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Sample records for cell maturation induced

  1. Cannabinoids induce incomplete maturation of cultured human leukemia cells

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    Murison, G.; Chubb, C.B.H.; Maeda, S.; Gemmell, M.A.; Huberman, E.

    1987-08-01

    Monocyte maturation markers were induced in cultured human myeloblastic ML-2 leukemia cells after treatment for 1-6 days with 0.03-30 ..mu..M ..delta../sup 9/-tetrahydrocannabinol (THC), the major psychoactive component of marijuana. After a 2-day or longer treatment, 2- to 5-fold increases were found in the percentages of cells exhibiting reactivity with either the murine OKM1 monoclonal antibody of the Leu-M5 monoclonal antibody, staining positively for nonspecific esterase activity, and displaying a promonocyte morphology. The increases in these differentiation markers after treatment with 0.03-1 ..mu..M THC were dose dependent. At this dose range, THC did not cause an inhibition of cell growth. The THC-induced cell maturation was also characterized by specific changes in the patterns of newly synthesized proteins. The THC-induced differentiation did not, however, result in cells with a highly developed mature monocyte phenotype. However, treatment of these incompletely matured cells with either phorbol 12-myristate 13-acetate of 1..cap alpha..,25-dihydroxycholecalciferol, which are inducers of differentiation in myeloid leukemia cells (including ML-2 cells), produced cells with a mature monocyte morphology. The ML-2 cell system described here may be a useful tool for deciphering critical biochemical events that lead to the cannabinoid-induced incomplete cell differentiation of ML-2 cells and other related cell types. Findings obtained from this system may have important implications for studies of cannabinoid effects on normal human bone-marrow progenitor cells.

  2. Heat Shock Protein 96 Induces Maturation of Dendritic Cells

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    Chunxia Cao; Wei Yang; Yonglie Chu; Qingguang Liu; Liang Yu; Cheng'en Pan

    2006-01-01

    Objective: Heat shock protein (HSP) has the promiscuous abilities to chaperone and present a broad repertoire of tumor antigens to antigen presenting cells including DCs. In this report, we analyzed the modulation of immature DC by HSP 96 (gp96).Method: Murine bone marrow-derived DC was induced by GM-CSF plus IL-4, which aped the immunostimulatory effects of DC.Cocultured DC and gp96-peptide complexes (gp96-PC) or inactivated H22 cells, the expression of MHC class Ⅱ, CD40, CD80 was quantified by flow cytometry. The concentration of IL-12 and TNF- in culture supernatants were determined by ELISA.[51] Cr release assay was used to test specific cytotoxic T cell. Results: Our study demonstrated that the extent of DC maturation induced by gp96-PC, which was reflected in surface density of costimulatory and MHC Ⅱ molecules, was correlated with the secretion of IL-12 and with the T cellactivating potential in vitro. Conclusion: Heat shock protein 96 could be isolated and purified from H22 cells and could induce maturation of dendritic cell. Our findings might be relevance to the use of DC vaccine in therapy of human tumors.

  3. Listeria monocytogenes protein fraction induces dendritic cells maturation and T helper 1 immune responses.

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    Azad Saei

    2014-02-01

    Full Text Available Fully mature dendritic cells (DCs play pivotal role in inducing immune responses and converting naïve T lymphocytes into functional Th1 cells. We aimed to evaluate Listeria Monocytogenes-derived protein fractions to induce DC maturation and stimulating T helper (Th1 immune responses.In the present study, we fractionated Listeria Monocytogenes-derived proteins by adding of ammonium sulfate in a stepwise manner. DCs were also generated from C57BL/6 mice bone marrow precursor cells. Then, the effects of protein fractions on bone marrow derived DC (BMDC maturation were evaluated. In addition, we assessed the capacity of activated DCs to induce cytokine production and proliferation of lymphocytes.Listeria-derived protein fractions induced fully mature DCs expressing high costimulatory molecules such as CD80, CD86 and CD40. DCs that were activated by selected F3 fraction had low capacity to uptake exogenous antigens while secreted high levels of Interleukine (IL-12. Moreover, lymphocytes cultured with activated BMDCs produced high amounts of IFN-γ and showed higher proliferation than control. Listeria derived protein fractions differently influenced DC maturation.In conclusion, Listeria protein activated-BMDCs can be used as a cell based vaccine to induce anti-tumor immune responses.

  4. Apoptosis of matured T lymphocytes induced by mouse sertoli cells in cocultures in vitro

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    LIU Yang; LIN Zi-hao; ZHU Xiao-hai; LIU Shan-rong

    2001-01-01

    Objective: To study whether mouse sertoli cells can induce the apoptosis of matured T lymphocytes in cocultures in vitro. Methods: With TUNEL, DNA electrophoresis, eleetro-mierography and flow cytometry, we examined the apoptosis and its rates of mouse matured T lymphocytes in control group (T lymphocytes only), group A (T lymphocytes + culture medium of sertoli cells), group B (T lymphocytes + sertoli cells). Results: Under electro-micrography, chromatin condensation, karyopyknosis, karyorhexis and apoptotic body were observed in some T lymphocytes in 3 groups; some nucleuses were stained dark blue with TUNEL; a typical DNA ladder was found with DNA electrophoresis. The apoptotic rates of T lymphocytes in group A and B were significantly higher than that in control group (P<0.01). The apoptotic rate of T lymphocytes in group B was significantly higher than that in group A (P<0.01). Conclusion: In coculture condition in vitro,mouse sertoli cells can induce the apoptosis of matured T lymphocytes.

  5. Hypoxia alters cell cycle regulatory protein expression and induces premature maturation of oligodendrocyte precursor cells.

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    Ravi Shankar Akundi

    Full Text Available BACKGROUND: Periventricular white matter injury (PWMI is a common form of brain injury sustained by preterm infants. A major factor that predisposes to PWMI is hypoxia. Because oligodendrocytes (OLs are responsible for myelination of axons, abnormal OL development or function may affect brain myelination. At present our understanding of the influences of hypoxia on OL development is limited. To examine isolated effects of hypoxia on OLs, we examined the influences of hypoxia on OL development in vitro. METHODOLOGY/FINDINGS: Cultures of oligodendrocyte precursor cells (OPCs were prepared from mixed glial cultures and were 99% pure. OPCs were maintained at 21% O(2 or hypoxia (1% or 4% O(2 for up to 7 days. We observed that 1% O(2 lead to an increase in the proportion of myelin basic protein (MBP-positive OLs after 1 week in culture, and a decrease in the proportion of platelet-derived growth factor receptor alpha (PDGFRalpha-positive cells suggesting premature OL maturation. Increased expression of the cell cycle regulatory proteins p27(Kip1 and phospho-cdc2, which play a role in OL differentiation, was seen as well. CONCLUSIONS: These results show that hypoxia interferes with the normal process of OL differentiation by inducing premature OPC maturation.

  6. Tumor cells prevent mouse dendritic cell maturation induced by TLR ligands.

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    Idoyaga, Juliana; Moreno, José; Bonifaz, Laura

    2007-08-01

    Tumor cells can evade the immune system through several mechanisms, one of which is to block DC maturation. It has been suggested that signaling via Toll-like receptors (TLR) may be involved in the induction of prophylactic anti-cancer immunity and in the treatment of established tumors. In the present study we found that high numbers of tumor cells interfere with BMDC activation induced by the TLR ligands LPS and poly IC. Tumor cells blocked TLR3- and TLR4-mediated induction of MHCII and the co-stimulatory molecules CD40 and CD86, as well as the cytokines IL-12, TNF-alpha and IL-6. Importantly, tumor cells induced inhibitory molecules (B7-DC, B7-H1 and CD80) on spleen DC in vivo and on BMDC, even in the presence of TLR ligands. Moreover, after a long exposure with tumor cells, purified BMDC were unable to respond to a second challenge with TLR ligands. The failure of tumor exposed-BMDC to express co-stimulatory molecules and cytokines in the presence of TLR ligands has implications for the future development of DC-based cancer immune therapies using TLR ligands as adjuvants for the activation of DC.

  7. Serine-Rich Repeat Adhesins Contribute to Streptococcus gordonii-Induced Maturation of Human Dendritic Cells

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    Ko, Eun Byeol; Kim, Sun Kyung; Seo, Ho Seong; Yun, Cheol-Heui; Han, Seung Hyun

    2017-01-01

    Dendritic cells (DCs) play a pivotal role in the induction of immunity by recognition, capture, process, and presentation of antigens from infectious microbes. Streptococcus gordonii is able to cause life-threatening systemic diseases such as infective endocarditis. Serine-rich repeat (SRR) glycoproteins of S. gordonii are sialic acid-binding adhesins mediating the bacterial adherence to the host and the development of infective endocarditis. Thus, the SRR adhesins are potentially involved in the bacterial adherence to DCs and the maturation and activation of DCs required for the induction of immunity to S. gordonii. Here, we investigated the phenotypic and functional changes of human monocyte-derived DCs treated with wild-type S. gordonii or the SRR adhesin-deficient mutant. The mutant poorly bound to DCs and only weakly increased the expression of CD83, CD86, MHC class II, and PD-L1 on DCs compared with the wild-type. In addition, the mutant induced lower levels of TNF-α, IL-6, and IL-12 than the wild-type in DCs. When DCs sensitized with the mutant were co-cultured with autologous T cells, they induced weaker proliferation and activation of T cells than DCs stimulated with the wild-type. Blockade of SRR adhesin with 3′-sialyllactose markedly reduced S. gordonii binding and internalization, causing attenuation of the bacterial immunostimulatory potency in DC maturation. Collectively, our results suggest that SRR adhesins of S. gordonii are important for maturation and activation of DCs.

  8. Mitoxantrone induces natural killer cell maturation in patients with secondary progressive multiple sclerosis.

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    Coralie Chanvillard

    Full Text Available Mitoxantrone is one of the few drugs approved for the treatment of progressive multiple sclerosis (MS. However, the prolonged use of this potent immunosuppressive agent is limited by the appearance of severe side effects. Apart from its general cytotoxic effect, the mode of action of mitoxantrone on the immune system is poorly understood. Thus, to develop safe therapeutic approaches for patients with progressive MS, it is essential to elucidate how mitoxantrone exerts it benefits. Accordingly, we initiated a prospective single-arm open-label study with 19 secondary progressive MS patients. We investigated long-term effects of mitoxantrone on patient peripheral immune subsets using flow cytometry. While we corroborate that mitoxantrone persistently suppresses B cells in vivo, we show for the first time that treatment led to an enrichment of neutrophils and immunomodulatory CD8(low T cells. Moreover, sustained mitoxantrone applications promoted not only persistent NK cell enrichment but also NK cell maturation. Importantly, this mitoxantrone-induced NK cell maturation was seen only in patients that showed a clinical response to treatment. Our data emphasize the complex immunomodulatory role of mitoxantrone, which may account for its benefit in MS. In particular, these results highlight the contribution of NK cells to mitoxantrone efficacy in progressive MS.

  9. Efficient and cost-effective generation of mature neurons from human induced pluripotent stem cells.

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    Badja, Cherif; Maleeva, Galyna; El-Yazidi, Claire; Barruet, Emilie; Lasserre, Manon; Tropel, Philippe; Binetruy, Bernard; Bregestovski, Piotr; Magdinier, Frédérique

    2014-12-01

    For years, our ability to study pathological changes in neurological diseases has been hampered by the lack of relevant models until the recent groundbreaking work from Yamanaka's group showing that it is feasible to generate induced pluripotent stem cells (iPSCs) from human somatic cells and to redirect the fate of these iPSCs into differentiated cells. In particular, much interest has focused on the ability to differentiate human iPSCs into neuronal progenitors and functional neurons for relevance to a large number of pathologies including mental retardation and behavioral or degenerative syndromes. Current differentiation protocols are time-consuming and generate limited amounts of cells, hindering use on a large scale. We describe a feeder-free method relying on the use of a chemically defined medium that overcomes the need for embryoid body formation and neuronal rosette isolation for neuronal precursors and terminally differentiated neuron production. Four days after induction, expression of markers of the neurectoderm lineage is detectable. Between 4 and 7 days, neuronal precursors can be expanded, frozen, and thawed without loss of proliferation and differentiation capacities or further differentiated. Terminal differentiation into the different subtypes of mature neurons found in the human brain were observed. At 6-35 days after induction, cells express typical voltage-gated and ionotrophic receptors for GABA, glycine, and acetylcholine. This specific and efficient single-step strategy in a chemically defined medium allows the production of mature neurons in 20-40 days with multiple applications, especially for modeling human pathologies.

  10. New model for the genesis and maturation of viroplasms induced by fijiviruses in insect vector cells.

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    Mao, Qianzhuo; Zheng, Shenglan; Han, Qingmei; Chen, Hongyan; Ma, Yuanyuan; Jia, Dongsheng; Chen, Qian; Wei, Taiyun

    2013-06-01

    Plant reoviruses are thought to replicate and assemble within cytoplasmic, nonmembranous structures called viroplasms. Here, we established continuous cell cultures of the white-backed planthopper (Sogatella furcifera Horváth) to investigate the mechanisms for the genesis and maturation of the viroplasm induced by Southern rice black-streaked dwarf virus (SRBSDV), a fijivirus in the family Reoviridae, during infection of its insect vector. Electron and confocal microscopy revealed that the viroplasm consisted of a granular region, where viral RNAs and nonstructural proteins P6 and P9-1 accumulated, and a filamentous region, where viral RNAs, progeny cores, viral particles, as well as nonstructural proteins P5 and P6 accumulated. Our results suggested that the filamentous viroplasm matrix was the site for the assembly of progeny virions. Because viral RNAs were produced by assembled core particles within the filamentous viroplasm matrix, we propose that these viral RNAs might be transported to the granular viroplasm matrix. P5 formed filamentous inclusions and P9-1 formed granular inclusions in the absence of viral infection, suggesting that the filamentous and granular viroplasm matrices were formed primarily by P5 and P9-1, respectively. P6 was apparently recruited in the whole viroplasm matrix by direct interaction with P9-1 and P5. Thus, the present results suggested that P5, P6, and P9-1 are collectively required for the genesis and maturation of the filamentous and granular viroplasm matrix induced by SRBSDV infection. Based on these results, we propose a new model to explain the genesis and maturation of the viroplasms induced by fijiviruses in insect vector cells.

  11. Proinsulin atypical maturation and disposal induces extensive defects in mouse Ins2+/Akita β-cells.

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    Qingxin Yuan

    Full Text Available Because of its low relative folding rate and plentiful manufacture in β-cells, proinsulin maintains a homeostatic balance of natively and plentiful non-natively folded states (i.e., proinsulin homeostasis, PIHO through the integration of maturation and disposal processes. PIHO is susceptible to genetic and environmental influences, and its disorder has been critically linked to defects in β-cells in diabetes. To explore this hypothesis, we performed polymerase chain reaction (PCR, metabolic-labeling, immunoblotting, and histological studies to clarify what defects result from primary disorder of PIHO in model Ins2(+/Akita β-cells. We used T antigen-transformed Ins2(+/Akita and control Ins2(+/+ β-cells established from Akita and wild-type littermate mice. In Ins2(+/Akita β-cells, we found no apparent defect at the transcriptional and translational levels to contribute to reduced cellular content of insulin and its precursor and secreted insulin. Glucose response remained normal in proinsulin biosynthesis but was impaired for insulin secretion. The size and number of mature insulin granules were reduced, but the size/number of endoplasmic reticulum, Golgi, mitochondrion, and lysosome organelles and vacuoles were expanded/increased. Moreover, cell death increased, and severe oxidative stress, which manifested as increased reactive oxygen species, thioredoxin-interacting protein, and protein tyrosine nitration, occurred in Ins2(+/Akita β-cells and/or islets. These data show the first clear evidence that primary PIHO imbalance induces severe oxidative stress and impairs glucose-stimulated insulin release and β-cell survival as well as producing other toxic consequences. The defects disclosed/clarified in model Ins2(+/Akita β-cells further support a role of the genetic and stress-susceptible PIHO disorder in β-cell failure and diabetes.

