamine superficiali del corno dorsale. Al contrario, dal lato operato, contenuto di entrambi i peptidi &egrave; stato ridotto di oltre la met&agrave; in lamine superficiali. C&#39;era un aumento compensatorio nel contenuto in lamine pi&ugrave; profonde dove nocicettivo peptidi non sono trovati solitamente. Ridistribuzione della sostanza P e la somatostatina pu&ograve; essere dovuto spuntano assonale, espressione aumentata del peptide di interneurons o aberrante espressione di peptidi nocicettivo dai neuroni normalmente mediando sensazione meccanica. La presenza di elevati livelli di peptidi nocicettivo nelle regioni del midollo spinale che mediano la sensazione innocuo pu&ograve; essere alla base di sviluppo di esso.

Transezione parziale nervo sciatico provoca la ridistribuzione dei peptidi correlati a dolore e abbassa la soglia di ritiro.

Sexual dimorphism of white matter has not been considered important, the assumption being that sex hormones are not essential for glial development. We recently showed exogenous hormones in vivo differentially regulate in male and female rodents the life span of oligodendrocytes (Olgs) and amount of myelin (Cerghet et al. [2006] J. Neurosci. 26:1439-1447). To determine which hormones regulate male and female Olg development, we prepared enriched Olg cultures grown in serum-free medium with estrogen (E2), progesterone (P2), and dihydrotestosterone (DHT) or their combinations. P2 significantly increased the number of Olgs in both sexes, but more so in females; E2 had minor effects on Olg numbers; and DHT reduced Olgs numbers in both sexes, but more so in females. Combinations of hormones affected Olg numbers differently from single hormones. The change in Olg numbers was due to changes not in proliferation but rather in survival. P2 increased pAKT by many-fold, but MAPK levels were unchanged, indicating that activation of the Akt pathway by P2 is sufficient to regulate Olg differentiation. DHT reduced pAkt in both sexes but differentially increased pMAPK in males and decreased it in females. Stressing Olgs reveals that both sexes are protected by P2, but females are slightly better protected than males. Females always showed greater differences than males regarding changes in Olg numbers and in signaling molecules. Given the greater fluctuation of neurosteroids in women than in men and the higher incidence of multiple sclerosis (MS) in women, these sexually dimorphic differences may contribute to differences in male and female MS lesions.

Sexual dimorphism of oligodendrocytes is mediated by differential regulation of signaling pathways.

Dimorphisme sexuel de la substance blanche n&#39;est pas jug&eacute;e important, l&#39;hypoth&egrave;se &eacute;tant que les hormones sexuelles ne sont pas essentiels pour le d&eacute;veloppement glial. Nous avons montr&eacute; r&eacute;cemment exog&egrave;nes hormones in vivo r&eacute;glementent diff&eacute;remment chez les rongeurs m&acirc;les et femelles la dur&eacute;e de vie des oligodendrocytes (paliers) et le montant de la my&eacute;line (Cerghet et al., [2006] J. Neurosci. Distributional-1447). Pour d&eacute;terminer quelles hormones r&eacute;gulent d&eacute;veloppement de Olg m&acirc;le et femelle, nous avons pr&eacute;par&eacute; enrichis Olg cultures cultiv&eacute;es dans un milieu sans s&eacute;rum avec les &oelig;strog&egrave;nes (E2), de progest&eacute;rone (P2) et de dihydrotestost&eacute;rone (DHT) ou de leurs combinaisons. P2 a consid&eacute;rablement augment&eacute; le nombre de paliers chez les deux sexes, mais encore plus chez les femelles ; E2 ont des effets mineurs sur les nombres de l&#39;Olg ; et DHT r&eacute;duit le nombre de paliers chez les deux sexes, mais encore plus chez les femelles. Combinaisons d&#39;hormones touch&eacute;s Olg num&eacute;ros diff&eacute;remment des hormones unique. Le changement de num&eacute;ros de l&#39;Olg est due aux changements pas dans la prolif&eacute;ration, mais plut&ocirc;t dans la survie. P2 pAKT augment&eacute; de plusieurs fois, mais des niveaux MAPK sont demeur&eacute;es inchang&eacute;es, indiquant que l&#39;activation de l&#39;Akt de P2 est suffisante pour r&eacute;guler la diff&eacute;renciation de l&#39;Olg. DHT r&eacute;duit pAkt chez les deux sexes mais pMAPK diff&eacute;rentiellement accrue chez les m&acirc;les et il a diminu&eacute; chez les femmes. Soulignant les paliers r&eacute;v&egrave;le que les deux sexes sont prot&eacute;g&eacute;s par P2, mais les femelles sont un peu mieux prot&eacute;g&eacute;es que les hommes. Les femelles ont toujours des diff&eacute;rences plus que les hommes au sujet des changements en nombre Olg et mol&eacute;cules de signalisation. Compte tenu de la plus grande fluctuation des neurosteroids chez les femmes que chez les hommes et l&#39;incidence plus &eacute;lev&eacute;e de la scl&eacute;rose en plaques (MS) chez les femmes, ces diff&eacute;rences de dimorphisme sexuel peuvent contribuer aux diff&eacute;rences entre m&acirc;les et femelles des l&eacute;sions de SP.

Le dimorphisme sexuel des oligodendrocytes est m&eacute;di&eacute; par la r&eacute;gulation diff&eacute;rentielle des voies de signalisation.

