In JoVE (1)
Other Publications (11)
- Neurochemical Research
- Experimental Neurology
- Journal of Neuroscience Research
- Journal of the Neurological Sciences
- In Vitro Cellular & Developmental Biology. Animal
- Cancer Investigation
- Cell Communication & Adhesion
- PLoS Pathogens
- Journal of Tissue Engineering and Regenerative Medicine
- Cell Adhesion & Migration
- Journal of Interferon & Cytokine Research : the Official Journal of the International Society for Interferon and Cytokine Research
Articles by Muthulekha Swamydas in JoVE
Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments Muthulekha Swamydas1, Michail S. Lionakis1 1Fungal Pathogenesis Unit, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.
Other articles by Muthulekha Swamydas on PubMed
Nerve Growth Factor (NGF) and Glial Cell Line-derived Neurotrophic Factor (GDNF) Regulate Substance P Release in Adult Spinal Sensory Neurons Neurochemical Research. Jun, 2003 | Pubmed ID: 12718437 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.
Partial Sciatic Nerve Transection Causes Redistribution of Pain-related Peptides and Lowers Withdrawal Threshold Experimental Neurology. Aug, 2004 | Pubmed ID: 15246843 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.
Sexual Dimorphism of Oligodendrocytes is Mediated by Differential Regulation of Signaling Pathways Journal of Neuroscience Research. Nov, 2009 | Pubmed ID: 19084904 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.  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 in the White Matter of Rodents Journal of the Neurological Sciences. Nov, 2009 | Pubmed ID: 19625027 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.
Matrix Compositions and the Development of Breast Acini and Ducts in 3D Cultures In Vitro Cellular & Developmental Biology. Animal. Sep, 2010 | Pubmed ID: 20585895 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.
The Endothelin Axis Stimulates the Expression of Pro-inflammatory Cytokines and Pro-migratory Molecules in Breast Cancer Cancer Investigation. Nov, 2010 | Pubmed ID: 20690805 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).
Progranulin Stimulated by LPA Promotes the Migration of Aggressive Breast Cancer Cells Cell Communication & Adhesion. Dec, 2011 | Pubmed ID: 22176685 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
Chemokine Receptor Ccr1 Drives Neutrophil-mediated Kidney Immunopathology and Mortality in Invasive Candidiasis PLoS Pathogens. 2012 | Pubmed ID: 22916017 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.
Increased Extracellular Matrix Density Decreases MCF10A Breast Cell Acinus Formation in 3D Culture Conditions Journal of Tissue Engineering and Regenerative Medicine. Feb, 2013 | Pubmed ID: 23404906 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.
Mesenchymal Stem Cell-derived CCL-9 and CCL-5 Promote Mammary Tumor Cell Invasion and the Activation of Matrix Metalloproteinases Cell Adhesion & Migration. May, 2013 | Pubmed ID: 23722213 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.
Soluble Tumor Necrosis Factor Receptors Shed by Breast Tumor Cells Inhibit Macrophage Chemotaxis Journal of Interferon & Cytokine Research : the Official Journal of the International Society for Interferon and Cytokine Research. Jun, 2013 | Pubmed ID: 23777205 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