  12. Mycobacterium tuberculosislpdC, Rv0462, induces dendritic cell maturation and Th1 polarization

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    Heo, Deok Rim [Department of Microbiology and Immunology, School of Medicine, Pusan National University, Beom-eo Ri, Mulgum Eop, Yangsan, Gyeongsangnam-do 626-770 (Korea, Republic of); Shin, Sung Jae; Kim, Woo Sik [Department of Microbiology, College of Medicine, Chungnam National University, Munwha-Dong, Jung-Ku, Daejeon 301-747 (Korea, Republic of); Noh, Kyung Tae; Park, Jin Wook; Son, Kwang Hee [Department of Microbiology and Immunology, School of Medicine, Pusan National University, Beom-eo Ri, Mulgum Eop, Yangsan, Gyeongsangnam-do 626-770 (Korea, Republic of); Park, Won Sun [Department of Physiology, Kangwon National University, School of Medicine, Chuncheon 200-701 (Korea, Republic of); Lee, Min-Goo [Department of Physiology, Korea University, College of Medicine, Anam-dong, Sungbuk-Gu, Seoul 136-705 (Korea, Republic of); Kim, Daejin [Department of Anatomy, Chung-Ang University, College of Medicine, 221 Heuksuk-Dong, Dongjak-Ku, Seoul 156-756 (Korea, Republic of); Shin, Yong Kyoo [Department of Pharmacology, Chung-Ang University, College of Medicine, 221 Heuksuk-Dong, Dongjak-Ku, Seoul 156-756 (Korea, Republic of); Jung, In Duk, E-mail: jungid@pusan.ac.kr [Department of Microbiology and Immunology, School of Medicine, Pusan National University, Beom-eo Ri, Mulgum Eop, Yangsan, Gyeongsangnam-do 626-770 (Korea, Republic of); Research Institute of Convergence of Biomedical Science and Technology, Pusan National University Yangsan Hospital, Beom-eo Ri, Mulgum Eop, Yangsan, Gyeongsangnam-do 626-770 (Korea, Republic of); Park, Yeong-Min, E-mail: immunpym@pusan.ac.kr [Department of Microbiology and Immunology, School of Medicine, Pusan National University, Beom-eo Ri, Mulgum Eop, Yangsan, Gyeongsangnam-do 626-770 (Korea, Republic of); Research Institute of Convergence of Biomedical Science and Technology, Pusan National University Yangsan Hospital, Beom-eo Ri, Mulgum Eop, Yangsan, Gyeongsangnam-do 626-770 (Korea, Republic of)

    2011-08-05

    Highlights: {yields} Treatment with Rv0462 induces the expression of surface molecules and the production of cytokines in DCs. {yields} Rv0462 induces the activation of MAPKs. {yields} Rv0462-treated DCs enhances the proliferation of CD4{sup +} T cells. -- Abstract: Mycobacterium tuberculosis, the etiological factor of pulmonary tuberculosis, causes significant morbidity and mortality worldwide. Activation of host immune responses for containment of mycobacterial infections involves participation of innate immune cells, such as dendritic cells (DCs). In this study, we demonstrated that the gene encoding lipoamide dehydrogenase C (lpdC) from M. tuberculosis, Rv0462, induce maturation and activation of DCs involved in the MAPKs signaling pathway. Moreover, Rv0462-treated DCs activated naive T cells, polarized CD4{sup +} and CD8{sup +} T cells to secrete IFN-{gamma} in syngeneic mixed lymphocyte reactions, which would be expected to contribute to Th1 polarization of the immune response. Our results suggest that Rv0462 can contribute to the innate and adaptive immune responses during tuberculosis infection, and thus modulate the clinical course of tuberculosis.

  13. Structural, phenotypic and functional maturation of bone marrow dendritic cells (BMDCs) induced by Chitosan (CTS).

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    Jia, Lihui; Gao, Xinghua; Wang, Yiqing; Yao, Na; Zhang, Xiaodong

    2014-11-01

    The objective of the present work was to explore the effect of CTS on structural, phenotypic and functional maturation of murine bone marrow derived dendritic cells (BMDCs). The maturity of BMDCs post treatment with CTS was evaluated using transmission electron microscopy (TEM) for structure changes, flow cytometry (FCM) for changes of key surface molecules, FITC-dextran bio-assay for phagocytosis, test of acid phosphatase activity (ACP) for biochemical changes and enzyme linked immunosorbent assay (ELISA) for cytokine level. We found that CTS downregulated the numbers of phagosomes inside the BMDCs, up-regulated the expression of MHC II, CD40, CD83, CD80 and CD86 molecules on BMDCs, decreased activity of ACP and phagocytosis by BMDCs, and induced production of higher levels of IL-12 and TNF-α. It was therefore confirmed that CTS could effectively promote the maturation of BMDCs. Our study provided more detailed evidence and rationale to support the application of CTS as an immune stimulator for enhancing host immunity and as an adjuvant in the design of DC-based vaccines.

  14. Dendritic morphology, synaptic transmission, and activity of mature granule cells born following pilocarpine-induced status epilepticus in the rat

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    Fei eGao

    2015-10-01

    Full Text Available To understand the potential role of enhanced hippocampal neurogenesis after pilocarpine-induced status epilepticus (SE in the development of epilepsy, we quantitatively analyzed the geometry of apical dendrites, synaptic transmission, and activation levels of normotopically distributed mature newborn granule cells in the rat.SE in male Sprague-Dawley rats lasting for more than 2 hours was induced by an intraperitoneal injection of pilocarpine. The complexity, spine density, miniature post-synaptic currents, and activity-regulated cytoskeleton-associated protein (Arc expression of granule cells born five days after SE were studied at least 10 weeks after CAG-GFP retroviral vector-mediated labeling.Mature granule cells born after SE had dendritic complexity similar to that of granule cells born naturally, but with denser mushroom-like spines in dendritic segments located in the outer molecular layer. Miniature inhibitory post-synaptic currents (mIPSCs were similar between the controls and rats subjected to SE; however, smaller miniature excitatory post-synaptic current (mEPSC amplitude with a trend toward less frequent was found in mature granule cells born after SE. After maturation, granule cells born after SE did not show denser Arc expression in the resting condition or after being activated by transient seizure activity than vicinal GFP-unlabeled granule cells.Thus our results suggest that normotopic granule cells born after pilocarpine-induced SE are no more active when mature than age-matched, naturally born granule cells.

  15. High-frequency stimulation induces gradual immediate early gene expression in maturing adult-generated hippocampal granule cells.

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    Jungenitz, Tassilo; Radic, Tijana; Jedlicka, Peter; Schwarzacher, Stephan W

    2014-07-01

    Increasing evidence shows that adult neurogenesis of hippocampal granule cells is advantageous for learning and memory. We examined at which stage of structural maturation and age new granule cells can be activated by strong synaptic stimulation. High-frequency stimulation of the perforant pathway in urethane-anesthetized rats elicited expression of the immediate early genes c-fos, Arc, zif268 and pCREB133 in almost 100% of mature, calbindin-positive granule cells. In contrast, it failed to induce immediate early gene expression in immature doublecortin-positive granule cells. Furthermore, doublecortin-positive neurons did not react with c-fos or Arc expression to mild theta-burst stimulation or novel environment exposure. Endogenous expression of pCREB133 was increasingly present in young cells with more elaborated dendrites, revealing a close correlation to structural maturation. Labeling with bromodeoxyuridine revealed cell age dependence of stimulation-induced c-fos, Arc and zif268 expression, with only a few cells reacting at 21 days, but with up to 75% of cells activated at 35-77 days of cell age. Our results indicate an increasing synaptic integration of maturing granule cells, starting at 21 days of cell age, but suggest a lack of ability to respond to activation with synaptic potentiation on the transcriptional level as long as immature cells express doublecortin.

  16. Interferon-γ Added During Bacillus Calmette-Guerin Induced Dendritic Cell Maturation Stimulates Potent Th1 Immune Responses

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    Pestano Linda A

    2003-10-01

    Full Text Available Abstract Dendritic cells (DC are increasingly prepared in vitro for use in immunotherapy trials. Mature DC express high levels of surface molecules needed for T cell activation and are superior at antigen-presentation than immature DC. Bacillus Calmette-Guerin (BCG is one of several products known to induce DC maturation, and interferon (IFN-γ has been shown to enhance the activity of DC stimulated with certain maturation factors. In this study, we investigated the use of IFN-γ in combination with the powerful maturation agent, BCG. The treatment of immature DC with IFN-γ plus BCG led to the upregulation of CD54, CD80, and CD86 in comparison with BCG treatment alone. In MLR or recall immune responses, the addition of IFN-γ at the time of BCG-treatment did not increase the number of antigen-specific T cells but enhanced the development of IFN-γ-producing Th1 cells. In primary immune responses, on the other hand, BCG and IFN-γ co-treated DC stimulated higher proportions of specific T cells as well as IFN-γ secretion by these T cells. Thus the use of IFN-γ during BCG-induced DC maturation differentially affects the nature of recall versus naïve antigen-specific T-cell responses. IFN-γ co-treatment with BCG was found to induce IL-12 and, in some instances, inhibit IL-10 secretion by DC. These findings greatly enhance the potential of BCG-matured dendritic cells for use in cancer immunotherapy.

  17. SMAC Mimetic BV6 Induces Cell Death in Monocytes and Maturation of Monocyte-Derived Dendritic Cells

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    Holtz, Philipp; Kapp, Markus; Grigoleit, Götz Ulrich; Schmuck, Carsten; Wajant, Harald; Siegmund, Daniela

    2011-01-01

    Background Compounds mimicking the inhibitory effect of SMAC / DIABLO on X-linked inhibitor of apoptosis (XIAP) have been developed with the aim to achieve sensitization for apoptosis of tumor cells resistant due to deregulated XIAP expression. It turned out that SMAC mimetics also have complex effects on the NFκB system and TNF signaling. In view of the overwhelming importance of the NFκB transcription factors in the immune system, we analyzed here the effects of the SMAC mimetic BV6 on immune cells. Principal Findings BV6 induced apoptotic and necrotic cell death in monocytes while T-cells, dendritic cells and macrophages were largely protected against BV6-induced cell death. In immature dendritic cells BV6 treatment resulted in moderate activation of the classical NFκB pathway, but it also diminished the stronger NFκB-inducing effect of TNF and CD40L. Despite its inhibitory effect on TNF- and CD40L signaling, BV6 was able to trigger maturation of immature DCs as indicated by upregulation of CD83, CD86 and IL12. Significance The demonstrated effects of SMAC mimetics on immune cells may complicate the development of tumor therapeutic concepts based on these compounds but also arise the possibility to exploit them for the development of immune stimulatory therapies. PMID:21738708

  18. SMAC mimetic BV6 induces cell death in monocytes and maturation of monocyte-derived dendritic cells.

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    Nicole Müller-Sienerth

    Full Text Available BACKGROUND: Compounds mimicking the inhibitory effect of SMAC/DIABLO on X-linked inhibitor of apoptosis (XIAP have been developed with the aim to achieve sensitization for apoptosis of tumor cells resistant due to deregulated XIAP expression. It turned out that SMAC mimetics also have complex effects on the NFκB system and TNF signaling. In view of the overwhelming importance of the NFκB transcription factors in the immune system, we analyzed here the effects of the SMAC mimetic BV6 on immune cells. PRINCIPAL FINDINGS: BV6 induced apoptotic and necrotic cell death in monocytes while T-cells, dendritic cells and macrophages were largely protected against BV6-induced cell death. In immature dendritic cells BV6 treatment resulted in moderate activation of the classical NFκB pathway, but it also diminished the stronger NFκB-inducing effect of TNF and CD40L. Despite its inhibitory effect on TNF- and CD40L signaling, BV6 was able to trigger maturation of immature DCs as indicated by upregulation of CD83, CD86 and IL12. SIGNIFICANCE: The demonstrated effects of SMAC mimetics on immune cells may complicate the development of tumor therapeutic concepts based on these compounds but also arise the possibility to exploit them for the development of immune stimulatory therapies.

  19. Cooperative transcription activation by Nurr1 and Pitx3 induces embryonic stem cell maturation to the midbrain dopamine neuron phenotype

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    Martinat, Cecile; Bacci, Jean-Jacques; Leete, Thomas

    2006-01-01

    , Pitx3, Lmx1b, Engrailed-1, and Engrailed-2. However, none of these factors appear sufficient alone to induce the mature midbrain DA neuron phenotype in ES cell cultures in vitro, suggesting a more complex regulatory network. Here we show that Nurr1 and Pitx3 cooperatively promote terminal maturation......Midbrain dopamine (DA) neurons play a central role in the regulation of voluntary movement, and their degeneration is associated with Parkinson's disease. Cell replacement therapies, and in particular embryonic stem (ES) cell-derived DA neurons, offer a potential therapeutic venue for Parkinson......'s disease. We sought to identify genes that can potentiate maturation of ES cell cultures to the midbrain DA neuron phenotype. A number of transcription factors have been implicated in the development of midbrain DA neurons by expression analyses and loss-of-function knockout mouse studies, including Nurr1...

  20. Farnesyl transferase inhibitors induce extended remissions in transgenic mice with mature B cell lymphomas

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    Refaeli Yosef

    2008-05-01

    Full Text Available Abstract Background We have used a mouse model based on overexpression of c-Myc in B cells genetically engineered to be self-reactive to test the hypothesis that farnesyl transferase inhibitors (FTIs can effectively treat mature B cell lymphomas. FTIs are undergoing clinical trials to treat both lymphoid and non-lymphoid malignancies and we wished to obtain evidence to support the inclusion of B cell lymphomas in future trials. Results We report that two FTIs, L-744,832 and SCH66336, blocked the growth of mature B cell lymphoma cells in vitro and in vivo. The FTI treatment affected the proliferation and survival of the transformed B cells to a greater extent than naïve B cells stimulated with antigen. In syngeneic mice transplanted with the transgenic lymphoma cells, L-744,832 treatment prevented the growth of the tumor cells and the morbidity associated with the resulting lymphoma progression. Tumors that arose from transplantation of the lymphoma cells regressed with as little as three days of treatment with L-744,832 or SCH66336. Treatment of these established lymphomas with L-744,832 for seven days led to long-term remission of the disease in approximately 25% of animals. Conclusion FTI treatment can block the proliferation and survival of self-reactive transformed B cells that overexpress Myc. In mice transplanted with mature B cell lymphomas, we found that FTI treatment led to regression of disease. FTIs warrant further consideration as therapeutic agents for mature B cell lymphomas and other lymphoid tumors.

  1. Maturation, reactivity and therapy induced changes of memory T-cells in rheumatic autoimmune diseases

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    Fritsch, R.D.E.

    2014-01-01

    This thesis covers different aspects of CD4+T-cells (maturational pathway, auto-reactive potential and differential reaction to glucocorticoid-therapy) in two auto-immune diseases: systemic lupus erythematosus (SLE) and Rheumatoid arthritis (RA). Immunological memory is important for an adequate res

  2. Matrine derivate MASM suppresses LPS-induced phenotypic and functional maturation of murine bone marrow-derived dendritic cells.

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    Xu, Jing; Qi, Yang; Xu, Wei-Heng; Liu, Ying; Qiu, Lie; Wang, Ke-Qi; Hu, Hong-Gang; He, Zhi-Gao; Zhang, Jun-Ping

    2016-07-01

    Dendritic cell (DC) maturation process is a crucial step for the development of T cell immune responses and immune tolerance. In this study, we evaluated MASM, a novel derivative of the natural compound matrine that possesses a significant anti-inflammatory and immune-regulating property, for its efficacy to inhibit lipopolysaccharides (LPS)-induced maturation of murine bone marrow-derived dendritic cells. Here we show that MASM profoundly suppresses LPS-induced phenotypic and functional DC maturation. MASM inhibited LPS-induced expression of costimulatory molecules CD80 and CD86 in a concentration-dependent manner. MASM also attenuated LPS-induced IL-12p70, TNF-α, IL-6 and NO release of DCs. The MASM-treated DCs were highly efficient at antigen capture via mannose receptor-mediated endocytosis but showed weak stimulatory capacity for allogeneic T cell proliferation. Furthermore, MASM inhibited LPS-induced PI3K/Akt, MAPK and NF-κB pathways. These novel findings provide new insight into the immunopharmacological role of MASM in impacting on the DCs.

  3. Potentially probiotic bacteria induce efficient maturation but differential cytokine production in human monocyte-derived dendritic cells

    Institute of Scientific and Technical Information of China (English)

    Sinikka Latvala; Taija E Pietil(a); Ville Veckman; Riina A Kekkonen; Soile Tynkkynen; Riitta Korpela; Ilkka Julkunen

    2008-01-01

    MM: To analyze the ability of nine different potentially probiotic bacteria to induce maturation and cytokine production in human monocyLe-derived dendritic cells (moDCs).METHODS: Cytokine production and maturation of moDCs in response to bacterial stimulation was analyzed with enzyme-linked immunosorbent assay (ELISA) and flow cytometric analysis (FACS),respectively.The kinetics of mRNA expression of cytokine genes was determined by Northern blotting.The involvement of different signaling pathways in cytokine gene expression was studied using specific pharmacological signaling inhibitors.RESULTS: All studied bacteria induced the maturation of moDCs in a dose-dependent manner.More detailed analysis with S.thermophilus THS,B.breve Bb99,and L.lactis subsp,cremoris ARH74 indicated that these bacteria induced the expression of moDC maturation markers HLA class II and CD86 as efficiently as pathogenic bacteria.However,these bacteria differed in their ability to induce moDC cytokine gene expression.S.therrnophilus induced the expression of pro-inflammatory (TNF-a,IL-12,IL-6,and CCL20)and Th1 type (IL-12 and IFN-y) cytokines,while B.breve and L.lactis were also potent inducers of antiinflammatory IL-10.Mitogen-activated protein kinase (MAPK) p38,phosphatidylinositol 3 (PI3) kinase,and nuclear factor-kappa B (NF-κB) signaling pathways were shown to be involved in bacteria-induced cytokine production.CONCLUSION: Our results indicate that potentially probiotic bacteria are able to induce moDC maturation,but their ability to induce cytokine gene expression varies significantly from one bacterial strain to another.

  4. A novel polysaccharide from the seeds of Plantago asiatica L. induces dendritic cells maturation through toll-like receptor 4.