Dimorfismo sessuale della materia bianca &egrave; non stato considerato importante, il presupposto &egrave; che gli ormoni sessuali non sono essenziali per lo sviluppo glia. Abbiamo recentemente mostrato esogeni ormoni in vivo differenzialmente regolano nel maschi e femmine roditori la durata della vita degli oligodendrociti (Olgs) e la quantit&agrave; di mielina (Cerghet et al [2006] J. Neurosci. 26:1439-1447). Per determinare quali ormoni regolano lo sviluppo di Olg maschio e femmina, abbiamo preparato arricchiti Olg colture coltivate in media serum-free con estrogeni (E2), progesterone (P2) e diidrotestosterone (DHT) o loro combinazioni. P2 significativamente aumentato il numero di Olgs in entrambi i sessi, ma ancora di pi&ugrave; nelle femmine; E2 aveva effetti minori sulla Olg numeri; e DHT ridotto Olgs numeri in entrambi i sessi, ma ancora di pi&ugrave; nelle femmine. Combinazioni di ormoni influenzati Olg numeri in modo diverso dal singoli ormoni. Il cambiamento di Olg numeri &egrave; stato a causa di modifiche non proliferazione ma piuttosto nella sopravvivenza. P2 aumentato patto di molti-piega, ma livelli di MAPK erano immutati, che indica che l&#39;attivazione del pathway di Akt da P2 &egrave; sufficiente a regolare la differenziazione Olg. DHT ridotto patto in entrambi i sessi ma pMAPK differenziale maggiore nei maschi e sono diminuiti nelle femmine. Sottolineando Olgs rivela che entrambi i sessi sono protetti dalla P2, ma le femmine sono leggermente meglio protetti rispetto ai maschi. Femmine sempre mostrate differenze maggiore rispetto ai maschi per quanto riguarda le modifiche in Olg numeri e molecole di segnalazione. Data la maggiore fluttuazione di neurosteroids nelle donne che negli uomini e la pi&ugrave; alta incidenza di sclerosi multipla (MS), nelle donne, queste differenze sessualmente dimorfici possono contribuire alle differenze nelle lesioni MS maschile e femmine.

Dimorfismo sessuale di oligodendrociti &egrave; mediata dal regolamento differenziale delle vie di segnalazione.

Sexual dimorphism of astrocytes and neurons is well documented in many brain and spinal cord structures. Sexual dimorphism of oligodendrocytes (Olgs) and myelin has received less attention. We recently showed that density of Olgs in corpus callosum, fornix, and spinal cord of wild-type male rodents is more densely packed than in females; myelin proteins and myelin gene expression are likewise greater in males than in female rodents. However, glial cell proliferation and cell death were two times greater in female corpus callosum. Endogenous sex hormones, specifically lack of androgens, produce an Olg female phenotype in castrated male mouse. In vitro studies using Olgs culture also showed differences between males and females Olg survival and signaling pathways in response to sexual hormones. Sexual dimorphism of white matter tracts and glia in rodents indicates the necessity for controlling gender in the experimental studies of neurodegenerative disorders. Most importantly, our studies suggest that hormones may contribute to sexual dimorphism observed in certain human diseases including multiple sclerosis.

Sexual dimorphism in the white matter of rodents.

Le dimorphisme sexuel des neurones et les astrocytes est bien document&eacute; dans nombreuses structures de cerveau et la moelle &eacute;pini&egrave;re. Le dimorphisme sexuel des oligodendrocytes (paliers) et la my&eacute;line a re&ccedil;u moins d&#39;attention. R&eacute;cemment, nous avons montr&eacute; que la densit&eacute; des paliers dans le corps calleux, fornix et la moelle &eacute;pini&egrave;re des rongeurs m&acirc;les type sauvage est plus dense que chez les femelles ; les prot&eacute;ines de la my&eacute;line et l&#39;expression g&eacute;nique de la my&eacute;line sont &eacute;galement plus &eacute;lev&eacute;s chez les hommes que chez les rongeurs femelles. Cependant, la prolif&eacute;ration des cellules gliales et mort cellulaire &eacute;taient deux fois plus &eacute;lev&eacute;s en femelle corps calleux. Hormones endog&egrave;nes de sexe, plus particuli&egrave;rement l&#39;absence d&#39;androg&egrave;nes, produire un ph&eacute;notype femelle Olg chez des souris m&acirc;les castr&eacute;s. &Eacute;tudes in vitro utilisant des paliers culture &eacute;galement a montr&eacute; des diff&eacute;rences entre m&acirc;les et femelles Olg survie et signaling pathways en r&eacute;ponse aux hormones sexuelles. Le dimorphisme sexuel des &eacute;tendues de la substance blanche et de la glie dans rongeurs indique la n&eacute;cessit&eacute; de contr&ocirc;le entre les sexes dans les &eacute;tudes exp&eacute;rimentales de maladies neurod&eacute;g&eacute;n&eacute;ratives. Plus important encore, nos &eacute;tudes sugg&egrave;rent que les hormones peuvent contribuer &agrave; dimorphisme sexuel observ&eacute; dans certaines maladies humaines, y compris la scl&eacute;rose en plaques.

Dimorphisme sexuel dans la substance blanche des rongeurs.

Dimorfismo sessuale di astrociti e neuroni &egrave; ben documentato in molte strutture del cervello e del midollo spinale. Dimorfismo sessuale degli oligodendrociti (Olgs) e guaina mielinica ha ricevuto meno attenzione. Abbiamo recentemente dimostrato che la densit&agrave; di Olgs nel corpo calloso, fornice e midollo spinale di roditori maschi wild-type &egrave; pi&ugrave; densamente compresso rispetto nelle femmine; proteine della mielina ed espressione genica mielina sono similarmente maggiore nei maschi rispetto femmine roditori. Tuttavia, proliferazione delle cellule gliali e cellule morte erano due volte maggiore nel sesso femmina del corpo calloso. Ormoni sessuali endogeni, in particolare la mancanza di androgeni, produrre un fenotipo femminile Olg nel topo maschio castrato. Studi in vitro utilizzando anche Olgs cultura ha mostrato differenze tra maschi e femmine Olg sopravvivenza e signaling pathways in risposta agli ormoni sessuali. Dimorfismo sessuale di tratti di sostanza bianca e glia in roditori indica la necessit&agrave; per il controllo di genere negli studi sperimentali di malattie neurodegenerative. Ancora pi&ugrave; importante, i nostri studi suggeriscono che gli ormoni possono contribuire a dimorfismo sessuale osservata in alcune malattie umane tra cui la sclerosi multipla.

Dimorfismo sessuale nella materia bianca dei roditori.