    Science.gov (United States)

    Huang, Danfei; Nie, Shaoping; Jiang, Leming; Xie, Mingyong

    2014-02-01

    In this study, we investigated the effect of a polysaccharide purified from the seeds of Plantago asiatica L. (PLP-2) on the phenotypic and functional maturation of murine bone marrow-derived dendritic cells (DCs) and relevant mechanisms. The results showed that PLP-2 increased the expression of maturation markers major histocompatibility complex II, CD86, CD80, and CD40 on DCs. Consistent with the changes in the phenotypic markers, functional assay for DCs maturation showed that PLP-2 decreased DCs endocytosis and increased intracellular interleukin (IL)-12 levels and allostimulatory activity. Furthermore, using a syngeneic T cell activation model, we found that PLP-2 treated DCs presented ovalbumin antigen to T cells more efficiently as demonstrated by increased T cell proliferation. In addition, the effects of PLP-2 on DCs were significantly impaired by treating the cells with anti-TLR4 antibody prior to PLP-2 treatment, implying direct interaction between PLP-2 and TLR4 on cell surface. These results suggested that PLP-2 may induce DCs maturation through TLR4. Our results may have important implications for our understanding on the molecular mechanisms of immunopotentiating action of the polysaccharides from plants.

  5. Defined MicroRNAs Induce Aspects of Maturation in Mouse and Human Embryonic-Stem-Cell-Derived Cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Desy S. Lee

    2015-09-01

    Full Text Available Pluripotent-cell-derived cardiomyocytes have great potential for use in research and medicine, but limitations in their maturity currently constrain their usefulness. Here, we report a method for improving features of maturation in murine and human embryonic-stem-cell-derived cardiomyocytes (m/hESC-CMs. We found that coculturing m/hESC-CMs with endothelial cells improves their maturity and upregulates several microRNAs. Delivering four of these microRNAs, miR-125b-5p, miR-199a-5p, miR-221, and miR-222 (miR-combo, to m/hESC-CMs resulted in improved sarcomere alignment and calcium handling, a more negative resting membrane potential, and increased expression of cardiomyocyte maturation markers. Although this could not fully phenocopy all adult cardiomyocyte characteristics, these effects persisted for two months following delivery of miR-combo. A luciferase assay demonstrated that all four miRNAs target ErbB4, and siRNA knockdown of ErbB4 partially recapitulated the effects of miR-combo. In summary, a combination of miRNAs induced via endothelial coculture improved ESC-CM maturity, in part through suppression of ErbB4 signaling.

  6. Novel analysis of maturation of murine bone-marrow-derived dendritic cells induced by Ginkgo Seed Polysaccharides.

    Science.gov (United States)

    Chen, Yinghan; Meng, Yiming; Cao, Yan; Wen, Hua; Luo, Hong; Gao, Xinghua; Shan, Fengping

    2015-01-01

    Our understanding of the mechanisms of effect of Ginkgo Seed Polysaccharides (GSPs) on the immune system remains unclear. The aim of this work was to investigate the effect of GSPs on the maturation and function of bone-marrow-derived dendritic cells (BMDCs). The results demonstrate that GSP could exert positive immune modulation on the maturation and functions of BMDCs. This effect was evidenced by decreased changes of phagosome number inside BMDCs, decreased activity of acidic phosphatase (ACP), decreased phagocytosis of BMDCs, and increased changes of key membrane molecules on BMDCs. Upregulated production of cytokines IL-12 and TNF-α also was confirmed. Therefore, it can be concluded that GSPs can efficiently induce the maturation of BMDCs. Our exploration provides direct data and a rationale for potential application of GSPs as an immune enhancer in improving immunity and as a potent adjuvant in the design of DC-based vaccines.

  7. Soluble Jagged 1/Fc chimera protein induces the differentiation and maturation of bone marrow-derived dendritic cells

    Institute of Scientific and Technical Information of China (English)

    XING FeiYue; LIU Jing; YU Zhe; JI YuHua

    2008-01-01

    A soluble Jagged 1/Fc chimera protein (Jagged 1/Fc) was directly used to induce differentiation and maturation of bone marrow-derived dendritic cells (DCs) in mice in vitro. A model of inducing and am-plifying DCs in vitro was established. The effect of Jagged 1/Fc on morphology of DCs induced by both rmGM-CSF and rmlL-4 was observed under a confocal microscope. A fluorescein-labeled monoclonal antibody staining combined with flow cytometry was applied to detect the effect of Jagged 1/Fc on the expression of CD11c, MHC-Ⅱ, CD86, CD80 and CD40 molecules on the surface of DCs. The results showed that Jagged 1/Fc did not affect the morphological properties of DC differentiation induced by both rmGM-CSF and rmlL-4. But it could promote the differentiation and maturation of DCs induced by both. The effect of it was strikingly different in the expression profile of co-stimulating molecules and the morphologic properties of DCs from lipopolysaccharide (LPS). The levels of MHC-Ⅱ and CD40 molecule expression on the surface of DCs stimulated by Jagged 1/Fc were significantly lower than those stimulated by LPS, and the level of CD80 expression on the surface of DCs induced by Jagged 1/Fc was near to that induced by LPS. Jagged 1/Fc had no influence on the expression of CD86 mole-cule on the surface of DCs. Jagged 1/Fc when used alone could not maintain the growth, differentiation and maturation of DCs. All the findings indicate that Jagged 1/Fc influences the differentiation and maturation of DCs, which is not markedly similar to LPS, providing important evidence for its devel-opment and application as a novel immunosuppressant.

  8. Short-term environmental enrichment exposure induces proliferation and maturation of doublecortin-positive cells in the prefrontal cortex

    Institute of Scientific and Technical Information of China (English)

    Chunling Fan; Mengqi Zhang; Lei Shang; Ngobe Akume Cynthia; Zhi Li; Zhenyu Yang; Dan Chen; Jufang Huang; Kun Xiong

    2014-01-01

    Previous studies have demonstrated that doublecortin-positive immature neurons exist pre-dominantly in the superficial layer of the cerebral cortex of adult mammals such as guinea pigs, and these neurons exhibit very weak properties of self-proliferation during adulthood under physiological conditions. To verify whether environmental enrichment has an impact on the proliferation and maturation of these immature neurons in the prefrontal cortex of adult guinea pigs, healthy adult guinea pigs were subjected to short-term environmental enrichment. Animals were allowed to play with various cognitive and physical stimulating objects over a period of 2 weeks, twice per day, for 60 minutes each. Immunolfuorescence staining results indicated that the number of doublecortin-positive cells in layer II of the prefrontal cortex was signiifcantly increased after short-term environmental enrichment exposure. In addition, these doublecortin-positive cells co-expressed 5-bromo-2-deoxyuridine (a marker of cell prolifera-tion), c-Fos (a marker of cell viability) and NeuN (a marker of mature neurons). Experimental ifndings showed that short-term environmental enrichment can induce proliferation, activation and maturation of doublecortin-positive cells in layer II of the prefrontal cortex of adult guinea pigs.

  9. NO2 inhalation induces maturation of pulmonary CD11c+ cells that promote antigenspecific CD4+ T cell polarization

    Directory of Open Access Journals (Sweden)

    Suratt Benjamin T

    2010-07-01

    Full Text Available Abstract Background Nitrogen dioxide (NO2 is an air pollutant associated with poor respiratory health, asthma exacerbation, and an increased likelihood of inhalational allergies. NO2 is also produced endogenously in the lung during acute inflammatory responses. NO2 can function as an adjuvant, allowing for allergic sensitization to an innocuous inhaled antigen and the generation of an antigen-specific Th2 immune response manifesting in an allergic asthma phenotype. As CD11c+ antigen presenting cells are considered critical for naïve T cell activation, we investigated the role of CD11c+ cells in NO2-promoted allergic sensitization. Methods We systemically depleted CD11c+ cells from transgenic mice expressing a simian diphtheria toxin (DT receptor under of control of the CD11c promoter by administration of DT. Mice were then exposed to 15 ppm NO2 followed by aerosolized ovalbumin to promote allergic sensitization to ovalbumin and were studied after subsequent inhaled ovalbumin challenges for manifestation of allergic airway disease. In addition, pulmonary CD11c+ cells from wildtype mice were studied after exposure to NO2 and ovalbumin for cellular phenotype by flow cytometry and in vitro cytokine production. Results Transient depletion of CD11c+ cells during sensitization attenuated airway eosinophilia during allergen challenge and reduced Th2 and Th17 cytokine production. Lung CD11c+ cells from wildtype mice exhibited a significant increase in MHCII, CD40, and OX40L expression 2 hours following NO2 exposure. By 48 hours, CD11c+MHCII+ DCs within the mediastinal lymph node (MLN expressed maturation markers, including CD80, CD86, and OX40L. CD11c+CD11b- and CD11c+CD11b+ pulmonary cells exposed to NO2 in vivo increased uptake of antigen 2 hours post exposure, with increased ova-Alexa 647+ CD11c+MHCII+ DCs present in MLN from NO2-exposed mice by 48 hours. Co-cultures of ova-specific CD4+ T cells from naïve mice and CD11c+ pulmonary cells from NO2

  10. Recombinant Lactobacillus plantarum induces immune responses to cancer testis antigen NY-ESO-1 and maturation of dendritic cells.

    Science.gov (United States)

    Mobergslien, Anne; Vasovic, Vlada; Mathiesen, Geir; Fredriksen, Lasse; Westby, Phuong; Eijsink, Vincent G H; Peng, Qian; Sioud, Mouldy

    2015-01-01

    Given their safe use in humans and inherent adjuvanticity, Lactic Acid Bacteria may offer several advantages over other mucosal delivery strategies for cancer vaccines. The objective of this study is to evaluate the immune responses in mice after oral immunization with Lactobacillus (L) plantarum WCFS1 expressing a cell-wall anchored tumor antigen NY-ESO-1. And to investigate the immunostimulatory potency of this new candidate vaccine on human dendritic cells (DCs). L. plantarum displaying NY-ESO-1 induced NY-ESO-1 specific antibodies and T-cell responses in mice. By contrast, L. plantarum displaying conserved proteins such as heat shock protein-27 and galectin-1, did not induce immunity, suggesting that immune tolerance to self-proteins cannot be broken by oral administration of L. plantarum. With respect to immunomodulation, immature DCs incubated with wild type or L. plantarum-NY-ESO-1 upregulated the expression of co-stimulatory molecules and secreted a large amount of interleukin (IL)-12, TNF-α, but not IL-4. Moreover, they upregulated the expression of immunosuppressive factors such as IL-10 and indoleamine 2,3-dioxygenase. Although L. plantarum-matured DCs expressed inhibitory molecules, they stimulated allogeneic T cells in-vitro. Collectively, the data indicate that L. plantarum-NY-ESO-1 can evoke antigen-specific immunity upon oral administration and induce DC maturation, raising the potential of its use in cancer immunotherapies.

  11. Protein-induced changes during the maturation process of human dendritic cells: A 2-D DIGE approach

    DEFF Research Database (Denmark)

    Ferreira, Gb; Overbergh, L; Hansen, Kasper Lage

    2008-01-01

    Dendritic cells (DCs) are unique antigen presenting cells, which upon maturation change from a specialized antigen-capturing cell towards a professional antigen presenting cells. In this study, a 2-D DIGE analysis of immature and mature DCs was performed, to identify proteins changing in expressi...

  12. Morphogenetic study on the maturation of osteoblastic cell as induced by inorganic polyphosphate.

    Directory of Open Access Journals (Sweden)

    Kaori Tsutsumi

    Full Text Available Since inorganic polyphosphates [poly(P] have an activity to induce bone differenciation in vitro and in vivo, we examined an effect of poly(P on organelle by light microscopy and electron microscopy in Murine MC3T3-E1 osteoblastic cells. The MC3T3-E1 cells were ultrastructurally observed to possess morphological characteristics of osteoblasts. Cells cultured with poly(P were strongly stained with an anti-collagen type I antibody but not in those cultured without poly(P. Ultrastructural analysis of cells cultured with poly(P revealed a well-developed Golgi apparatus, swollen and elongated rough endoplasmic reticulum, large mitochondria and many coated pits. Since MC3T3-E1 cells can be transformed from a resting phase to an active blastic cell phase after supplementation with poly(P, it implies that poly(P can be an effective material for bone regeneration.

  13. Concanavalin-A induces granulosa cell death and inhibits FSH-mediated follicular growth and ovarian maturation in female rats.

    Science.gov (United States)

    Velasquez, Ethel V; Ríos, Mariana; Ortiz, María Elena; Lizama, Carlos; Nuñez, Elizabeth; Abramovich, Dalhia; Orge, Felipe; Oliva, Barbara; Orellana, Renán; Villalon, Manuel; Moreno, Ricardo D; Tesone, Marta; Rokka, Anne; Corthals, Garry; Croxatto, Horacio B; Parborell, Fernanda; Owen, Gareth I

    2013-05-01

    Reproductive success stems from a finely regulated balance between follicular maturation and atresia, in which the role of carbohydrate structure is poorly understood. Here, we describe for the first time a fraction of purified recombinant human FSH that is capable of bringing about the cell death of granulosa cells and preventing follicular maturation in a rat model. Further analysis by mass spectrometry revealed the presence of the lectin Concanavalin-A (Con-A) within this fraction of recombinant FSH. Using both the fractionated FSH and Con-A, the observed cell death was predominantly located to the granulosa cells. Ex vivo culture of rat follicles demonstrated that follicle degeneration occurred and resulted in the release of a denuded and deteriorated oocyte. Moreover, in vivo experiments confirmed an increase in atresia and a corresponding reduction confined to follicle in early antral stage. As a mechanism of action, Con-A reduces ovarian proliferation, Von Willebrand staining, and angiogenesis. Based on the observation that Con-A may induce granulosa cell death followed by follicle death, our results further demonstrate that follicular carbohydrate moiety is changing under the influence of FSH, which may allow a carbohydrate-binding lectin to increase granulosa cell death. The physiological consequences of circulating lectin-like molecules remain to be determined. However, our results suggest a potential exploitation of carbohydrate binding in fertility and ovarian cancer treatment. This work may shed light on a key role of carbohydrates in the still obscure physiological process of follicular selection and atresia.

  14. Influenza Virus–induced Dendritic Cell Maturation Is Associated with the Induction of Strong T Cell Immunity to a Coadministered, Normally Nonimmunogenic Protein

    Science.gov (United States)

    Brimnes, Marie K.; Bonifaz, Laura; Steinman, Ralph M.; Moran, Thomas M.

    2003-01-01

    We evaluated the proposal that during microbial infection, dendritic cells (DCs) undergo maturation and present a mixture of peptides derived from the microbe as well as harmless environmental antigens. Mice were exposed to an aerosol of endotoxin free ovalbumin (OVA) in the absence or presence of influenza virus. In its absence, OVA failed to induce B and T cell responses and even tolerized, but with influenza, OVA-specific antibodies and CD8+ cytolytic T lymphocytes developed. With or without infection, OVA was presented selectively in the draining mediastinal lymph nodes, as assessed by the comparable proliferation of infused, CD8+ and CD4+, TCR transgenic T cells. In the absence of influenza, these OVA-specific T cells produced little IL-2, IL-4, IL-10, and IFN-γ, but with infection, both CD4+ and CD8+ T cells made high levels of IL-2 and IFN-γ. The OVA plus influenza-treated mice also showed accelerated recovery to a challenge with recombinant vaccinia OVA virus. CD11c+ DCs from the mediastinal lymph nodes of infected mice selectively stimulated both OVA- and influenza-specific T cells and underwent maturation, with higher levels of MHC class II, CD80, and CD86 molecules. The relatively slow (2–3 d) kinetics of maturation correlated closely to the time at which OVA inhalation elicited specific antibodies. Therefore respiratory infection can induce DC maturation and simultaneously B and T cell immunity to an innocuous antigen inhaled concurrently. PMID:12847140

  15. Cryptococcus gattii is killed by dendritic cells, but evades adaptive immunity by failing to induce dendritic cell maturation.

    Science.gov (United States)

    Huston, Shaunna M; Li, Shu Shun; Stack, Danuta; Timm-McCann, Martina; Jones, Gareth J; Islam, Anowara; Berenger, Byron M; Xiang, Richard F; Colarusso, Pina; Mody, Christopher H

    2013-07-01

    During adaptive immunity to pathogens, dendritic cells (DCs) capture, kill, process, and present microbial Ags to T cells. Ag presentation is accompanied by DC maturation driven by appropriate costimulatory signals. However, current understanding of the intricate regulation of these processes remains limited. Cryptococcus gattii, an emerging fungal pathogen in the Pacific Northwest of Canada and the United States, fails to stimulate an effective immune response in otherwise healthy hosts leading to morbidity or death. Because immunity to fungal pathogens requires intact cell-mediated immunity initiated by DCs, we asked whether C. gattii causes dysregulation of DC functions. C. gattii was efficiently bound and internalized by human monocyte-derived DCs, trafficked to late phagolysosomes, and killed. Yet, even with this degree of DC activation, the organism evaded pathways leading to DC maturation. Despite the ability to recognize and kill C. gattii, immature DCs failed to mature; there was no increased expression of MHC class II, CD86, CD83, CD80, and CCR7, or decrease of CD11c and CD32, which resulted in suboptimal T cell responses. Remarkably, no increase in TNF-α was observed in the presence of C. gattii. However, addition of recombinant TNF-α or stimulation that led to TNF-α production restored DC maturation and restored T cell responses. Thus, despite early killing, C. gattii evades DC maturation, providing a potential explanation for its ability to infect immunocompetent individuals. We have also established that DCs retain the ability to recognize and kill C. gattii without triggering TNF-α, suggesting independent or divergent activation pathways among essential DC functions.