Breast epithelial cells develop into polarized and highly organized acinar and ductal structures in response to stromal cues, including extracellular matrix composition and density, which can in part be reproduced in 3D culture conditions. Here, we present the effects of various 3D in vitro stroma compositions (termed "matrices" or "substrates") on the ability of heterotypic cultures of epithelial and mesenchymal stem cells to organize into acinar and tubular structures. Normal murine mammary gland (NMuMG) cells were cultured, either alone or in combination (30:70) with mouse mesenchymal stem cells (D1), in 3D matrices generated by agarose, collagen, and Matrigel alone or by a combination thereof. After 3-5 d in culture, cell distribution, organization, and the presence of acinus-like and tubule-like structures were determined. The number of acinar structures was significantly higher in cultures grown in combination matrices of agarose with Matrigel or collagen I when compared with cultures grown in Matrigel or collagen I alone (p < 0.05). No tubular structures were formed when agarose was included in the matrix, regardless of the combination. In Matrigel, but not in collagen I/Matrigel microenvironment, the number of tubular structures was significantly increased in NMuMG/D1 coculture when compared with culture of NMuMG cells alone (p < 0.05). By immunohistochemical analysis, NMuMG cells cocultured with D1 cells were shown to form acinar structures with the NMuMG epithelial cells surrounding a lumen composed of dead cells while the D1 cells were mostly peripheral. Immunostaining for laminin indicated the presence of basement membrane when NMuMG cells were grown in Matrigel alone or cocultured with D1 cells in a combination of Matrigel and collagen I. These results indicate that the physical and biochemical properties of the matrix and cellular composition alter the organization of the mammary gland.

Matrix compositions and the development of breast acini and ducts in 3D cultures.

Les cellules &eacute;pith&eacute;liales mammaires se d&eacute;veloppent en structures acineuses et canalaires polaris&eacute;es et hautement organis&eacute;es, en r&eacute;action aux signaux stromales, y compris la composition de la matrice extracellulaire et la densit&eacute;, qui peut en partie &ecirc;tre reproduite dans des conditions de culture 3D. Nous pr&eacute;sentons ici les effets de diverses compositions de stroma in vitro 3D (appel&eacute;es &laquo; matrices &raquo; ou &laquo; substrats &raquo;) sur la capacit&eacute; des cultures h&eacute;t&eacute;rotypiques des cellules souches &eacute;pith&eacute;liales et m&eacute;senchymateuses d&#39;organiser en structures acineuses et tubulaires. Les cellules de la glande mammaire murine normale (NMuMG) ont &eacute;t&eacute; cultiv&eacute;s, seul ou en combinaison (30: 70) avec la souris des cellules souches m&eacute;senchymateuses (D1), dans des matrices 3D g&eacute;n&eacute;r&eacute;es par d&#39;agarose, collag&egrave;ne et Matrigel seul ou par une combinaison des deux. Apr&egrave;s 3-5 jours en culture, cellule distribution, organisation et la pr&eacute;sence de structures de type tubule et acinus ont &eacute;t&eacute; d&eacute;termin&eacute;es. Le nombre de structures acineuses &eacute;tait significativement plus &eacute;lev&eacute; dans les cultures dans des matrices de combinaison d&#39;agarose avec Matrigel ou collag&egrave;ne I comparativement aux cultures en Matrigel ou collag&egrave;ne je seul (p &lt; 0,05). Pas de structures tubulaires ont &eacute;t&eacute; form&eacute;es quand agarose est incluse dans la matrice, quelle que soit la combinaison. Matrigel, mais pas dans le collag&egrave;ne I / Matrigel microenvironnement, le nombre de structures tubulaires a augment&eacute; significativement en co-culture NMuMG/D1 par rapport &agrave; la culture de cellules NMuMG seuls (p &lt; 0,05). Par analyse immunohistochimique, NMuMG cellules co-cultiv&eacute;es avec des cellules de D1 ont &eacute;t&eacute; montr&eacute;s pour former des structures acineuses avec la NMuMG des cellules &eacute;pith&eacute;liales entourant une lumi&egrave;re compos&eacute;es de cellules mortes tandis que les cellules D1 &eacute;taient pour la plupart p&eacute;riph&eacute;riques. Immunostaining pour laminine a r&eacute;v&eacute;l&eacute; la pr&eacute;sence de la membrane basale quand les cellules de NMuMG ont &eacute;t&eacute; cultiv&eacute;s en Matrigel seuls ou co-cultiv&eacute;es avec des cellules de D1 dans une combinaison de Matrigel et collag&egrave;ne I. Ces r&eacute;sultats indiquent que les propri&eacute;t&eacute;s physiques et biochimiques de la composition de la matrice et cellulaire modifient l&#39;organisation de la glande mammaire.

Compositions de matrice et le d&eacute;veloppement des acini mammaires et conduits dans des cultures 3D.

Cellule epiteliali mammarie si sviluppano in strutture acinose e duttale polarizzate e altamente organizzate in risposta a segnali stromale, compresa la composizione della matrice extracellulare e densit&agrave;, che in parte pu&ograve; essere riprodotta in condizioni di coltura 3D. Presentiamo qui, gli effetti delle varie composizioni 3D stroma in vitro (chiamati &quot;matrici&quot; o &quot;substrati&quot;) sulla capacit&agrave; di heterotypic culture di cellule staminali mesenchimali ed epiteliali di organizzare in strutture acinose e tubolari. Le cellule normali murino mammaria ghiandola (NMuMG) erano coltivate, da soli o in combinazione (30: 70) con cellule staminali mesenchimali del mouse (D1), in matrici 3D generati da agarosio, collagene e Matrigel da sola o da una combinazione di questi. Dopo 3-5 d in cultura, distribuzione delle cellule, l&#39;organizzazione e la presenza di strutture simil-uva e tubulo-come sono stati determinati. Il numero di strutture acinose era significativamente pi&ugrave; alto nelle colture coltivate in matrici di combinazione di agarosio con Matrigel o collagene quando confrontato con colture coltivate in Matrigel o collagene da solo (p &lt; 0,05). No strutture tubolari si formarono quando dell&#39;agarosi &egrave; stata inclusa nella matrice, indipendentemente dalla combinazione. In Matrigel, ma non nel collagene I / Matrigel microambiente, il numero di strutture tubolari era significativamente aumentato in NMuMG/D1 coculture quando confrontato con la coltura delle cellule di NMuMG da solo (p &lt; 0,05). Analisi immunoistochimica, NMuMG cellule cocultured con cellule D1 sono state mostrate a formare strutture acinose con NMuMG cellule epiteliali che circonda un lume composto da cellule morte mentre le cellule D1 erano per lo pi&ugrave; periferiche. Immunocolorazione per laminina indicato la presenza della membrana basale quando le cellule NMuMG sono state coltivate in Matrigel da solo o cocultured con cellule D1 in una combinazione di Matrigel e collagene I. Questi risultati indicano che le propriet&agrave; fisiche e biochimiche della composizione matrice e cellulari alterano l&#39;organizzazione della ghiandola mammaria.

Composizioni di matrice e lo sviluppo degli acini mammella e condotti nelle culture 3D.

We investigated the effects of the endothelin-1 (ET-1) receptor dual antagonist (BosentanÂ®) on the inflammatory cytokines and the chemoattractant molecules associated with breast cancer growth and the development of tumor infiltration in bone explants. Immunocompetent mice implanted with the murine mammary carcinoma 4T1 cells in a skin-fold chamber and treated with BosentanÂ® had reduced tumor growth (p < .05). ET-1 promoted the secretion of the anti-inflammatory soluble tumor necrosis factor (TNF) receptor and IL12 p40 in vitro. The BosentanÂ® treatment in vivo was associated with a local increase of the anti-inflammatory IL-1Î± cytokine concentration and decrease of the pro-inflammatory TNF-Î± and IL-17 cytokine concentrations (p < .05).

The endothelin axis stimulates the expression of pro-inflammatory cytokines and pro-migratory molecules in breast cancer.

Nous avons &eacute;tudi&eacute; les effets de l&#39;antagoniste double de l&#39;endoth&eacute;line-1 (ET-1) r&eacute;cepteur (Bosentan &reg;) sur les cytokines inflammatoires et les mol&eacute;cules de facteur chimiotactique associ&eacute;es &agrave; la croissance du cancer du sein et le d&eacute;veloppement d&#39;infiltration de la tumeur osseuse des explants. Souris immunocomp&eacute;tentes implant&eacute; les cellules 4 t 1 de carcinome mammaire murin dans une chambre de pli de la peau et trait&eacute;s par Bosentan &reg; avaient r&eacute;duit la croissance tumorale (p &lt;.05. ET-1 promu la s&eacute;cr&eacute;tion du receveur anti-inflammatoire soluble du facteur de n&eacute;crose de tumeur (TNF) et IL12 p40 in vitro. Le Bosentan &reg; traitement in vivo a &eacute;t&eacute; associ&eacute; &agrave; une augmentation locale de la concentration de cytokine IL-1&alpha; anti-inflammatoires et diminution de la concentration de cytokines pro-inflammatoires TNF-&alpha; et IL-17 (p &lt;.05.

L&#39;axe de l&#39;endoth&eacute;line stimule l&#39;expression des cytokines pro-inflammatoires et des mol&eacute;cules pro-migrateurs dans le cancer du sein.

Abbiamo studiato gli effetti dell&#39;endotelina-1 (ET-1) del recettore dual antagonista (Bosentan &reg;) sulle citochine infiammatorie e molecole chemioattrattanti associate con la crescita del cancro al seno e lo sviluppo di infiltrazione tumorale in osso espianti. Immunitarie topi impiantati con cellule carcinoma mammario murino 4T1 in una camera di inoculazione e trattati con Bosentan &reg; avevano ridotto la crescita del tumore (p &lt;,05 dentellatura ET-1 promosso la secrezione del recettore anti-infiammatori solubile fattore di necrosi tumorale (TNF) e IL12 p40 in vitro. Il Bosentan &reg; trattamento in vivo &egrave; stata associata con un aumento locale della concentrazione di citochine IL-1&alpha; anti-infiammatorio e diminuire le concentrazioni di citochine pro-infiammatorie TNF-&alpha; e IL-17 (p &lt;,05 dentellatura

L&#39;asse delle endoteline stimola l&#39;espressione di citochine pro-infiammatorie e molecole pro-migratori nel cancro della mammella.

Activator and inhibitor roles for the 88-kDa-secreted glycoprotein progranulin (PGRN) have been demonstrated in ovarian cancer cells. Here, we investigated the effects of PGRN in breast cancer migration. Testing MCF7, MDA-MB-453, and MDA-MB-231 human breast cancer cells and the MCF10A breast epithelial cell line, we demonstrate that LPA-induced PGRN stimulation led to a significant increase in cell invasion of MDA-MB-453 and MDA-MB-231 cells only (p<0.05). Moreover, incubation with an anti-PGRN antibody, an inhibitor of the ERK pathway (PD98059) or both in combination inhibited the ability of MDA-MB-231 cells to invade. Furthermore, the expression of focal adhesion kinases promoted by LPA-induced PGRN was also inhibited by PD98059 alone or in combination with an anti-PGRN antibody (p<0.05). Taken together, these results suggest that the LPA activation of PGRN involving the ERK pathway is critical to promote MDA-MB-231 breast cancer cell invasion.

Progranulin stimulated by LPA promotes the migration of aggressive breast cancer cells.

R&ocirc;les activateur et inhibiteur de la glycoprot&eacute;ine s&eacute;cr&eacute;t&eacute;e 88-kDa progranuline (PGRN) ont &eacute;t&eacute; d&eacute;montr&eacute;es dans des cellules de cancer de l&#39;ovaire. Ici, nous avons &eacute;tudi&eacute; les effets du PGRN dans migration du cancer du sein. Essai MCF7, MDA-MB-453 et les cellules de cancer du sein humain MDA-MB-231 et la lign&eacute;e de cellules &eacute;pith&eacute;liales mammaires MCF10A, nous d&eacute;montrons que la stimulation induite par l&#39;APL PGRN a conduit &agrave; une augmentation significative de l&#39;invasion des cellules des cellules MDA-MB-453 et MDA-MB-231 seulement (p &lt; 0,05). En outre, incubation avec un anticorps anti-PGRN, un inhibiteur de la voie ERK (le PD98059) ou les deux en combinaison inhibe la capacit&eacute; des cellules MDA-MB-231 &agrave; envahir. En outre, l&#39;expression des kinases d&#39;adh&eacute;rence focale promue par PGRN induite par le LPA est aussi inhib&eacute;e par le PD98059 seul ou en combinaison avec un anticorps anti-PGRN (p &lt; 0,05). Pris ensemble, ces r&eacute;sultats sugg&egrave;rent que l&#39;activation de l&#39;APL du PGRN impliquant la voie ERK est essentielle pour promouvoir l&#39;invasion des cellules MDA-MB-231 breast cancer.