  16. AKT induces erythroid-cell maturation of JAK2-deficient fetal liver progenitor cells and is required for Epo regulation of erythroid-cell differentiation.

    Science.gov (United States)

    Ghaffari, Saghi; Kitidis, Claire; Zhao, Wei; Marinkovic, Dragan; Fleming, Mark D; Luo, Biao; Marszalek, Joseph; Lodish, Harvey F

    2006-03-01

    AKT serine threonine kinase of the protein kinase B (PKB) family plays essential roles in cell survival, growth, metabolism, and differentiation. In the erythroid system, AKT is known to be rapidly phosphorylated and activated in response to erythropoietin (Epo) engagement of Epo receptor (EpoR) and to sustain survival signals in cultured erythroid cells. Here we demonstrate that activated AKT complements EpoR signaling and supports erythroid-cell differentiation in wild-type and JAK2-deficient fetal liver cells. We show that erythroid maturation of AKT-transduced cells is not solely dependent on AKT-induced cell survival or proliferation signals, suggesting that AKT transduces also a differentiation-specific signal downstream of EpoR in erythroid cells. Down-regulation of expression of AKT kinase by RNA interference, or AKT activity by expression of dominant negative forms, inhibits significantly fetal liver-derived erythroid-cell colony formation and gene expression, demonstrating that AKT is required for Epo regulation of erythroid-cell maturation.

  17. Mature adipocyte-derived cells, dedifferentiated fat cells (DFAT), promoted functional recovery from spinal cord injury-induced motor dysfunction in rats.

    Science.gov (United States)

    Ohta, Yuki; Takenaga, Mitsuko; Tokura, Yukie; Hamaguchi, Akemi; Matsumoto, Taro; Kano, Koichiro; Mugishima, Hideo; Okano, Hideyuki; Igarashi, Rie

    2008-01-01

    Transplantation of mature adipocyte-derived cells (dedifferentiated fat cells) led to marked functional recovery from spinal cord injury (SCI)-induced motor dysfunction in rats. When mature adipocytes were isolated from rat adipose tissue and grown in ceiling culture, transformation into fibroblast-like cells without lipid droplets occurred. These fibroblast-like cells, termed dedifferentiated fat cells (DFAT), could proliferate and could also differentiate back into adipocytes. DFAT expressed neural markers such as nestin, betaIII tubulin, and GFAP. Allografting of DFAT into SCI-induced rats led to significant recovery from hindlimb dysfunction. Grafted cells were detected at the injection site, and some of these cells expressed betaIII tubulin. DFAT expressed neurotrophic factors such as BDNF and GDNF prior to transplantation, and grafted cells were also positive for these factors. Therefore, these neurotrophic factors derived from grafted DFAT might have contributed to the promotion of functional recovery. These findings also suggest that mature adipocytes could become a new source for cell replacement therapy to treat central nervous system disorders.

  18. Novel plant virus-based vaccine induces protective cytotoxic T-lymphocyte-mediated antiviral immunity through dendritic cell maturation.

    Science.gov (United States)

    Lacasse, Patrick; Denis, Jérôme; Lapointe, Réjean; Leclerc, Denis; Lamarre, Alain

    2008-01-01

    Currently used vaccines protect mainly through the production of neutralizing antibodies. However, antibodies confer little or no protection for a majority of chronic viral infections that require active involvement of cytotoxic T lymphocytes (CTLs). Virus-like particles (VLPs) have been shown to be efficient inducers of cell-mediated immune responses, but administration of an adjuvant is generally required. We recently reported the generation of a novel VLP system exploiting the self-assembly property of the papaya mosaic virus (PapMV) coat protein. We show here that uptake of PapMV-like particles by murine splenic dendritic cells (DCs) in vivo leads to their maturation, suggesting that they possess intrinsic adjuvant-like properties. DCs pulsed with PapMV-like particles displaying the lymphocytic choriomeningitis virus (LCMV) p33 immunodominant CTL epitope (PapMV-p33) efficiently process and cross-present the viral epitope to p33-specific transgenic T cells. Importantly, the CTL epitope is also properly processed and presented in vivo, since immunization of p33-specific T-cell receptor transgenic mice with PapMV-p33 induces the activation of large numbers of specific CTLs. C57BL/6 mice immunized with PapMV-p33 VLPs in the absence of adjuvant develop p33-specific effector CTLs that rapidly expand following LCMV challenge and protect vaccinated mice against LCMV infection in a dose-dependent manner. These results demonstrate the efficiency of this novel plant virus-based vaccination platform in inducing DC maturation leading to protective CTL responses.

  19. ERK1 and ERK2 are involved in recruitment and maturation of human mesenchymal stem cells induced to adipogenic differentiation

    Institute of Scientific and Technical Information of China (English)

    Elisabetta Donzelli; Caterina Lucchini; Elisa Ballarini; Arianna Scuteri; Fabrizio Carini; Giovanni Tredici; Mariarosaria Miloso

    2011-01-01

    Adipocytes' biology and the mechanisms that control adipogenesis have gained importance because of the need to develop therapeutic strategies to control obesity and the related pathologies. Human mesenchymal stem cells (hMSCs), undifferentiated stem cells present in the bone marrow that are physiological precursors of adipocytes, were induced to adipogenic differentiation. The molecular mechanisms on the basis of the adipogenesis were evaluated, focusing on the MAPKinases ERK1 and ERK2, which are involved in many biological and cellular processes. ERK1 and ERK2 phosphorylation was reduced with different timing and intensity for the two isoforms in treated hMSCs in comparison with control cells until day 10 and then at 14-28 days, it reached the level of untreated cultures. The total amount of ERK1 was also decreased up to day 10 and then was induced to the level of untreated cultures, whereas the expression of ERK2 was not changed following adipogenic induction. Treatment with the specific ERK1/2 inhibitor U0126 during the whole differentiation period hampered hMSCs' adipogenic differentiation, as lipid droplets appeared in very few cells and were reduced in number and size. When U0126 was administered only during the initial phase of differentiation, the number of hMSCs recruited to adipogenesis was reduced while, when it was administered later, hMSCs did not acquire a mature adipocytic phenotype. ERK1 and ERK2 are important for hMSC adipogenic differentiation since any alteration to the correct timing of their phosphorylation affects either the recruitment into the differentiation program and the extent of their maturation.

  20. Complement protein C1q induces maturation of human dendritic cells

    DEFF Research Database (Denmark)

    Cosmor, E; Bajtay, Z; Sándor, N;

    2007-01-01

    in the absence of antibodies, we undertook to investigate whether this complement protein has an impact on various functions of human DCs. Maturation of monocyte-derived immature DCs (imMDCs) cultured on immobilized C1q was followed by monitoring expression of CD80, CD83, CD86, MHCII and CCR7. The functional...

  1. Unconventional maturation of dendritic cells induced by particles from the laminated layer of larval Echinococcus granulosus.

    Science.gov (United States)

    Casaravilla, Cecilia; Pittini, Alvaro; Rückerl, Dominik; Seoane, Paula I; Jenkins, Stephen J; MacDonald, Andrew S; Ferreira, Ana M; Allen, Judith E; Díaz, Alvaro

    2014-08-01

    The larval stage of the cestode parasite Echinococcus granulosus causes hydatid disease in humans and livestock. This infection is characterized by the growth in internal organ parenchymae of fluid-filled structures (hydatids) that elicit surprisingly little inflammation in spite of their massive size and persistence. Hydatids are protected by a millimeter-thick layer of mucin-based extracellular matrix, termed the laminated layer (LL), which is thought to be a major factor determining the host response to the infection. Host cells can interact both with the LL surface and with materials that are shed from it to allow parasite growth. In this work, we analyzed the response of dendritic cells (DCs) to microscopic pieces of the native mucin-based gel of the LL (pLL). In vitro, this material induced an unusual activation state characterized by upregulation of CD86 without concomitant upregulation of CD40 or secretion of cytokines (interleukin 12 [IL-12], IL-10, tumor necrosis factor alpha [TNF-α], and IL-6). When added to Toll-like receptor (TLR) agonists, pLL-potentiated CD86 upregulation and IL-10 secretion while inhibiting CD40 upregulation and IL-12 secretion. In vivo, pLL also caused upregulation of CD86 and inhibited CD40 upregulation in DCs. Contrary to expectations, oxidation of the mucin glycans in pLL with periodate did not abrogate the effects on cells. Reduction of disulfide bonds, which are known to be important for LL structure, strongly diminished the impact of pLL on DCs without altering the particulate nature of the material. In summary, DCs respond to the LL mucin meshwork with a "semimature" activation phenotype, both in vitro and in vivo.

  2. Immature and maturation-resistant human dendritic cells generated from bone marrow require two stimulations to induce T cell anergy in vitro.

    Directory of Open Access Journals (Sweden)

    Thomas G Berger

    Full Text Available Immature dendritic cells (DC represent potential clinical tools for tolerogenic cellular immunotherapy in both transplantation and autoimmunity. A major drawback in vivo is their potential to mature during infections or inflammation, which would convert their tolerogenicity into immunogenicity. The generation of immature DC from human bone marrow (BM by low doses of GM-CSF (lowGM in the absence of IL-4 under GMP conditions create DC resistant to maturation, detected by surface marker expression and primary stimulation by allogeneic T cells. This resistence could not be observed for BM-derived DC generated with high doses of GM-CSF plus IL-4 (highGM/4, although both DC types induced primary allogeneic T cell anergy in vitro. The estabishment of the anergic state requires two subsequent stimulations by immature DC. Anergy induction was more profound with lowGM-DC due to their maturation resistance. Together, we show the generation of immature, maturation-resistant lowGM-DC for potential clinical use in transplant rejection and propose a two-step-model of T cell anergy induction by immature DC.

  3. Glycans from Fasciola hepatica Modulate the Host Immune Response and TLR-Induced Maturation of Dendritic Cells.

    Science.gov (United States)

    Rodríguez, Ernesto; Noya, Verónica; Cervi, Laura; Chiribao, María Laura; Brossard, Natalie; Chiale, Carolina; Carmona, Carlos; Giacomini, Cecilia; Freire, Teresa

    2015-12-01

    Helminths express various carbohydrate-containing glycoconjugates on their surface, and they release glycan-rich excretion/secretion products that can be very important in their life cycles, infection and pathology. Recent evidence suggests that parasite glycoconjugates could play a role in the evasion of the immune response, leading to a modified Th2-polarized immune response that favors parasite survival in the host. Nevertheless, there is limited information about the nature or function of glycans produced by the trematode Fasciola hepatica, the causative agent of fasciolosis. In this paper, we investigate whether glycosylated molecules from F. hepatica participate in the modulation of host immunity. We also focus on dendritic cells, since they are an important target of immune-modulation by helminths, affecting their activity or function. Our results indicate that glycans from F. hepatica promote the production of IL-4 and IL-10, suppressing IFNγ production. During infection, this parasite is able to induce a semi-mature phenotype of DCs expressing low levels of MHCII and secrete IL-10. Furthermore, we show that parasite glycoconjugates mediate the modulation of LPS-induced maturation of DCs since their oxidation restores the capacity of LPS-treated DCs to secrete high levels of the pro-inflammatory cytokines IL-6 and IL-12/23p40 and low levels of the anti-inflammatory cytokine IL-10. Inhibition assays using carbohydrates suggest that the immune-modulation is mediated, at least in part, by the recognition of a mannose specific-CLR that signals by recruiting the phosphatase Php2. The results presented here contribute to the understanding of the role of parasite glycosylated molecules in the modulation of the host immunity and might be useful in the design of vaccines against fasciolosis.

  4. Mycobacterium avium subspecies impair dendritic cell maturation.

    Science.gov (United States)

    Basler, Tina; Brumshagen, Christina; Beineke, Andreas; Goethe, Ralph; Bäumer, Wolfgang

    2013-10-01

    Mycobacterium avium ssp. paratuberculosis (MAP) causes Johne's disease, a chronic, granulomatous enteritis of ruminants. Dendritic cells (DC) of the gut are ideally placed to combat invading mycobacteria; however, little is known about their interaction with MAP. Here, we investigated the interaction of MAP and the closely related M. avium ssp. avium (MAA) with murine DC and the effect of infected macrophages on DC maturation. The infection of DC with MAP or MAA induced DC maturation, which differed to that of LPS as maturation was accompanied by higher production of IL-10 and lower production of IL-12. Treatment of maturing DC with supernatants from mycobacteria-infected macrophages resulted in impaired DC maturation, leading to a semi-mature, tolerogenic DC phenotype expressing low levels of MHCII, CD86 and TNF-α after LPS stimulation. Though the cells were not completely differentiated they responded with an increased IL-10 and a decreased IL-12 production. Using recombinant cytokines we provide evidence that the semi-mature DC phenotype results from a combination of secreted cytokines and released antigenic mycobacterial components of the infected macrophage. Our results indicate that MAP and MAA are able to subvert DC function directly by infecting and indirectly via the milieu created by infected macrophages.

  5. Skin irritants and contact sensitizers induce Langerhans cell migration and maturation at irritant concentration

    NARCIS (Netherlands)

    Jacobs, J.J.L.; Lehé, C.L.; Hasegawa, H.; Elliott, G.R.; Das, P.K.

    2006-01-01

    Skin irritants and contact allergens reduce the number of Langerhans cells (LCs). It has been assumed that this reduction is due their migration to the draining lymph node (LN) for initiating immune sensitization in a host. Skin irritation, however, as opposed to contact allergy is not considered to

  6. Maturation-Based Model of Arrhythmogenic Right Ventricular Dysplasia Using Patient-Specific Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Wen, Jian-Yan; Wei, Chuan-Yu; Shah, Khooshbu; Wong, Johnson; Wang, Cheng; Chen, Huei-Sheng Vincent

    2015-01-01

    Cellular reprogramming of somatic cells to patient-specific induced pluripotent stem cells (iPSCs) enables in-vitro modeling of human cardiac disorders for pathogenic and therapeutic investigations. However, using iPSC-derived cardiomyocytes (iPSC-CMs) to model an adult-onset heart disease remains challenging because of the uncertainty regarding the ability of relatively immature iPSC-CMs to fully recapitulate adult disease phenotypes. Arrhythmogenic right ventricular dysplasia (ARVD) is an inherited cardiomyopathy characterized by pathological fibrofatty infiltration and cardiomyocyte (CM) loss predominantly in the right ventricle (RV), leading to heart failure and lethal arrhythmias. Over 50% of affected individuals have desmosome gene mutations, most commonly inPKP2encoding plakophilin-2. Using Yamanaka's pluripotent factors, we generated iPSC lines from ARVD patients withPKP2mutations. We first developed a method to induce metabolic maturation of iPSC-CMs and showed that induction of adult-like metabolic energetics from an embryonic/glycolytic state is essential to model an adult-onset cardiac disease using patient-specific iPSCs. Furthermore, we showed that coactivation of normal peroxisome proliferator-activated receptor (PPAR)-α and abnormal PPARγ pathways in ARVD iPSC-CMs resulted in exaggerated CM lipogenesis, CM apoptosis, Na(+)channel downregulation and defective intracellular calcium handling, recapitulating the pathological signatures of ARVD. Using this model, we revealed novel pathogenic insights that metabolic derangement in an adult-like metabolic milieu underlies ARVD pathologies, enabling us to propose novel disease-modifying therapeutic strategies.

  7. Regulated assembly of vacuolar ATPase is increased during cluster disruption-induced maturation of dendritic cells through a phosphatidylinositol 3-kinase/mTOR-dependent pathway.

    Science.gov (United States)

    Liberman, Rachel; Bond, Sarah; Shainheit, Mara G; Stadecker, Miguel J; Forgac, Michael

    2014-01-17

    The vacuolar (H(+))-ATPases (V-ATPases) are ATP-driven proton pumps composed of a peripheral V1 domain and a membrane-embedded V0 domain. Regulated assembly of V1 and V0 represents an important regulatory mechanism for controlling V-ATPase activity in vivo. Previous work has shown that V-ATPase assembly increases during maturation of bone marrow-derived dendritic cells induced by activation of Toll-like receptors. This increased assembly is essential for antigen processing, which is dependent upon an acidic lysosomal pH. Cluster disruption of dendritic cells induces a semi-mature phenotype associated with immune tolerance. Thus, semi-mature dendritic cells are able to process and present self-peptides to suppress autoimmune responses. We have investigated V-ATPase assembly in bone marrow-derived, murine dendritic cells and observed an increase in assembly following cluster disruption. This increased assembly is not dependent upon new protein synthesis and is associated with an increase in concanamycin A-sensitive proton transport in FITC-loaded lysosomes. Inhibition of phosphatidylinositol 3-kinase with wortmannin or mTORC1 with rapamycin effectively inhibits the increased assembly observed upon cluster disruption. These results suggest that the phosphatidylinositol 3-kinase/mTOR pathway is involved in controlling V-ATPase assembly during dendritic cell maturation.

  8. α-MSH inhibits TNF-α-induced maturation of human dendritic cells in vitro through the up-regulation of ANXA1

    Institute of Scientific and Technical Information of China (English)

    Yan Min; Deping Han; Zhanjiang Fu; Honghai Wang; Lirong Liu; Yeping Tian

    2011-01-01

    α-Melanocyte-stimulating hormone(α-MSH),an antiinflammatory and immunomodulatory neuropeptide.has been shown to be effective in the experimental treatment of autoimmune diseases and allograft rejection.However,its regulatory mechanism is still unclear.Mature dendritic cells(DCs)are pivotal initiators of immune response and inflammation.We hypothesized that the regulatory role ofα-MSH in DC maturation would contribute to the effects of α-MSH in immune-response-mediated disease models.It was found that α-MSH inhibited tumor necrosis factor-alpha(TNF-α)-induced maturation of human peripheral-monocyte-derived DCs(MoDCs),both phenotypicaily and functionally.This occurred through the down-regulation of the expression of co-stimulatory molecules CD83 and CD86,the production Of IL-12,the promotion Of IL-10secretion,and the MoDC phagocytic activity,suggesting that the inhibition Of DC maturation by α-MSH could contribute to the anti-inflammatory effect Of this neuro-peptide.Furthermore.increased expression of annexin A1(ANXA1)was found to be responsible for the α-MSH inhibiting effect on TNF-α-induced MoDC maturation.which could be abolished by the treatment of MoDCs with specific,small interfering RNAs targeting ANXA1(ANXA1-siRNA),suggesting that α-MSH-induced ANXA1mediates the inhibition.Therefore,α-MSH inhibits TNF-α-induced maturation of human DCs through α-MSH-upregulated ANXAI,suggesting that inhibition of the maturation of DCs by α-MSH could mediate the antiinflammatory effect of the neuropeptide.Furthermore,ANXA1 could be identified as a new therapeutic drug target based on the role Of DCs In immune-mediated Inflammatory diseases.

  9. Danhong inhibits oxidized low-density lipoprotein-induced immune maturation of dentritic cells via a peroxisome proliferator activated receptor γ-mediated pathway.

    Science.gov (United States)

    Liu, Hongying; Wang, Shijun; Sun, Aijun; Huang, Dong; Wang, Wei; Zhang, Chunyu; Shi, Dazhuo; Chen, Keji; Zou, Yunzeng; Ge, Junbo

    2012-01-01

    Danhong injection (DHI), a Chinese Materia Medica standardized product extracted from Radix Salviae miltiorrhizae and Flos Carthami tinctorii, is effective in the treatment of atherosclerosis (AS)-related diseases. It is widely recognized that AS is a complex inflammatory disease of the arterial wall and the dendritic cells (DCs) is a major player in the pathogenesis of AS via mediating atherosclerotic antigen presenting and T lymphocytes. Here, we determined the effect and possible mechanism of DHI on oxidized low-density lipoprotein (ox-LDL)-induced maturation and immune function of DCs. Human monocyte-derived DCs were incubated with DHI or ciglitazone and were subsequently stimulated with ox-LDL to induce maturation. Similar to ciglitazone, a peroxisome proliferator activated receptor (PPAR) γ agonist, DHI, could significantly reduce ox-LDL-induced expressions of mature markers, enhance the endocytotic function, and inhibit secretions of cytokine on DCs. These effects of DHI could be partly reversed by silencing the PPARγ. In conclusion, DHI could inhibit ox-LDL-induced maturation of DCs partly through activating a PPARγ-mediated signaling pathway.

  10. Activation of natural killer T cells by alpha-galactosylceramide rapidly induces the full maturation of dendritic cells in vivo and thereby acts as an adjuvant for combined CD4 and CD8 T cell immunity to a coadministered protein.

    Science.gov (United States)

    Fujii, Shin-Ichiro; Shimizu, Kanako; Smith, Caroline; Bonifaz, Laura; Steinman, Ralph M

    2003-07-21

    The maturation of dendritic cells (DCs) allows these antigen-presenting cells to initiate immunity. We pursued this concept in situ by studying the adjuvant action of alpha-galactosylceramide (alphaGalCer) in mice. A single i.v. injection of glycolipid induced the full maturation of splenic DCs, beginning within 4 h. Maturation was manifest by marked increases in costimulator and major histocompatibility complex class II expression, interferon (IFN)-gamma production, and stimulation of the mixed leukocyte reaction. These changes were not induced directly by alphaGalCer but required natural killer T (NKT) cells acting independently of the MyD88 adaptor protein. To establish that DC maturation was responsible for the adjuvant role of alphaGalCer, mice were given alphaGalCer together with soluble or cell-associated ovalbumin antigen. Th1 type CD4+ and CD8+ T cell responses developed, and the mice became resistant to challenge with ovalbumin-expressing tumor. DCs from mice given ovalbumin plus adjuvant, but not the non-DCs, stimulated ovalbumin-specific proliferative responses and importantly, induced antigen-specific, IFN-gamma producing, CD4+ and CD8+ T cells upon transfer into naive animals. In the latter instance, immune priming did not require further exposure to ovalbumin, alphaGalCer, NKT, or NK cells. Therefore a single dose of alphaGalCer i.v. rapidly stimulates the full maturation of DCs in situ, and this accounts for the induction of combined Th1 CD4+ and CD8+ T cell immunity to a coadministered protein.

  11. Activation of Natural Killer T Cells by α-Galactosylceramide Rapidly Induces the Full Maturation of Dendritic Cells In Vivo and Thereby Acts as an Adjuvant for Combined CD4 and CD8 T Cell Immunity to a Coadministered Protein

    Science.gov (United States)

    Fujii, Shin-ichiro; Shimizu, Kanako; Smith, Caroline; Bonifaz, Laura; Steinman, Ralph M.

    2003-01-01

    The maturation of dendritic cells (DCs) allows these antigen-presenting cells to initiate immunity. We pursued this concept in situ by studying the adjuvant action of α-galactosylceramide (αGalCer) in mice. A single i.v. injection of glycolipid induced the full maturation of splenic DCs, beginning within 4 h. Maturation was manifest by marked increases in costimulator and major histocompatibility complex class II expression, interferon (IFN)-γ production, and stimulation of the mixed leukocyte reaction. These changes were not induced directly by αGalCer but required natural killer T (NKT) cells acting independently of the MyD88 adaptor protein. To establish that DC maturation was responsible for the adjuvant role of αGalCer, mice were given αGalCer together with soluble or cell-associated ovalbumin antigen. Th1 type CD4+ and CD8+ T cell responses developed, and the mice became resistant to challenge with ovalbumin-expressing tumor. DCs from mice given ovalbumin plus adjuvant, but not the non-DCs, stimulated ovalbumin-specific proliferative responses and importantly, induced antigen-specific, IFN-γ producing, CD4+ and CD8+ T cells upon transfer into naive animals. In the latter instance, immune priming did not require further exposure to ovalbumin, αGalCer, NKT, or NK cells. Therefore a single dose of αGalCer i.v. rapidly stimulates the full maturation of DCs in situ, and this accounts for the induction of combined Th1 CD4+ and CD8+ T cell immunity to a coadministered protein. PMID:12874260

  12. Boosting high-intensity focused ultrasound-induced anti-tumor immunity using a sparse-scan strategy that can more effectively promote dendritic cell maturation

    Directory of Open Access Journals (Sweden)

    Zhong Pei

    2010-01-01

    Full Text Available Abstract Background The conventional treatment protocol in high-intensity focused ultrasound (HIFU therapy utilizes a dense-scan strategy to produce closely packed thermal lesions aiming at eradicating as much tumor mass as possible. However, this strategy is not most effective in terms of inducing a systemic anti-tumor immunity so that it cannot provide efficient micro-metastatic control and long-term tumor resistance. We have previously provided evidence that HIFU may enhance systemic anti-tumor immunity by in situ activation of dendritic cells (DCs inside HIFU-treated tumor tissue. The present study was conducted to test the feasibility of a sparse-scan strategy to boost HIFU-induced anti-tumor immune response by more effectively promoting DC maturation. Methods An experimental HIFU system was set up to perform tumor ablation experiments in subcutaneous implanted MC-38 and B16 tumor with dense- or sparse-scan strategy to produce closely-packed or separated thermal lesions. DCs infiltration into HIFU-treated tumor tissues was detected by immunohistochemistry and flow cytometry. DCs maturation was evaluated by IL-12/IL-10 production and CD80/CD86 expression after co-culture with tumor cells treated with different HIFU. HIFU-induced anti-tumor immune response was evaluated by detecting growth-retarding effects on distant re-challenged tumor and tumor-specific IFN-γ-secreting cells in HIFU-treated mice. Results HIFU exposure raised temperature up to 80 degrees centigrade at beam focus within 4 s in experimental tumors and led to formation of a well-defined thermal lesion. The infiltrated DCs were recruited to the periphery of lesion, where the peak temperature was only 55 degrees centigrade during HIFU exposure. Tumor cells heated to 55 degrees centigrade in 4-s HIFU exposure were more effective to stimulate co-cultured DCs to mature. Sparse-scan HIFU, which can reserve 55 degrees-heated tumor cells surrounding the separated lesions, elicited an

  13. TLR4-mediated podosome loss discriminates gram-negative from gram-positive bacteria in their capacity to induce dendritic cell migration and maturation.

    Science.gov (United States)

    van Helden, Suzanne F G; van den Dries, Koen; Oud, Machteld M; Raymakers, Reinier A P; Netea, Mihai G; van Leeuwen, Frank N; Figdor, Carl G

    2010-02-01

    Chronic infections are caused by microorganisms that display effective immune evasion mechanisms. Dendritic cell (DC)-dependent T cell-mediated adaptive immunity is one of the mechanisms that have evolved to prevent the occurrence of chronic bacterial infections. In turn, bacterial pathogens have developed strategies to evade immune recognition. In this study, we show that gram-negative and gram-positive bacteria differ in their ability to activate DCs and that gram-negative bacteria are far more effective inducers of DC maturation. Moreover, we observed that only gram-negative bacteria can induce loss of adhesive podosome structures in DCs, a response necessary for the induction of effective DC migration. We demonstrate that the ability of gram-negative bacteria to trigger podosome turnover and induce DC migration reflects their capacity to selectively activate TLR4. Examining mice defective in TLR4 signaling, we show that this DC maturation and migration are mainly Toll/IL-1 receptor domain-containing adaptor-inducing IFNbeta-dependent. Furthermore, we show that these processes depend on the production of PGs by these DCs, suggesting a direct link between TLR4-mediated signaling and arachidonic metabolism. These findings demonstrate that gram-positive and gram-negative bacteria profoundly differ in their capacity to activate DCs. We propose that this inability of gram-positive bacteria to induce DC maturation and migration is part of the armamentarium necessary for avoiding the induction of an effective cellular immune response and may explain the frequent involvement of these pathogens in chronic infections.

  14. Ebola and Marburg Viruses Replicate in Monocyle- Derived Dendritic Cells without Inducing the Production of Cytokines and Full Maturation

    Science.gov (United States)

    2007-11-02

    secretion in response to a second IFN-inducing stimulus ( replication -defective alphavirus ) was also potently inhibited by both viruses. From these data...1630 • JID 2003:188 (1 December) • Bosio et al. M A J O R A R T I C L E Ebola and Marburg Viruses Replicate in Monocyte- Derived Dendritic Cells...immune responses. We demonstrate that EBOV and MARV infected and replicated in primary human DCs without inducing cytokine secretion. Infected DC

  15. Parathyroid Hormone Induces Bone Cell Motility and Loss of Mature Osteocyte Phenotype through L-Calcium Channel Dependent and Independent Mechanisms.

    Directory of Open Access Journals (Sweden)

    Matthew Prideaux

    Full Text Available Parathyroid Hormone (PTH can exert both anabolic and catabolic effects on the skeleton, potentially through expression of the PTH type1 receptor (PTH1R, which is highly expressed in osteocytes. To determine the cellular and molecular mechanisms responsible, we examined the effects of PTH on osteoblast to osteocyte differentiation using primary osteocytes and the IDG-SW3 murine cell line, which differentiate from osteoblast to osteocyte-like cells in vitro and express GFP under control of the dentin matrix 1 (Dmp1 promoter. PTH treatment resulted in an increase in some osteoblast and early osteocyte markers and a decrease in mature osteocyte marker expression. The gene expression profile of PTH-treated Day 28 IDG-SW3 cells was similar to PTH treated primary osteocytes. PTH treatment induced striking changes in the morphology of the Dmp1-GFP positive cells in IDG-SW3 cultures and primary cells from Dmp1-GFP transgenic mice. The cells changed from a more dendritic to an elongated morphology and showed increased cell motility. E11/gp38 has been shown to be important for cell migration, however, deletion of the E11/gp38/podoplanin gene had no effect on PTH-induced motility. The effects of PTH on motility were reproduced using cAMP, but not with protein kinase A (PKA, exchange proteins activated by cAMP (Epac, protein kinase C (PKC or phosphatidylinositol-4,5-bisphosphonate 3-kinase (Pi3K agonists nor were they blocked by their antagonists. However, the effects of PTH were mediated through calcium signaling, specifically through L-type channels normally expressed in osteoblasts but decreased in osteocytes. PTH was shown to increase expression of this channel, but decrease the T-type channel that is normally more highly expressed in osteocytes. Inhibition of L-type calcium channel activity attenuated the effects of PTH on cell morphology and motility but did not prevent the downregulation of mature osteocyte marker expression. Taken together, these

  16. IL-1β induces hypomyelination in the periventricular white matter through inhibition of oligodendrocyte progenitor cell maturation via FYN/MEK/ERK signaling pathway in septic neonatal rats.

    Science.gov (United States)

    Xie, Di; Shen, Fengcai; He, Shaoru; Chen, Mengmeng; Han, Qianpeng; Fang, Ming; Zeng, Hongke; Chen, Chunbo; Deng, Yiyu

    2016-04-01

    Neuroinflammation elicited by microglia plays a key role in periventricular white matter (PWM) damage (PWMD) induced by infectious exposure. This study aimed to determine if microglia-derived interleukin-1β (IL-1β) would induce hypomyelination through suppression of maturation of oligodendrocyte progenitor cells (OPCs) in the developing PWM. Sprague-Dawley rats (1-day old) were injected with lipopolysaccharide (LPS) (1 mg/kg) intraperitoneally, following which upregulated expression of IL-1β and IL-1 receptor 1 (IL-1R1 ) was observed. This was coupled with enhanced apoptosis and suppressed proliferation of OPCs in the PWM. The number of PDGFR-α and NG2-positive OPCs was significantly decreased in the PWM at 24 h and 3 days after injection of LPS, whereas it was increased at 14 days and 28 days. The protein expression of Olig1, Olig2, and Nkx2.2 was significantly reduced, and mRNA expression of Tcf4 and Axin2 was upregulated in the developing PWM after LPS injection. The expression of myelin basic protein (MBP) and 2',3'-cyclic-nucleotide 3"-phosphodiesterase (CNPase) was downregulated in the PWM at 14 days and 28 days after LPS injection; this was linked to reduction of the proportion of myelinated axons and thinner myelin sheath as revealed by electron microscopy. Primary cultured OPCs treated with IL-1β showed the failure of maturation and proliferation. Furthermore, FYN/MEK/ERK signaling pathway was involved in suppression of maturation of primary OPCs induced by IL-1β administration. Our results suggest that following LPS injection, microglia are activated and produce IL-1β in the PWM in the neonatal rats. Excess IL-1β inhibits the maturation of OPCs via suppression of FYN/MEK/ERK phosphorylation thereby leading to axonal hypomyelination.

  17. Peroxisome proliferator-activated receptor α agonist attenuates oxidized-low density lipoprotein induced immune maturation of human monocyte-derived dendritic cells

    Institute of Scientific and Technical Information of China (English)

    SHI Hong-yu; CAO Xue-tao; GE Jun-bo; FANG Wei-yi; YAO Kang; SUN Ai-jun; HUANG Rong-chong; JIA Qing-zhe; WANG Ke-qiang; ZOU Yun-zeng

    2008-01-01

    @@ Accumulating evidence suggests that the Th1 immune response induced by various antigens such as oxidized low density lipoprotein (ox-LDL) and heat shock proteins (HSPs) play a key role in the process of atherosclerosis.1 Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) in the body with the unique ability to initiate a primary immune response to certain antigens by the activation of "naive" T cells.2 The maturation of DC with the upregulation of costimulatory molecules such as CD83,CD40,CD86,and major histocompatibility complex (MHC) class molecules such as human leukocyte antigen (HLA)-DR,is required for DC to activate T cells.

  18. CD8 T cell response maturation defined by anentropic specificity and repertoire depth correlates with SIVΔnef-induced protection.

    Directory of Open Access Journals (Sweden)

    Sama Adnan

    2015-02-01

    Full Text Available The live attenuated simian immunodeficiency virus (LASIV vaccine SIVΔnef is one of the most effective vaccines in inducing protection against wild-type lentiviral challenge, yet little is known about the mechanisms underlying its remarkable protective efficacy. Here, we exploit deep sequencing technology and comprehensive CD8 T cell epitope mapping to deconstruct the CD8 T cell response, to identify the regions of immune pressure and viral escape, and to delineate the effect of epitope escape on the evolution of the CD8 T cell response in SIVΔnef-vaccinated animals. We demonstrate that the initial CD8 T cell response in the acute phase of SIVΔnef infection is mounted predominantly against more variable epitopes, followed by widespread sequence evolution and viral escape. Furthermore, we show that epitope escape expands the CD8 T cell repertoire that targets highly conserved epitopes, defined as anentropic specificity, and generates de novo responses to the escaped epitope variants during the vaccination period. These results correlate SIVΔnef-induced protection with expanded anentropic specificity and increased response depth. Importantly, these findings render SIVΔnef, long the gold standard in HIV/SIV vaccine research, as a proof-of-concept vaccine that highlights the significance of the twin principles of anentropic specificity and repertoire depth in successful vaccine design.