Progranuline stimul&eacute;e par la LPA favorise la migration des cellules canc&eacute;reuses du sein agressif.

Attivatore e inibitore di ruoli per la glicoproteina secreta 88-kDa progranulin (PGRN) sono stati dimostrati in cellule di carcinoma ovarico. Qui, abbiamo studiato gli effetti di PGRN in migrazione di cancro al seno. Test MCF7, MDA-MB-453 e le cellule di cancro al seno umano MDA-MB-231 e la linea di cellule epiteliali del seno MCF10A, dimostriamo che la stimolazione indotta da LPA PGRN ha portato a un aumento significativo di invasione delle cellule delle cellule MDA-MB-453 e MDA-MB-231 solo (p &lt; 0,05). Inoltre, incubazione con un anticorpo anti-PGRN, un inibitore della via del ERK (PD98059) o entrambe in combinazione inibito la capacit&agrave; di invadere le cellule MDA-MB-231. Inoltre, l&#39;espressione della chinasi di adesione focale promosso da PGRN LPA-indotta &egrave; stata inibita anche da PD98059 da solo o in combinazione con un anticorpo anti-PGRN (p &lt; 0,05). Presi insieme, questi risultati suggeriscono che l&#39;attivazione di LPA di PGRN via del ERK &egrave; fondamentale per promuovere l&#39;invasione di MDA-MB-231 breast cancer cell.

PROGRANULIN stimolata da LPA promuove la migrazione delle cellule di cancro al seno aggressivo.

Invasive candidiasis is the 4(th) leading cause of nosocomial bloodstream infection in the US with mortality that exceeds 40% despite administration of antifungal therapy; neutropenia is a major risk factor for poor outcome after invasive candidiasis. In a fatal mouse model of invasive candidiasis that mimics human bloodstream-derived invasive candidiasis, the most highly infected organ is the kidney and neutrophils are the major cellular mediators of host defense; however, factors regulating neutrophil recruitment have not been previously defined. Here we show that mice lacking chemokine receptor Ccr1, which is widely expressed on leukocytes, had selectively impaired accumulation of neutrophils in the kidney limited to the late phase of the time course of the model; surprisingly, this was associated with improved renal function and survival without affecting tissue fungal burden. Consistent with this, neutrophils from wild-type mice in blood and kidney switched from Ccr1(lo) to Ccr1(high) at late time-points post-infection, when Ccr1 ligands were produced at high levels in the kidney and were chemotactic for kidney neutrophils ex vivo. Further, when a 1âˆ¶1 mixture of Ccr1(+/+) and Ccr1(-/-) donor neutrophils was adoptively transferred intravenously into Candida-infected Ccr1(+/+) recipient mice, neutrophil trafficking into the kidney was significantly skewed toward Ccr1(+/+) cells. Thus, neutrophil Ccr1 amplifies late renal immunopathology and increases mortality in invasive candidiasis by mediating excessive recruitment of neutrophils from the blood to the target organ.

Chemokine receptor Ccr1 drives neutrophil-mediated kidney immunopathology and mortality in invasive candidiasis.

Candidose invasive est la 4(th), principale cause de l&#39;infection nosocomiale circulation sanguine aux &Eacute;tats-Unis avec la mortalit&eacute; qui d&eacute;passe 40 % malgr&eacute; l&#39;administration d&#39;un traitement antifongique ; la neutrop&eacute;nie est un facteur de risque majeur pour les mauvais pronostic apr&egrave;s la candidose invasive. Dans un mod&egrave;le de souris fatale de candidose invasive qui imite la candidose invasive &agrave; d&eacute;riv&eacute; de sang humaine, l&#39;organe plus fortement infect&eacute; est le rein et les neutrophiles sont les principaux m&eacute;diateurs cellulaires de d&eacute;fense de l&#39;h&ocirc;te ; Cependant, facteurs r&eacute;gulant les neutrophil recrutement n&#39;ont pas &eacute;t&eacute; pr&eacute;c&eacute;demment d&eacute;finis. Ici, nous montrons que les souris d&eacute;pourvues du r&eacute;cepteur de chimiokine Ccr1, largement exprim&eacute; sur les leucocytes, avaient alt&eacute;r&eacute;e s&eacute;lectivement accumulation de neutrophiles dans le rein, limit&eacute; &agrave; la fin de la phase de l&#39;&eacute;volution temporelle du mod&egrave;le ; &eacute;tonnamment, c&#39;&eacute;tait associ&eacute;e de l&#39;am&eacute;lioration de la fonction r&eacute;nale et de la survie sans affecter le fardeau fongique de tissu. Conform&eacute;ment &agrave; cette, neutrophiles de souris de type sauvage dans le sang et les reins, pass&eacute; de Ccr1(lo) &agrave; Ccr1(high) &agrave; la note de fin-points dans le temps, quand Ccr1 ligands ont &eacute;t&eacute; produits &agrave; des niveaux &eacute;lev&eacute;s dans les reins et sont chimiotactiques pour les polynucl&eacute;aires neutrophiles rein ex vivo. En outre, quand un m&eacute;lange de 1&#x2236;1 de Ccr1(+/+) et Ccr1(-/-) neutrophiles donneur a &eacute;t&eacute; transf&eacute;r&eacute; Moutschen par voie intraveineuse dans les b&eacute;n&eacute;ficiaires souris infect&eacute;es par Candida Ccr1(+/+), traite des neutrophiles dans le rein a &eacute;t&eacute; consid&eacute;rablement biais&eacute;e vers Ccr1(+/+) cellules. Ainsi, neutrophiles Ccr1 amplifie fin immunopathologie r&eacute;nale et augmente la mortalit&eacute; dans la candidose invasive par m&eacute;diation recrutement excessif des neutrophiles dans le sang &agrave; l&#39;organe cible.

R&eacute;cepteur de chimiokine Ccr1 disques immunopathologie r&eacute;nale m&eacute;di&eacute;e par les neutrophiles et la mortalit&eacute; dans la candidose invasive.