  19. Addition of interferon-alpha to a standard maturation cocktail induces CD38 up-regulation and increases dendritic cell function

    DEFF Research Database (Denmark)

    Trepiakas, Redas; Pedersen, Anders Elm; Met, Ozcan;

    2009-01-01

    differs from sDC maturation: The major phenotypic change after IFN-alpha maturation was dose-dependent up-regulation of CD38 but not CD83, while sDCs expressed the opposite profile with low CD38 and high CD83 expression. Similarly, maturation by Poly I:C leads to CD38high, CD83low DCs indicating...... other changes in DC phenotype and cytokine secretion. Our observations suggest that IFN-alpha could be included in maturation protocols for clinical grade DCs used for immunotherapy against cancer and should be included if DCs are used for CD8+ T-cell stimulation in vitro.......Monocyte-derived dendritic cells (DCs) are used as adjuvant cells in cancer immunotherapy and have shown promising results. In order to obtain full functional capacity, these DCs need to be maturated, and the current "gold standard" for this process is maturation with TNF-alpha, IL-1beta, IL-6...

  20. Murine dendritic cells generated under serum-free conditions have a mature phenotype and efficiently induce primary immune responses.

    Science.gov (United States)

    Warncke, Max; Dodero, Anna; Dierbach, Heide; Follo, Marie; Veelken, Hendrik

    2006-03-20

    Vaccination with in vitro-generated dendritic cells (DC) that present tumor-associated antigens is a promising approach for immunotherapy of malignant tumors. For optimization of DC-based vaccination protocols, preclinical tumor models that mimic the clinical situation closely are highly desirable. Strong non-specific T cell activation was observed in experimental immunization of mice with syngeneic DC generated in standard FCS-supplemented culture medium. To avoid deviation of the immune response to FCS-derived antigens, a serum-free culture protocol for in vitro generation of murine DC from bone marrow progenitor cells was developed. In comparison to DC differentiated with FCS supplementation, DC generated under serum-free conditions (sfDC) have a more homogeneous phenotype with higher expression of IL-12 and the differentiation and activation markers CD11c, CD40, CD80, CD83, CD86, DEC-205, and MHC class II. Demonstration of strong uptake of protein and carbohydrate antigens and analysis of the in vivo migration behaviour of sfDC also indicated excellent APC function. Vaccination of mice with peptide-pulsed sfDC efficiently induced an antigen-specific T cell response as assessed by MHC tetramer staining, IFN-gamma ELISPOT and in vivo cytotoxicity assay. sfDC may therefore represent a valuable tool to improve active tumor immunotherapy in animal models.

  1. Mycobacterium tuberculosis PE25/PPE41 protein complex induces activation and maturation of dendritic cells and drives Th2-biased immune responses.

    Science.gov (United States)

    Chen, Wei; Bao, Yige; Chen, Xuerong; Burton, Jeremy; Gong, Xueli; Gu, Dongqing; Mi, Youjun; Bao, Lang

    2016-04-01

    Mycobacterium tuberculosis evades innate host immune responses by parasitizing macrophages and causes significant morbidity and mortality around the world. A mycobacterial antigen that can activate dendritic cells (DCs) and elicit effective host innate immune responses will be vital to the development of an effective TB vaccine. The M. tuberculosis genes PE25/PPE41 encode proteins which have been associated with evasion of the host immune response. We constructed a PE25/PPE41 complex gene via splicing by overlapping extension and expressed it successfully in E. coli. We investigated whether this protein complex could interact with DCs to induce effective host immune responses. The PE25/PPE41 protein complex induced maturation of isolated mouse DCs in vitro, increasing expression of cell surface markers (CD80, CD86 and MHC-II), thereby promoting Th2 polarization via secretion of pro-inflammatory cytokines IL-4 and IL-10. In addition, PE25/PPE41 protein complex-activated DCs induced proliferation of mouse CD4(+) and CD8(+) T cells, and a strong humoral response in immunized mice. The sera of five TB patients were also highly reactive to this antigen. These findings suggest that interaction of the PE25/PPE41 protein complex with DCs may be of great immunological significance.

  2. Lactobacillus crispatus strain SJ-3C-US induces human dendritic cells (DCs) maturation and confers an anti-inflammatory phenotype to DCs.

    Science.gov (United States)

    Eslami, Solat; Hadjati, Jamshid; Motevaseli, Elahe; Mirzaei, Reza; Farashi Bonab, Samad; Ansaripour, Bita; Khoramizadeh, Mohammad Reza

    2016-08-01

    Lactobacillus crispatus is one of the most predominant species in the healthy vagina microbiota. Nevertheless, the interactions between this commensal bacterium and the immune system are largely unknown. Given the importance of the dendritic cells (DCs) in the regulation of the immunity, this study was performed to elucidate the influence of vaginal isolated L. crispatus SJ-3C-US from healthy Iranian women on DCs, either directly by exposure of DCs to ultraviolet-inactivated (UVI) and heat-killed (HK) L. crispatus SJ-3C-US or indirectly to its cell-free supernatant (CFS), and the outcomes of immune response. In this work we showed that L. crispatus SJ-3C-US induced strong dose-dependent activation of dendritic cells and production of high levels of IL-10, whereas IL-12p70 production was induced at low level in an inverse dose-dependent manner. This stimulation skewed T cells polarization toward CD4(+) CD25(+) FOXP3(+) Treg cells and production of IL-10 in a dose-dependent manner in mixed leukocyte reaction (MLR) test. The mode of bacterial inactivation did not affect the DCs activation pattern, upon encounter with L. crispatus SJ-3C-US. Moreover, while DCs stimulated with CFS showed moderate phenotypic maturation and IL-10 production, it failed to skew T cells polarization toward CD4(+) CD25(+) FOXP3(+) regulatory T cells (Treg) and production of IL-10. This study showed that L. crispatus SJ-3C-US confers an anti-inflammatory phenotype to DCs through up-regulation of anti-inflammatory/regulatory IL-10 cytokine production and induction of CD4(+) CD25(+) FOXP3(+) T cells at optimal dosage. Our findings suggest that L. crispatus SJ-3C-US could be a potent candidate as protective probiotic against human immune-mediated pathologies, such as chronic inflammation, vaginitis or pelvic inflammatory disease (PID).

  3. Generation of mature hematopoietic cells from human pluripotent stem cells.

    Science.gov (United States)

    Togarrati, Padma Priya; Suknuntha, Kran

    2012-06-01

    A number of malignant and non-malignant hematological disorders are associated with the abnormal production of mature blood cells or primitive hematopoietic precursors. Their capacity for continuous self-renewal without loss of pluripotency and the ability to differentiate into adult cell types from all three primitive germ layers make human embryonic stem cells and induced pluripotent stem cells (hiPSCs) attractive complementary cell sources for large-scale production of transfusable mature blood cell components in cell replacement therapies. The generation of patient-specific hematopoietic stem/precursor cells from iPSCs by the regulated manipulation of various factors involved in reprograming to ensure complete pluripotency, and developing innovative differentiation strategies for generating unlimited supply of clinically safe, transplantable, HLA-matched cells from hiPSCs to outnumber the inadequate source of hematopoietic stem cells obtained from cord blood, bone marrow and peripheral blood, would have a major impact on the field of regenerative and personalized medicine leading to translation of these results from bench to bedside.

  4. Evaluation of the lipopolysaccharide-induced transcription of the human TREM-1 gene in vitamin D3-matured THP-1 macrophage-like cells.

    Science.gov (United States)

    Hosoda, Hiroshi; Tamura, Hiroshi; Nagaoka, Isao

    2015-11-01

    Triggering receptor expressed on myeloid cells-1 (TREM-1) plays a role in inflammation by augmenting inflammatory responses through the production of pro-inflammatory cytokines. TREM-1 is expressed in mature macrophages, and is upregulated by stimulation with bacterial components, such as lipopolysaccharide (LPS). In the present study, the regulatory mechanisms responsible for the transcription of the human TREM-1 gene were examined using a human monocytic cell line (THP-1 cells). Reverse transcription-polymerase chain reaction (RT-PCR) revealed that TREM-1 mRNA was constitutively expressed at a low level in resting cells, and that its expression was upregulated by treatment with vitamin D3 (VitD3), but not by LPS. Importantly, TREM-1 mRNA expression was further upregulated by stimulation of the VitD3‑treated THP-1 cells with LPS. In addition, a luciferase reporter assay revealed that the serum response element (SRE) was involved in VitD3-induced promoter activity, whereas the activator protein-1 (AP-1) sites participated in the VitD3- and LPS-induced promoter activity. Of note, the CCAAT-enhancer-binding protein (C/EBP) site contributed not only to basal, but also to VitD3- and LPS-induced promoter activity. Transfection with transcription factor oligodeoxynucleotide (ODN) decoys indicated that transcription factors of the C/EBP and AP-1 families are likely involved in the basal, as well as in the VitD3- and LPS-induced TREM-1 transcription. Western blot analysis indicated that, of the members of the C/EBP family, C/EBPα was constitutively expressed in resting cells; its expression was enhanced by treatment with VitD3 and was further increased by treatment with VitD3 and LPS. Moreover, the expression of c-Fos and c-Jun (members of the AP-1 family) was augmented by treatment with both VitD3 and LPS. These observations indicate that members of the C/EBP family participate not only in basal, but also in the VitD3- and LPS-induced promoter activity of the human

  5. Physiological maturation and drug responses of human induced pluripotent stem cell-derived cortical neuronal networks in long-term culture.

    Science.gov (United States)

    Odawara, A; Katoh, H; Matsuda, N; Suzuki, I

    2016-05-18

    The functional network of human induced pluripotent stem cell (hiPSC)-derived neurons is a potentially powerful in vitro model for evaluating disease mechanisms and drug responses. However, the culture time required for the full functional maturation of individual neurons and networks is uncertain. We investigated the development of spontaneous electrophysiological activity and pharmacological responses for over 1 year in culture using multi-electrode arrays (MEAs). The complete maturation of spontaneous firing, evoked responses, and modulation of activity by glutamatergic and GABAergic receptor antagonists/agonists required 20-30 weeks. At this stage, neural networks also demonstrated epileptiform synchronized burst firing (SBF) in response to pro-convulsants and SBF suppression using clinical anti-epilepsy drugs. Our results reveal the feasibility of long-term MEA measurements from hiPSC-derived neuronal networks in vitro for mechanistic analyses and drug screening. However, developmental changes in electrophysiological and pharmacological properties indicate the necessity for the international standardization of culture and evaluation procedures.

  6. Whole inactivated avian Influenza H9N2 viruses induce nasal submucosal dendritic cells to sample luminal viruses via transepithelial dendrites and trigger subsequent DC maturation.

    Science.gov (United States)

    Qin, Tao; Yin, Yinyan; Wang, Xiaoqing; Liu, Haofei; Lin, Jian; Yu, Qinghua; Yang, Qian

    2015-03-10

    Nasal mucosal barrier is a key impediment for the absorption of influenza whole inactivated virus (WIV) intranasal vaccine. Yet it is still unclear how WIV cross the epithelial cells (ECs) in nasal cavity. Here, in vitro, a coculture system was well established, consisting of surrogate nasal ECs (Calu-3) and dendritic cells (DCs). After adding H9N2 WIV on the apical side of ECs, we found that submucosal DCs extended their transepithelial dendrites (TEDs) and sampled luminal viruses. However, ECs were not involved in the transepithelial transport of viruses. Subsequently, the phenotypic and functional maturation of DCs were also enhanced, whereas they were attenuated after blocking of TED formation by anti-JAM1 antibody. In vivo, we confirmed that H9N2 WIV were capable of inducing nasal submucosal DCs to sample luminal viruses via TEDs in the nasal passage but not nasal-associated lymphoid tissue (NALT). CD103(+) and CD103(-) DC subsets participated in this process. Of note, chemokine CCL20, released from the H9N2 WIV-induced ECs, played a vital role in DC recruitment and TED formation. Taken together, our findings indicated that TEDs played a critical role in facilitating viral transport across the epithelial barrier, which may guide the design of novel nasal mucosal vaccine strategies.

  7. Delta Inulin Adjuvant Enhances Plasmablast Generation, Expression of Activation-Induced Cytidine Deaminase and B-Cell Affinity Maturation in Human Subjects Receiving Seasonal Influenza Vaccine.

    Directory of Open Access Journals (Sweden)

    Lei Li

    Full Text Available There is a major need for new adjuvants to improve the efficacy of seasonal and pandemic influenza vaccines. Advax is a novel polysaccharide adjuvant based on delta inulin that has been shown to enhance the immunogenicity of influenza vaccine in animal models and human clinical trials. To better understand the mechanism for this enhancement, we sought to assess its effect on the plasmablast response in human subjects. This pilot study utilised cryopreserved 7 day post-vaccination (7dpv peripheral blood mononuclear cell samples obtained from a subset of 25 adult subjects from the FLU006-12 trial who had been immunized intramuscularly with a standard dose of 2012 trivalent inactivated influenza vaccine (TIV alone (n=9 subjects or combined with 5mg (n=8 or 10mg (n=8 of Advax adjuvant. Subjects receiving Advax adjuvant had increased 7dpv plasmablasts, which in turn exhibited a 2-3 fold higher rate of non-silent mutations in the B-cell receptor CDR3 region associated with higher expression of activation-induced cytidine deaminase (AID, the major enzyme controlling BCR affinity maturation. Together, these data suggest that Advax adjuvant enhances influenza immunity in immunized subjects via multiple mechanisms including increased plasmablast generation, AID expression and CDR3 mutagenesis resulting in enhanced BCR affinity maturation and increased production of high avidity antibody. How Advax adjuvant achieves these beneficial effects on plasmablasts remains the subject of ongoing investigation.Australia New Zealand Clinical Trials Register ACTRN12612000709842 https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=362709.

  8. Simulation of B Cell Affinity Maturation Explains Enhanced Antibody Cross-Reactivity Induced by the Polyvalent Malaria Vaccine AMA1

    Science.gov (United States)

    2014-07-01

    other Ags this may not be the case. For example, in dengue virus, subneutralizing cross-reactive Abs and cross- reactive memory B cells are thought to...HLA-driven CD8 + T- cell escape mutations and covarying HLA-independent polymorphisms. J. Virol. 85: 1384–1390. 33. Fischer, W., V. V. Ganusov, E. E...Y. Lee, K. L. Artiles, S. Zompi, M. J. Vargas, B. B. Simen, et al. 2013. Convergent antibody signatures in human dengue . Cell Host Microbe 13: 691–700

  9. The Major Immediate-Early Protein IE2 of Human Cytomegalovirus Is Sufficient to Induce Proteasomal Degradation of CD83 on Mature Dendritic Cells

    Science.gov (United States)

    Heilingloh, Christiane S.; Grosche, Linda; Kummer, Mirko; Mühl-Zürbes, Petra; Kamm, Lisa; Scherer, Myriam; Latzko, Melanie; Stamminger, Thomas; Steinkasserer, Alexander

    2017-01-01

    Human cytomegalovirus (HCMV) is the prototypic beta-herpesvirus and widespread throughout the human population. While infection is asymptomatic in healthy individuals, it can lead to high morbidity and mortality in immunocompromised persons. Importantly, HCMV evolved multiple strategies to interfere with immune cell function in order to establish latency in infected individuals. As mature DCs (mDCs) are antigen-presenting cells able to activate naïve T cells they play a crucial role during induction of effective antiviral immune responses. Interestingly, earlier studies demonstrated that the functionally important mDC surface molecule CD83 is down-regulated upon HCMV infection resulting in a reduced T cell stimulatory capacity of the infected cells. However, the viral effector protein and the precise mechanism of HCMV-mediated CD83 reduction remain to be discovered. Using flow cytometric analyses, we observed significant down-modulation of CD83 surface expression becoming significant already 12 h after HCMV infection. Moreover, Western bot analyses revealed that, in sharp contrast to previous studies, loss of CD83 is not restricted to the membrane-bound molecule, but also occurs intracellularly. Furthermore, inhibition of the proteasome almost completely restored CD83 surface expression during HCMV infection. Results of infection kinetics and cycloheximide-actinomycin D-chase experiments, strongly suggested that an HCMV immediate early gene product is responsible for the induction of CD83 down-modulation. Consequently, we were able to identify the major immediate early protein IE2 as the viral effector protein that induces proteasomal CD83 degradation. PMID:28203230

  10. Technical advance: Langerhans cells derived from a human cell line in a full-thickness skin equivalent undergo allergen-induced maturation and migration.

    Science.gov (United States)

    Ouwehand, Krista; Spiekstra, Sander W; Waaijman, Taco; Scheper, Rik J; de Gruijl, Tanja D; Gibbs, Susan

    2011-11-01

    In this report, the construction of a functional, immunocompetent, full-thickness skin equivalent (SE) is described, consisting of an epidermal compartment containing keratinocytes, melanocytes, and human LCs derived from the MUTZ-3 cell line (MUTZ-LC) and a fibroblast-populated dermal compartment. The CD1a(+)Langerin(+)HLA-DR(+) MUTZ-LCs populate the entire epidermis at a similar density to that found in native skin. Exposure of the SE to subtoxic concentrations of the allergens NiSO(4) and resorcinol resulted in LC migration out of the epidermis toward the fibroblast-populated dermal compartment. A significant dose-dependent up-regulation of the DC maturation-related CCR7 and IL-1β transcripts and of CD83 at the protein level upon epidermal exposure to both allergens was observed, indicative of maturation and migration of the epidermally incorporated LC. We have thus successfully developed a reproducible and functional full-thickness SE model containing epidermal MUTZ-LC. This model offers an alternative to animal testing for identifying potential chemical sensitizers and for skin-based vaccination strategies and provides a unique research tool to study human LC biology in situ under controlled in vitro conditions.