Candidiasi invasiva &egrave; la 4(th) che porta la causa di infezione nosocomiale flusso sanguigno negli Stati Uniti con mortalit&agrave; che supera il 40% nonostante la somministrazione della terapia antimicotica; neutropenia &egrave; un importante fattore di rischio per una prognosi sfavorevole dopo candidiasi invasiva. In un modello murino fatale di candidiasi invasiva che imita la candidiasi invasiva derivati da sangue umana, l&#39;organo pi&ugrave; altamente infetto &egrave; il rene e i neutrofili sono i principali mediatori cellulari di difesa ospite; Tuttavia, fattori che regolano il reclutamento dei neutrofili non sono stati precedentemente definiti. Qui dimostriamo che i topi privi di chemokine receptor Ccr1, che &egrave; ampiamente espresso sui leucociti, selettivamente avevano alterato accumulo di neutrofili nel rene limitato per la fine della fase del decorso del modello; Sorprendentemente, questo &egrave; stato associato con miglioramento della funzione renale e la sopravvivenza senza intaccare l&#39;onere fungine dei tessuti. Coerentemente con questo, i neutrofili da topi wild-type nel sangue e dei reni, passato da Ccr1(lo) a Ccr1(high) a fine tempo-punti post-infection, quando Ccr1 ligandi sono state prodotte ad alti livelli nel rene e furono chemiotattiche per neutrofili Rene ex vivo. Ulteriormente, quando una miscela di 1&#x2236;1 di Ccr1(+/+) e Ccr1(-/-) neutrofili donatore adoptively fu trasferita per via endovenosa in topi destinatari Ccr1(+/+) infetti da Candida, traffico dei neutrofili nel rene era notevolmente inclinata verso le cellule Ccr1(+/+). Cos&igrave;, neutrofili Ccr1 amplifica tardo Immunopatologia renale e aumenta la mortalit&agrave; nella candidiasi invasiva di mediazione eccessivo reclutamento dei neutrofili dal sangue all&#39;organo bersaglio.

Chemokine receptor Ccr1 unit&agrave; Immunopatologia renale mediata da neutrofili e mortalit&agrave; nella candidiasi invasiva.

The extracellular matrix (ECM) contributes to the generation and dynamic of normal breast tissue, in particular to the generation of polarized acinar and ductal structures.â€‰In vitro 3D culture conditions, including variations in the composition of the ECM, have been shown to directly influence the formation and organization of acinus-like and duct-like structures. Furthermore, the density of the ECM appears to also play a role in the normal mammary tissue and tumour formation. Here we show that the density of the ECM directly influences the number, organization and function of breast acini. Briefly, non-malignant human breast MCF10A cells were incubated in increasing densities of a MatrigelÂ®-collagen I matrix. Elastic moduli near and distant to the acinus structures were measured by atomic force microscopy, and the number of acinus structures was determined. Immunochemistry was used to investigate the expression levels of E-cadherin, laminin, matrix metalloproteinase-14 and ÃŸ-casein in MCF10A cells. The modulus of the ECM was significantly increased near the acinus structures and the number of acinus structures decreased with the increase in Matrigel-collagen I density. As evaluated by the expression of laminin, the organization of the acinus structures present was altered as the density of the ECM increased. Increases in both E-cadherin and MMP14 expression by MCF10A cells as ECM density increased were also observed. In contrast, MCF10A cells expressed lower ÃŸ-casein levels as the ECM density increased. Taken together, these observations highlight the key role of ECM density in modulating the number, organization and function of breast acini. Copyright Â© 2013 John Wiley & Sons, Ltd.

Increased extracellular matrix density decreases MCF10A breast cell acinus formation in 3D culture conditions.

La matrice extracellulaire (ECM) contribue &agrave; la g&eacute;n&eacute;ration dynamique du tissu mammaire normal et en particulier &agrave; la cr&eacute;ation des structures acineuses et canalaire polaris&eacute;e.&thinsp;Des conditions de culture in vitro de 3D, y compris les variations dans la composition de l&#39;ECM, montrent d&#39;influencer directement la formation et l&#39;Organisation des structures conduit-type et acinus. En outre, la densit&eacute; de l&#39;ECM semble &eacute;galement jouer un r&ocirc;le dans le tissu mammaire normal et de la formation de tumeurs. Ici, nous montrons que la densit&eacute; de l&#39;ECM influe directement sur le nombre, l&#39;organisation et la fonction des acini mammaires. En bref, les b&eacute;nignes mammaires humains MCF10A cellules ont &eacute;t&eacute; incub&eacute;es en augmentant la densit&eacute; d&#39;un collag&egrave;ne de Matrigel &reg; j&#39;ai matrice. Modules d&#39;&eacute;lasticit&eacute; proches et lointains aux structures acinus ont &eacute;t&eacute; mesur&eacute;es par microscopie &agrave; force atomique, et le nombre de structures de l&#39;acinus a &eacute;t&eacute; d&eacute;termin&eacute;. Immunochimie a permis d&#39;&eacute;tudier les niveaux d&#39;expression de la E-cadh&eacute;rine, laminine, matrix metalloproteinase-14 et &szlig;-cas&eacute;ine dans les cellules MCF10A. Le module de l&#39;ECM a &eacute;t&eacute; significativement augment&eacute; pr&egrave;s les structures de l&#39;acinus, et le nombre de structures de l&#39;acinus diminue avec l&#39;augmentation de Matrigel-collag&egrave;ne I densit&eacute;. Telle qu&#39;&eacute;valu&eacute;e par l&#39;expression de la laminine, l&#39;Organisation des structures acinus actuelles a &eacute;t&eacute; modifi&eacute;e comme la densit&eacute; de l&#39;ECM a augment&eacute;. Augmentations de la E-cadh&eacute;rine et MMP14 expression par les cellules de MCF10A comme l&#39;augmentation de la densit&eacute; ECM ont aussi &eacute;t&eacute; observ&eacute;es. En revanche, les cellules MCF10A a exprim&eacute; des niveaux inf&eacute;rieurs de &szlig;-cas&eacute;ine comme l&#39;augmentation de la densit&eacute; de l&#39;ECM. Pris ensemble, ces observations mettent en &eacute;vidence le r&ocirc;le cl&eacute; de la densit&eacute; de l&#39;ECM en modulant le nombre, l&#39;organisation et la fonction des acini mammaires. Copyright &copy; 2013 John Wiley &amp; Sons, Ltd.