  11. Domoic Acid-Induced Neurotoxicity Is Mainly Mediated by the AMPA/KA Receptor: Comparison between Immature and Mature Primary Cultures of Neurons and Glial Cells from Rat Cerebellum

    Directory of Open Access Journals (Sweden)

    Helena T. Hogberg

    2011-01-01

    Full Text Available Domoic acid (DomA is a naturally occurring shellfish toxin that can induce brain damage in mammalians. Neonates have shown increased sensitivity to DomA-induced toxicity, and prenatal exposure has been associated with e.g. decreased brain GABA levels, and increased glutamate levels. Here, we evaluated DomA-induced toxicity in immature and mature primary cultures of neurons and glial cells from rat cerebellum by measuring the mRNA levels of selected genes. Moreover, we assessed if the induced toxicity was mediated by the activation of the AMPA/KA and/or the NMDA receptor. The expression of all studied neuronal markers was affected after DomA exposure in both immature and mature cultures. However, the mature cultures seemed to be more sensitive to the treatment, as the effects were observed at lower concentrations and at earlier time points than for the immature cultures. The DomA effects were completely prevented by the antagonist of the AMPA/KA receptor (NBQX, while the antagonist of the NMDA receptor (APV partly blocked the DomA-induced effects. Interestingly, the DomA-induced effect was also partly prevented by the neurotransmitter GABA. DomA exposure also affected the mRNA levels of the astrocytic markers in mature cultures. These DomA-induced effects were reduced by the addition of NBQX, APV, and GABA.

  12. 体外诱导成熟树突状细胞的研究%Research of mature dendritic cells induced in vitro

    Institute of Scientific and Technical Information of China (English)

    林苹; 张洁; 陆燕蓉; 周宏远; 宁其志; 黄孝忠

    2001-01-01

    目的:探讨在体外从干细胞中诱导出成熟DC的适宜环境。方法:分别将人胎肝、骨髓和脾细胞以及小鼠骨髓和脾细胞在体外用GM-CSF和TNF-α诱导,观察了第3、5、7天DC的收获情况。进一步用S-P免疫细胞化学染色检测了mBmDC和fLDC有关分子表达。结果:在体外通过GM-CSF和TNF-α的作用,小鼠骨髓、人胎肝、骨髓细胞呈典型的毛刺状胞浆突起,第5天的收获率分别为:39.5%、67.2%、12.9%,而基本不能从脾细胞中诱导出成熟DC,获得的DC能与MAbCD80、MAbCD40呈强阳性反应。结论:在GM-CSF和TNF-α的共同作用下,能在体外从人胎肝、骨髓和小鼠骨髓干细胞中获得成熟DC,其DC能表达高水平的B7-1和CD40分子,这为大量获取DC提供了一种简便可行的手段,为研究DC的生物学特征及其抗原提呈功能提供了丰富的材料,为开展以DC为基础的各种免疫治疗研究奠定了良好的基础。%Objective: To study the suitable condition of inducing maturedendritic ce lls (DC) from stem cells in vitro.Methods: With GM-CSF and TNF-α, the cells of liver, bone marrow, spleen from human fetals and bone marrow, spleen from mice were cultured. The rates of harvest DC were counted on 3 d, 5 d and 7 d of culture. Some molecular expression of the mice bone marrow DC (mBmDC) and fetal liver DC (fLDC) were detected by S-P immunocytochemical stain.Results:By GM-CSF and TN F-α affecting, the mBmDC, fLDC and fetal bone marrow DC (fBm DC) grow as typic a l DC which cytoplasm is protrusion like polythoorn. The 5d rates of harvest fro m mBmDC, fLDC and fBmDC were 39.5%. 67.2% and 12.9%, but there were few mature DCs induced from spleen cells. The mBmDC, fLDC and fBmDC were strong positive wit h MAb CD80 and MAb CD40.Conclusion: Mature DCs can be induced in vitro from the stem cells of fetal liver, bone marrow and mice bone marrow with cooperate with GM-CSF and TNF-α. The high lever of B7-1 and CD40

  13. The Polyunsaturated Fatty Acids Arachidonic Acid and Docosahexaenoic Acid Induce Mouse Dendritic Cells Maturation but Reduce T-Cell Responses In Vitro.

    Science.gov (United States)

    Carlsson, Johan A; Wold, Agnes E; Sandberg, Ann-Sofie; Östman, Sofia M

    2015-01-01

    Long-chain polyunsaturated fatty acids (PUFAs) might regulate T-cell activation and lineage commitment. Here, we measured the effects of omega-3 (n-3), n-6 and n-9 fatty acids on the interaction between dendritic cells (DCs) and naïve T cells. Spleen DCs from BALB/c mice were cultured in vitro with ovalbumin (OVA) with 50 μM fatty acids; α-linolenic acid, arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid or oleic acid and thereafter OVA-specific DO11.10 T cells were added to the cultures. Fatty acids were taken up by the DCs, as shown by gas chromatography analysis. After culture with arachidonic acid or DHA CD11c+ CD11b+ and CD11c+ CD11bneg DCs expressed more CD40, CD80, CD83, CD86 and PDL-1, while IAd remained unchanged. However, fewer T cells co-cultured with these DCs proliferated (CellTrace Violet low) and expressed CD69 or CD25, while more were necrotic (7AAD+). We noted an increased proportion of T cells with a regulatory T cell (Treg) phenotype, i.e., when gating on CD4+ FoxP3+ CTLA-4+, CD4+ FoxP3+ Helios+ or CD4+ FoxP3+ PD-1+, in co-cultures with arachidonic acid- or DHA-primed DCs relative to control cultures. The proportion of putative Tregs was inversely correlated to T-cell proliferation, indicating a suppressive function of these cells. With arachidonic acid DCs produced higher levels of prostaglandin E2 while T cells produced lower amounts of IL-10 and IFNγ. In conclusion arachidonic acid and DHA induced up-regulation of activation markers on DCs. However arachidonic acid- and DHA-primed DCs reduced T-cell proliferation and increased the proportion of T cells expressing FoxP3, indicating that these fatty acids can promote induction of regulatory T cells.

  14. Inflammation-induced preterm lung maturation: lessons from animal experimentation.

    Science.gov (United States)

    Moss, Timothy J M; Westover, Alana J

    2016-10-20

    Intrauterine inflammation, or chorioamnionitis, is a major contributor to preterm birth. Prematurity per se is associated with considerable morbidity and mortality resulting from lung immaturity but exposure to chorioamnionitis reduces the risk of neonatal respiratory distress syndrome (RDS) in preterm infants. Animal experiments have identified that an increase in pulmonary surfactant production by the preterm lungs likely underlies this decreased risk of RDS in infants exposed to chorioamnionitis. Further animal experimentation has shown that infectious or inflammatory agents in amniotic fluid exert their effects on lung development by direct effects within the developing respiratory tract, and probably not by systemic pathways. Differences in the effects of intrauterine inflammation and glucocorticoids demonstrate that canonical glucocorticoid-mediated lung maturation is not responsible for inflammation-induced changes in lung development. Animal experimentation is identifying alternative lung maturational pathways, and transgenic animals and cell culture techniques will allow identification of novel mechanisms of lung maturation that may lead to new treatments for the prevention of RDS.

  15. Ajulemic acid, a nonpsychoactive cannabinoid acid, suppresses osteoclastogenesis in mononuclear precursor cells and induces apoptosis in mature osteoclast-like cells.

    Science.gov (United States)

    George, Kerri L; Saltman, Laura H; Stein, Gary S; Lian, Jane B; Zurier, Robert B

    2008-03-01

    Oral administration of ajulemic acid (AjA), a cannabinoid acid devoid of psychoactivity, prevents joint tissue injury in rats with adjuvant induced arthritis. Because activation of osteoclasts is central to the pathogenesis of bone erosion in patients with rheumatoid arthritis (RA), we investigated the influence of AjA on osteoclast differentiation and survival. Osteoclast cultures were established by stimulation of RAW264.7 cells and primary mouse bone marrow cultures with receptor activator of NF-kappaB ligand (RANKL). Simultaneous addition of AjA (15 and 30 microM) and RANKL to both culture systems significantly suppressed development of multinucleated osteoclasts (osteoclastogenesis) in a dose dependent manner, as determined by quantification of multinuclear, tartrate-resistant acid phosphatase (TRAP)-positive cells. AjA impaired growth of RAW264.7 monocytes and prevented further osteoclast formation in cultures in which osteoclastogenesis had already begun. Reduction by AjA of both monocyte growth and osteoclast formation was associated with apoptosis, assayed by annexin V and propidium iodide staining, and caspase activity. The anti-osteoclastogenic effects of AjA did not require the continuous presence of AjA in the cell cultures. Based on these findings, we propose that AjA or other nonpsychoactive synthetic analogs of Cannabis constituents may be useful therapy for diseases such as RA and osteoporosis in which bone resorption is a central feature.

  16. Predicting the influence of liposomal lipid composition on liposome size, zeta potential and liposome-induced dendritic cell maturation using a design of experiments approach.

    Science.gov (United States)

    Soema, Peter C; Willems, Geert-Jan; Jiskoot, Wim; Amorij, Jean-Pierre; Kersten, Gideon F

    2015-08-01

    In this study, the effect of liposomal lipid composition on the physicochemical characteristics and adjuvanticity of liposomes was investigated. Using a design of experiments (DoE) approach, peptide-containing liposomes containing various lipids (EPC, DOPE, DOTAP and DC-Chol) and peptide concentrations were formulated. Liposome size and zeta potential were determined for each formulation. Moreover, the adjuvanticity of the liposomes was assessed in an in vitro dendritic cell (DC) model, by quantifying the expression of DC maturation markers CD40, CD80, CD83 and CD86. The acquired data of these liposome characteristics were successfully fitted with regression models, and response contour plots were generated for each response factor. These models were applied to predict a lipid composition that resulted in a liposome with a target zeta potential. Subsequently, the expression of the DC maturation factors for this lipid composition was predicted and tested in vitro; the acquired maturation responses corresponded well with the predicted ones. These results show that a DoE approach can be used to screen various lipids and lipid compositions, and to predict their impact on liposome size, charge and adjuvanticity. Using such an approach may accelerate the formulation development of liposomal vaccine adjuvants.

  17. Transcriptional profiling of dendritic cells matured in different osmolarities

    Directory of Open Access Journals (Sweden)

    Federica Chessa

    2016-03-01

    Full Text Available Tissue-specific microenvironments shape the fate of mononuclear phagocytes [1–3]. Interstitial osmolarity is a tissue biophysical parameter which considerably modulates the phenotype and function of dendritic cells [4]. In the present report we provide a detailed description of our experimental workflow and bioinformatic analysis applied to our gene expression dataset (GSE72174, aiming to investigate the influence of different osmolarity conditions on the gene expression signature of bone marrow-derived dendritic cells. We established a cell culture system involving murine bone marrow cells, cultured under different NaCl-induced osmolarity conditions in the presence of the dendritic cell growth factor GM-CSF. Gene expression analysis was applied to mature dendritic cells (day 7 developed in different osmolarities, with and without prior stimulation with the TLR2/4 ligand LPS.

  18. Mannoproteins from Cryptococcus neoformans promote dendritic cell maturation and activation.

    Science.gov (United States)

    Pietrella, Donatella; Corbucci, Cristina; Perito, Stefano; Bistoni, Giovanni; Vecchiarelli, Anna

    2005-02-01

    Our previous data show that mannoproteins (MPs) from Cryptococcus neoformans are able to induce protective responses against both C. neoformans and Candida albicans. Here we provide evidence that MPs foster maturation and activation of human dendritic cells (DCs). Maturation was evaluated by the ability of MPs to facilitate expression of costimulatory molecules such as CD40, CD86, CD83, and major histocompatibility complex classes I and II and to inhibit receptors such as CD14, CD16, and CD32. Activation of DCs was measured by the capacity of MPs to promote interleukin-12 and tumor necrosis factor alpha secretion. DC-induced maturation and interleukin-12 induction are largely mediated by engagement of mannose receptors and presume MP internalization and degradation. DC activation leads to IkappaBalpha phosphorylation, which is necessary for nuclear factor kappaB transmigration into the nucleus. MP-loaded DCs are efficient stimulators of T cells and show a remarkable capacity to promote CD4 and CD8 proliferation. In conclusion, we have evidenced a novel regulatory role of MPs that promotes their candidacy as a vaccine against fungi.

  19. Vesicle associated membrane protein (VAMP)-7 and VAMP-8, but not VAMP-2 or VAMP-3, are required for activation-induced degranulation of mature human mast cells.

    Science.gov (United States)

    Sander, Leif E; Frank, Simon P C; Bolat, Seza; Blank, Ulrich; Galli, Thierry; Bigalke, Hans; Bischoff, Stephan C; Lorentz, Axel

    2008-03-01

    Mediator release from mast cells (MC) is a crucial step in allergic and non-allergic inflammatory disorders. However, the final events in response to activation leading to membrane fusion and thereby facilitating degranulation have hitherto not been analyzed in human MC. Soluble N-ethyl-maleimide-sensitive factor attachment protein receptors (SNARE) represent a highly conserved family of proteins that have been shown to mediate intracellular membrane fusion events. Here, we show that mature MC isolated from human intestinal tissue express soluble N-ethylmaleide sensitive factor attachment protein (SNAP)-23, Syntaxin (STX)-1B, STX-2, STX-3, STX-4, and STX-6 but not SNAP-25. Furthermore, we found that primary human MC express substantial amounts of vesicle associated membrane protein (VAMP)-3, VAMP-7 and VAMP-8 and, in contrast to previous reports about rodent MC, only low levels of VAMP-2. Furthermore, VAMP-7 and VAMP-8 were found to translocate to the plasma membrane and interact with SNAP-23 and STX-4 upon activation. Inhibition of SNAP-23, STX-4, VAMP-7 or VAMP-8, but not VAMP-2 or VAMP-3, resulted in a markedly reduced high-affinity IgE receptor-mediated histamine release. In summary, our data show that mature human MC express a specific pattern of SNARE and that VAMP-7 and VAMP-8, but not VAMP-2, are required for rapid degranulation.

  20. The Influence of Ouabain on Human Dendritic Cells Maturation

    Directory of Open Access Journals (Sweden)

    C. R. Nascimento

    2014-01-01

    Full Text Available Although known as a Na,K-ATPase inhibitor, several other cellular and systemic actions have been ascribed to the steroid Ouabain (Oua. Particularly in the immune system, our group showed that Ouabain acts on decreasing lymphocyte proliferation, synergizing with glucocorticoids in spontaneous thymocyte apoptosis, and also lessening CD14 expression and blocking CD16 upregulation on human monocytes. However, Ouabain effects on dendritic cells (DCs were not explored so far. Considering the peculiar plasticity and the importance of DCs in immune responses, the aim of our study was to investigate DC maturation under Ouabain influence. To generate immature DCs, human monocytes were cultured with IL-4 and GM-CSF (5 days. To investigate Ouabain role on DC activation, DCs were stimulated with TNF-α for 48 h in the presence or absence of Ouabain. TNF-induced CD83 expression and IL-12 production were abolished in DCs incubated with 100 nM Ouabain, though DC functional capacity concerning lymphocyte activation remained unaltered. Nevertheless, TNF-α-induced antigen capture downregulation, another maturation marker, occurred even in the presence of Ouabain. Besides, Ouabain increased HLA-DR and CD86 expression, whereas CD80 expression was maintained. Collectively, our results suggest that DCs respond to Ouabain maturating into a distinct category, possibly contributing to the balance between immunity and tolerance.

  1. Manganese-induced integrin affinity maturation promotes recruitment of alpha V beta 3 integrin to focal adhesions in endothelial cells: evidence for a role of phosphatidylinositol 3-kinase and Src.

    Science.gov (United States)

    Dormond, Olivier; Ponsonnet, Lionel; Hasmim, Meriem; Foletti, Alessandro; Rüegg, Curzio

    2004-07-01

    Integrin activity is controlled by changes in affinity (i.e. ligand binding) and avidity (i.e. receptor clustering). Little is known, however, about the effect of affinity maturation on integrin avidity and on the associated signaling pathways. To study the effect of affinity maturation on integrin avidity, we stimulated human umbilical vein endothelial cells (HUVEC) with MnCl(2) to increase integrin affinity and monitored clustering of beta 1 and beta 3 integrins. In unstimulated HUVEC, beta 1 integrins were present in fibrillar adhesions, while alpha V beta 3 was detected in peripheral focal adhesions. Clustered beta 1 and beta 3 integrins expressed high affinity/ligand-induced binding site (LIBS) epitopes. MnCl(2)-stimulation promoted focal adhesion and actin stress fiber formation at the basal surface of the cells, and strongly enhanced mAb LM609 staining and expression of beta 3 high affinity/LIBS epitopes at focal adhesions. MnCl(2)-induced alpha V beta 3 clustering was blocked by a soluble RGD peptide, by wortmannin and LY294002, two pharmacological inhibitors of phosphatidylinositol 3-kinase (PI 3-K), and by over-expressing a dominant negative PI 3-K mutant protein. Conversely, over-expression of active PI 3-K and pharmacological inhibiton of Src with PP2 and CGP77675, enhanced basal and manganese-induced alpha V beta 3 clustering. Transient increased phosphorylation of protein kinase B/Akt, a direct target of PI 3K, occurred upon manganese stimulation. MnCl(2) did not alter beta 1 integrin distribution or beta1 high-affinity/LIBS epitope expression. Based on these results, we conclude that MnCl(2)-induced alpha V beta 3 integrin affinity maturation stimulates focal adhesion and actin stress fiber formation, and promotes recruitment of high affinity alpha V beta 3 to focal adhesions. Affinity-modulated alpha V beta 3 clustering requires PI3-K signaling and is negatively regulate by Src.