La densit&eacute; accrue de la matrice extracellulaire diminue MCF10A du sein cellule acinus formation dans des conditions de culture 3D.

La matrice extracellulare (ECM) contribuisce alla generazione e dinamico del tessuto mammario normale, in particolare per la generazione di strutture acinose e duttale polarizzate.&thinsp;Condizioni di coltura in vitro 3D, comprese variazioni nella composizione dell&#39;ECM, hanno dimostrate di influenzare direttamente la formazione e l&#39;organizzazione di uva-come e -dotto-come le strutture. Inoltre, la densit&agrave; di ECM sembra anche svolgere un ruolo nel tessuto mammario normale e di tumori. Qui vi mostriamo che la densit&agrave; di ECM influenza direttamente il numero, l&#39;organizzazione e la funzione degli acini al seno. Brevemente, le cellule non maligne del seno umano MCF10A sono state incubate in aumentando la densit&agrave; di un collagene Matrigel &reg; I matrix. Moduli elastici vicino e da lontani con le strutture di uva sono stati misurati mediante microscopia a forza atomica, ed &egrave; stato determinato il numero di strutture di uva. Immunochimica &egrave; stato utilizzato per indagare i livelli di espressione di E-caderina, laminina, matrix metalloproteinase-14 e &szlig;-caseina nelle cellule MCF10A. Il modulo di ECM &egrave; stato aumentato significativamente vicino le strutture di uva e il numero di strutture di uva diminuita con l&#39;aumento di collagene Matrigel sono densit&agrave;. Come valutato dall&#39;espressione della laminina, l&#39;organizzazione delle strutture di uva presenti &egrave; stato alterato come la densit&agrave; di ECM &egrave; aumentato. Inoltre sono stati osservati aumenti nell&#39;espressione sia E-caderina e MMP14 dalle cellule MCF10A come ECM densit&agrave; aumentata. In contrasto, le cellule MCF10A espresso pi&ugrave; bassi livelli di &szlig;-caseina come la densit&agrave; di ECM &egrave; aumentata. Prese insieme, queste osservazioni evidenziano il ruolo chiave della densit&agrave; ECM nel modulare il numero, l&#39;organizzazione e la funzione degli acini al seno. Copyright &copy; 2013 John Wiley &amp; Sons, Ltd.

Matrice extracellulare maggiore densit&agrave; diminuisce MCF10A seno cella uva formazione in condizioni di coltura 3D.

Stromal chemokine gradients within the breast tissue microenvironment play a critical role in breast cancer cell invasion, a prerequisite to metastasis. To elucidate which chemokines and mechanisms are involved in mammary cell migration we determined whether mesenchymal D1 stem cells secreted specific chemokines that differentially promoted the invasion of mammary tumor cells in vitro. Results indicate that mesenchymal D1 cells produced concentrations of CCL5 and CCL9 4- to 5-fold higher than the concentrations secreted by 4T1 tumor cells (P < 0.01). Moreover, 4T1 tumor cell invasion toward D1 mesenchymal stem cell conditioned media (D1CM), CCL5 alone, CCL9 alone or a combination CCL5 and CCL9 was observed. The invasion of 4T1 cells toward D1 mesenchymal stem CM was dose-dependently suppressed by pre-incubation with the CCR1/CCR5 antagonist met-CCL5 (P < 0.01). Furthermore, the invasion of 4T1 cells toward these chemokines was prevented by incubation with the broad-spectrum MMP inhibitor GM6001. Additionally, the addition of specific MMP9/MMP13 and MMP14 inhibitors prevented the MMP activities of supernatants collected from 4T1 cells incubated with D1CM, CCL5 or CCL9. Taken together these data highlight the role of CCL5 and CCL9 produced by mesenchymal stem cells in mammary tumor cell invasion.

Mesenchymal stem cell-derived CCL-9 and CCL-5 promote mammary tumor cell invasion and the activation of matrix metalloproteinases.

Chemokine stromales d&eacute;grad&eacute;s dans le microenvironnement de tissu mammaire jouent un r&ocirc;le critique dans l&#39;invasion des cellules de cancer dans la poitrine, une condition sine qua non de m&eacute;tastases. Afin d&#39;&eacute;lucider les chimiokines et m&eacute;canismes sont impliqu&eacute;s dans la migration de cellules mammaires nous avons d&eacute;termin&eacute; si les cellules de tige mesenchymal D1 s&eacute;cr&eacute;t&eacute;e chimiokines sp&eacute;cifiques qui promus diff&eacute;rentiellement l&#39;invasion des cellules tumorales mammaires in vitro. Les r&eacute;sultats indiquent que les cellules m&eacute;senchymateuses D1 produisent des concentrations de CCL5 et CCL9 4 - 5 fois plus &eacute;lev&eacute;es que les concentrations s&eacute;cr&eacute;t&eacute;es par les cellules tumorales 4 t 1 (P &lt; 0,01). En outre, invasion des cellules 4 t 1 tumeur &agrave; cellules souches m&eacute;senchymateuses de D1 conditionn&eacute; m&eacute;dias (D1CM), CCL5 seul, CCL9 seul ou une combinaison de CCL5 et CCL9 a &eacute;t&eacute; observ&eacute;e. L&#39;invasion des cellules 4 t 1 vers D1 tige mesenchymal CM a &eacute;t&eacute; supprim&eacute;e de mani&egrave;re dose-d&eacute;pendante de pr&eacute;-incubation avec le CCR1/CCR5 antagoniste rencontr&eacute;-CCL5 (P &lt; 0,01). En outre, l&#39;invasion des cellules 4 t 1 vers ces chimiokines a &eacute;t&eacute; emp&ecirc;ch&eacute;e par incubation avec l&#39;inhibiteur MMP &agrave; large spectre GM6001. De plus, l&#39;addition d&#39;inhibiteurs sp&eacute;cifiques de MMP9/MMP13 et MMP14 ont emp&ecirc;ch&eacute; les activit&eacute;s MMP des surnageants pr&eacute;lev&eacute;s dans les cellules 4 t 1 incub&eacute;es avec D1CM, CCL5 ou CCL9. Pris ensemble ces donn&eacute;es mettent en &eacute;vidence le r&ocirc;le de CCL5 et CCL9 produites par les cellules souches m&eacute;senchymateuses dans l&#39;invasion des cellules tumorales mammaires.

D&eacute;riv&eacute; de cellules souches m&eacute;senchymateuses CCL-9 et CCL-5 promouvoir l&#39;invasion des cellules tumorales mammaires et l&#39;activation des m&eacute;talloprot&eacute;inases matricielles.

Breast tumor cells alter their microenvironment in part through the expression of protumor molecules that influence macrophages during tumor progression and metastasis. Macrophage recruitment is stimulated by chemotactic factors, including tumor necrosis factor alpha (TNF-Î±), which also stimulates the cytotoxic/tumor cell killing macrophage phenotype. Through TNF-Î± converting enzyme (TACE/ADAM17) activities, breast tumor cells shed membrane-bound proteins, including their TNF receptors (sTNFR1/2), which serve as decoys sequestering TNF-Î± and preventing TNF-Î±-driven apoptosis of tumor cells, thereby decreasing TNF-Î± bioavailability. Here we investigated the levels of sTNFRs shed by breast tumor cells and determined the effects of shed sTNFRs on macrophage migration toward TNF-Î±. TNF-Î± and sTNFRs concentrations were measured in murine normal epithelial, stromal, and mammary tumor cells. The migration of murine macrophages towards TNF-Î± in the presence of tumor derived soluble factors (TDSFs) shed by TACE was determined. TNF-Î± concentrations secreted by tumor and normal epithelial cells were below the detection limit contrasting with stromal cells, especially macrophages, which expressed higher levels of TNF-Î± (P<0.001). Regardless of the cell tested, treatment with the TACE inhibitor TAPI-0 led to a significant decrease in sTNFR2 shed (P<0.05). The dose-dependent macrophage migration toward TNF-Î± prevented by incubation with TDSFs was not observed with TDSFs collected following TAPI-0 treatment (P<0.05). Furthermore, the TNF-Î±-driven increased pAkt expression in macrophage was inhibited by TACE shed TDSFs (P<0.05). These results highlight the role of tumor-shed sTNFRs in TNF-Î± -driven macrophage chemotaxis.

Soluble Tumor Necrosis Factor Receptors Shed by Breast Tumor Cells Inhibit Macrophage Chemotaxis.

Les cellules de tumeur du sein modifient leur microenvironnement en partie gr&acirc;ce &agrave; l&#39;expression de mol&eacute;cules protumor qui influencent les macrophages au cours de la progression tumorale et les m&eacute;tastases. Recrutement de macrophages est stimul&eacute;e par des facteurs chimiotactiques, y compris le facteur de n&eacute;crose tumorale alpha (TNF-&alpha;), qui stimule &eacute;galement la cellule cytotoxique/tumeur tuant ph&eacute;notype macrophage. &Agrave; travers des activit&eacute;s enzymatiques (TACE/ADAM17) conversion de TNF-&alpha;, tumeur du sein cellules hangar membranaires prot&eacute;ines, y compris leurs r&eacute;cepteurs TNF (sTNFR1/2), qui servent de leurres s&eacute;questrant de TNF-&alpha; et de pr&eacute;vention ax&eacute;e sur les TNF-&alpha; de l&#39;apoptose des cellules tumorales, diminuant ainsi la biodisponibilit&eacute; de TNF-&alpha;. Ici, nous avons &eacute;tudi&eacute; les niveaux de sTNFRs hangar par les cellules tumorales mammaires et d&eacute;termin&eacute; les effets du hangar sTNFRs sur la migration des macrophages vers le TNF-&alpha;. Les concentrations de TNF-&alpha; et sTNFRs ont &eacute;t&eacute; mesur&eacute;es dans les cellules tumorales murines d&#39;&eacute;pith&eacute;lial, stromal et mammaires normales. La migration des macrophages de souris vers le TNF-&alpha; en pr&eacute;sence de tumeur d&eacute;riv&eacute;e des facteurs solubles (TDSFs) remise par TACE a &eacute;t&eacute; d&eacute;termin&eacute;e. Les concentrations de TNF-&alpha; s&eacute;cr&eacute;t&eacute;es par la tumeur et les cellules &eacute;pith&eacute;liales normales &eacute;taient inf&eacute;rieures au seuil de d&eacute;tection, contrastant avec les cellules du stroma, surtout des macrophages, qui a exprim&eacute; des niveaux plus &eacute;lev&eacute;s de TNF-&alpha; (P &lt; 0,001). Quelle que soit la cellule test&eacute;e, traitement avec l&#39;inhibiteur TACE TAPI-0 a entra&icirc;n&eacute; une diminution significative dans le hangar de sTNFR2 (P &lt; 0,05). La migration des macrophages dose-d&eacute;pendante vers TNF-&alpha; a emp&ecirc;ch&eacute; par incubation avec TDSFs n&#39;a pas &eacute;t&eacute; observ&eacute;e avec TDSFs recueillies suite &agrave; un traitement de TAPI-0 (P &lt; 0,05). En outre, l&#39;expression de TNF-&alpha;-driven accrue pAkt en macrophage est inhib&eacute;e par TACE hangar TDSFs (P &lt; 0,05). Ces r&eacute;sultats mettent en &eacute;vidence le r&ocirc;le de sTNFRs de la tumeur-hangar en TNF-&alpha;-pilot&eacute; par chimiotactisme des macrophages.

Muthulekha Swamydas

Fungal Pathogenesis Unit,
Laboratory of Clinical Infectious Diseases,
Fungal Pathogenesis Unit, Laboratory of Clinical Infectious Diseases

National Institute of Allergy and Infectious Diseases, NIH

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Muthulekha Swamydas

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Fungal Pathogenesis Unit,Laboratory of Clinical Infectious Diseases,Fungal Pathogenesis Unit, Laboratory of Clinical Infectious Diseases

National Institute of Allergy and Infectious Diseases, NIH

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Fungal Pathogenesis Unit,
Laboratory of Clinical Infectious Diseases,
Fungal Pathogenesis Unit, Laboratory of Clinical Infectious Diseases