  2. Cigarette smoke differentially modulates dendritic cell maturation and function in time

    NARCIS (Netherlands)

    Givi, Masoumeh Ezzati; Folkerts, Gert; Wagenaar, Gerry T M; Redegeld, Frank A; Mortaz, Esmaeil

    2015-01-01

    BACKGROUND: Dendritic cells (DCs) as professional antigen presenting cells (APCs) play a critical role in the regulation of host immune responses. DCs evolve from immature, antigen-capturing cells, to mature antigen-presenting cells. The relative contribution of DCs to cigarette smoke-induced inflam

  3. Mechanisms behind functional avidity maturation in T cells

    DEFF Research Database (Denmark)

    von Essen, Marina Rode; Kongsbak, Martin; Geisler, Carsten

    2012-01-01

    During an immune response antigen-primed B-cells increase their antigen responsiveness by affinity maturation mediated by somatic hypermutation of the genes encoding the antigen-specific B-cell receptor (BCR) and by selection of higher-affinity B cell clones. Unlike the BCR, the T-cell receptor...... (TCR) cannot undergo affinity maturation. Nevertheless, antigen-primed T cells significantly increase their antigen responsiveness compared to antigen-inexperienced (naïve) T cells in a process called functional avidity maturation. This paper covers studies that describe differences in T-cell antigen...... responsiveness during T-cell differentiation along with examples of the mechanisms behind functional avidity maturation in T cells....

  4. Estradiol and progesterone regulate the migration of mast cells from the periphery to the uterus and induce their maturation and degranulation.

    Directory of Open Access Journals (Sweden)

    Federico Jensen

    Full Text Available BACKGROUND: Mast cells (MCs have long been suspected as important players for implantation based on the fact that their degranulation causes the release of pivotal factors, e.g., histamine, MMPs, tryptase and VEGF, which are known to be involved in the attachment and posterior invasion of the embryo into the uterus. Moreover, MC degranulation correlates with angiogenesis during pregnancy. The number of MCs in the uterus has been shown to fluctuate during menstrual cycle in human and estrus cycle in rat and mouse indicating a hormonal influence on their recruitment from the periphery to the uterus. However, the mechanisms behind MC migration to the uterus are still unknown. METHODOLOGY/PRINCIPAL FINDINGS: We first utilized migration assays to show that MCs are able to migrate to the uterus and to the fetal-maternal interface upon up-regulation of the expression of chemokine receptors by hormonal changes. By using a model of ovariectomized animals, we provide clear evidences that also in vivo, estradiol and progesterone attract MC to the uterus and further provoke their maturation and degranulation. CONCLUSION/SIGNIFICANCE: We propose that estradiol and progesterone modulate the migration of MCs from the periphery to the uterus and their degranulation, which may prepare the uterus for implantation.

  5. Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells

    Institute of Scientific and Technical Information of China (English)

    Donghui Zhang; Wei Jiang; Meng Liu; Xin Sui; Xiaolei Yin; Song Chen; Yan Shi; Hongkui Deng

    2009-01-01

    Human pluripotent stem cells represent a potentially unlimited source of functional pancreatic endocrine lineage cells. Here we report a highly efficient approach to induce human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells to differentiate into mature insulin-producing cells in a chemical-defined culture system. The differentiated human ES cells obtained by this approach comprised nearly 25% insulin-positive cells as assayed by flow cytometry analysis, which released insulin/C-peptide in response to glucose stimuli in a manner comparable to that of adult human islets. Most of these insulin-producing cells co-expressed mature β cell-specific markers such as NKX6-1 and PDX1, indicating a similar gene expression pattern to adult islet β cells in vivo. In this study, we also demonstrated that EGF facilitates the expansion of PDX1-positive pancreatic progenitors. Moreover, our protocol also succeeded in efficiently inducing human iPS cells to differentiate into insulin-producing cells. Therefore, this work not only provides a new model to study the mechanism of human pancreatic specialization and maturation in vitro, but also enhances the possibility of utilizing patient-specific iPS cells for the treatment of diabetes.

  6. Mesenchymal stem cell conditioning promotes rat oligodendroglial cell maturation.

    Directory of Open Access Journals (Sweden)

    Janusz Joachim Jadasz

    Full Text Available Oligodendroglial progenitor/precursor cells (OPCs represent the main cellular source for the generation of new myelinating oligodendrocytes in the adult central nervous system (CNS. In demyelinating diseases such as multiple sclerosis (MS myelin repair activities based on recruitment, activation and differentiation of resident OPCs can be observed. However, the overall degree of successful remyelination is limited and the existence of an MS-derived anti-oligodendrogenic milieu prevents OPCs from contributing to myelin repair. It is therefore of considerable interest to understand oligodendroglial homeostasis and maturation processes in order to enable the development of remyelination therapies. Mesenchymal stem cells (MSC have been shown to exert positive immunomodulatory effects, reduce demyelination, increase neuroprotection and to promote adult neural stem cell differentiation towards the oligodendroglial lineage. We here addressed whether MSC secreted factors can boost the OPC's oligodendrogenic capacity in a myelin non-permissive environment. To this end, we analyzed cellular morphologies, expression and regulation of key factors involved in oligodendroglial fate and maturation of primary rat cells upon incubation with MSC-conditioned medium. This demonstrated that MSC-derived soluble factors promote and accelerate oligodendroglial differentiation, even under astrocytic endorsing conditions. Accelerated maturation resulted in elevated levels of myelin expression, reduced glial fibrillary acidic protein expression and was accompanied by downregulation of prominent inhibitory differentiation factors such as Id2 and Id4. We thus conclude that apart from their suggested application as potential anti-inflammatory and immunomodulatory MS treatment, these cells might also be exploited to support endogenous myelin repair activities.

  7. Tea Tree (Melaleuca alternifolia) Oil induced Phenotypic and Functional Maturation of Murine Bone Marrow-derived Dendritic Cells%茶树油调控树突状细胞的表型和功能

    Institute of Scientific and Technical Information of China (English)

    唐永富; 李积华; 陈家翠; 林丽静; 韩志萍; 曹玉坡

    2012-01-01

    The effect of tea tree oil on the phenotypic and functional maturation of dendritic cell was investigated in vitro. Dendritic cells treated with tea tree oil (0.005 μL/mL), were enlarged, suspended, and displayed typical stellate and aggregates like cells treated with LPS (positive controls). The levels of surface marker expression were monitored throughout the culture by flow cytometry using CD1 1c-PEcy7, I-Ab-FITC, CD86-FITC, and H-2Db-PE. Dendritic cells treated with tea bee oil expressed higher level of H-2Db (48.20%), costimulatory molecule I-Ab (6.60%) and CD86 (41.00%) with the cells gated by CD11c, being of 1.28,3.40 and 1.28-fold higher for H-2Db, I-Ab and CD86, respectiely compared with untreated cells. It was also found that tea tree oil could enhance the dendritic cell to induce proliferation of T lymphocyte. And it was reduced the endocytic activity of dendritic cell by tea tree oil. These results showed that tea tree oil had significant immunoenhancing activity by inducing the phenotypic and functional maturation of dendritic cells.%本文主要探讨了茶树油对树突状细胞表型和功能的影响.采用茶树油(0.005μL/mL)干预未成熟的树突状细胞48h后,流式细胞仪检测细胞表型,MTT法检测刺激淋巴细胞增殖能力.结果发现经茶树油干预后,以CD11c设门,树突状细胞高表达H-2Db(48.20%),I-Ab(6.60%)和CD86 (41.00%),和空白对照组比较分别达到1.28倍,3.40倍和1.28倍的增长.混合淋巴培养实验发现茶树油能够增强DC刺激淋巴细胞增殖的能力,降低树突状细胞的吞噬功能.初步表明茶树油可以促进树突状细胞的表型和功能成熟.

  8. Early cytoskeletal rearrangement during dendritic cell maturation enhances synapse formation and Ca(2+) signaling in CD8(+) T cells.

    Science.gov (United States)

    Averbeck, Marco; Braun, Thorsten; Pfeifer, Gunther; Sleeman, Jonathan; Dudda, Jan; Martin, Stefan F; Kremer, Bernhard; Aktories, Klaus; Simon, Jan C; Termeer, Christian

    2004-10-01

    The interplay between dendritic cells (DC) and T cells is a dynamic process critically depending on DC maturation. Ca(2+) influx is one of the initial events occurring during DC/T cell contacts. To determine how DC maturation influences DC/T cell contacts, time-lapse video microscopy was established using TCR-transgenic CD8(+) T cells from P14 mice. DC maturation shifted DC/T cell contacts from short-lived interactions with transient Ca(2+) influx in T cells to long-lasting interactions and sustained Ca(2+) influx of 30 min and more. Follow-up of DC/T cell interactions after 2 h using confocal microscopy revealed that long-lasting Ca(2+) responses in T cells were preferentially associated with the formation of an immunological synapse involving CD54 and H2-K(b) at the DC/T cell interface. Such synapse formation preceded MHC or B7 up-regulation, since DC developed into potent Ca(2+) stimulators 7 h after initiation of maturation. Instead, the enhanced capacity of 7 h-matured DC to induce sustained Ca(2+) responses in CD8(+) T cells is critically dependent on the polarization and rearrangement of the cytoskeleton, as shown by Clostridium difficile toxin B inhibitor experiments. These data indicate that already very early after receiving a maturation stimulus, DC display enhanced cytoskeletal activity resulting in the rapid formation of immunological synapses and effective CD8(+) T cell stimulation.

  9. Endotoxin-induced maturation of monocytes in preterm fetal sheep lung.

    Science.gov (United States)

    Kramer, Boris W; Joshi, Shubhada N; Moss, Timothy J M; Newnham, John P; Sindelar, Richard; Jobe, Alan H; Kallapur, Suhas G

    2007-08-01

    The fetal lung normally contains immature monocytes and very few mature macrophages. The chorioamnionitis frequently associated with preterm birth induces monocyte influx into the fetal lung. Previous studies demonstrated that monocytes in the developing lung can mediate lung injury responses that resemble BPD in humans. We hypothesized that chorioamnionitis would induce maturation of immature monocytes in the fetal lung. Groups of three to seven time-mated ewes received saline or 10 mg of endotoxin (Escherichia coli 055:B5) in saline by intra-amniotic injection for intervals from 1 to 14 days before operative delivery at 124 days of gestational age. Monocytic cells from lung tissue were recovered using Percoll gradients. Monocytic cells consistent with macrophages were identified morphologically and by myosin heavy chain class II expression. An increase in macrophages was preceded by induction of granulocyte-macrophage colony-stimulating factor in the lung and subsequent activation of the transcription factor PU.1. The production of IL-6 by monocytes/macrophages in response to endotoxin challenge in vitro increased 7 and 14 days after exposure to intra-amniotic endotoxin. Recombinant TNF-alpha induced IL-6 production by lung monocytic cells exposed to intra-amniotic endotoxin but not in control cells. Monocytic phagocytosis of apoptotic neutrophils also increased 7 and 14 days after exposure to intra-amniotic endotoxin. Intra-amniotic endotoxin induced lung monocytes to develop into functionally mature cells consistent with macrophages. These findings have implications for lung immune responses after exposure to chorioamnionitis.

  10. Mature adipocyte-derived dedifferentiated fat cells exhibit multilineage potential.

    Science.gov (United States)

    Matsumoto, Taro; Kano, Koichiro; Kondo, Daisuke; Fukuda, Noboru; Iribe, Yuji; Tanaka, Nobuaki; Matsubara, Yoshiyuki; Sakuma, Takahiro; Satomi, Aya; Otaki, Munenori; Ryu, Jyunnosuke; Mugishima, Hideo

    2008-04-01

    When mature adipocytes are subjected to an in vitro dedifferentiation strategy referred to as ceiling culture, these mature adipocytes can revert to a more primitive phenotype and gain cell proliferative ability. We refer to these cells as dedifferentiated fat (DFAT) cells. In the present study, we examined the multilineage differentiation potential of DFAT cells. DFAT cells obtained from adipose tissues of 18 donors exhibited a fibroblast-like morphology and sustained high proliferative activity. Flow cytometric analysis revealed that DFAT cells comprised a highly homogeneous cell population compared with that of adipose-derived stem/stromal cells (ASCs), although the cell-surface antigen profile of DFAT cells was very similar to that of ASCs. DFAT cells lost expression of mature adipocytes marker genes but retained or gained expression of mesenchymal lineage-committed marker genes such as peroxisome proliferator-activated receptor gamma (PPARgamma), RUNX2, and SOX9. In vitro differentiation analysis revealed that DFAT cells could differentiate into adipocytes, chondrocytes, and osteoblasts under appropriate culture conditions. DFAT cells also formed osteoid matrix when implanted subcutaneously into nude mice. In addition, clonally expanded porcine DFAT cells showed the ability to differentiate into multiple mesenchymal cell lineages. These results indicate that DFAT cells represent a type of multipotent progenitor cell. The accessibility and ease of culture of DFAT cells support their potential application for cell-based therapies.

  11. Immunophotoaffinity labeling of binders of 1-methyladenine, the oocyte maturation-inducing hormone of starfish.

    Science.gov (United States)

    Toraya, Tetsuo; Kida, Tetsuo; Kuyama, Atsushi; Matsuda, Shinjiro; Tanaka, Seiichi; Komatsu, Yo; Tsurukai, Taro

    2017-02-04

    Starfish oocytes are arrested at the prophase stage of the first meiotic division in the ovary and resume meiosis by the stimulus of 1-methyladenine (1-MeAde), the oocyte maturation-inducing hormone of starfish. Putative 1-MeAde receptors on the oocyte surface have been suggested, but not yet been biochemically characterized. Immunophotoaffinity labeling, i.e., photoaffinity labeling combined with immunochemical detection, was attempted to detect unknown 1-MeAde binders including putative maturation-inducing hormone receptors in starfish oocytes. When the oocyte crude membrane fraction or its Triton X-100/EDTA extract was incubated with N(6)-[6-(5-azido-2-nitrobenzoyl)aminohexyl]carboxamidomethyl-1-methyladenine and then photo-irradiated, followed by western blotting with antibody that was raised against a 1-MeAde hapten, a single band with Mr of 47.5 K was detected. The band was lost when extract was heated at 100 °C. A similar 47.5 K band was detected in the crude membrane fraction of testis as well. Upon labeling with whole cells, this band was detected in immature and maturing oocytes, but only faintly in mature oocytes. As judged from these results, this 1-MeAde binder might be a possible candidate of the starfish maturation-inducing hormone receptors.

  12. Mechanosensitivity of dental pulp stem cells is related to their osteogenic maturity

    DEFF Research Database (Denmark)

    Kraft, David C E; Bindslev, Dorthe A; Melsen, Birte

    2010-01-01

    For engineering bone tissue, mechanosensitive cells are needed for bone (re)modelling. Local bone mass and architecture are affected by mechanical loading, which provokes a cellular response via loading-induced interstitial fluid flow. We studied whether human dental pulp-derived mesenchymal stem...... cells (PDSCs) portraying mature (PDSC-mature) or immature (PDSC-immature) bone cell characteristics are responsive to pulsating fluid flow (PFF) in vitro. We also assessed bone formation by PDSCs on hydroxyapatite-tricalcium phosphate granules after subcutaneous implantation in mice. Cultured PDSC...... expression was higher than in PDSC-immature. Implantation of PDSC-mature resulted in more osteoid deposition and lamellar bone formation than PDSC-immature. We conclude that PDSCs with a mature osteogenic phenotype are more responsive to pulsating fluid shear stress than osteogenically immature PDSCs...

  13. Acute myeloid leukemia cells inhibit the differentiation and maturation of dendritic cells and induce the generation of regulatory T cells%急性髓系白血病细胞对树突状细胞分化、成熟和调节性T细胞生成的影响

    Institute of Scientific and Technical Information of China (English)

    Xingbing Wang; Xin Chen; Jun Liu; Zimin Sun; Shiang Huang

    2008-01-01

    Objective: To investigate the effects of soluble factors secreted by acute myeloid leukemia (AML) cells on the phenotypical and functional properties of DCs derived from normal mononuclear cells. Methods: Mononuclear cells were cultured with interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF), in the presence or absence of 24 h culture supernatants from fresh primary AML cells, to generate immature DCs. The maturation of DCs was induced by cytokines IL-1beta, IL-6, tumor necrosis factor-alpha (TNF-alpha), and prostaglandin-2 (PGE-2). The phenotypic alterations of DCs and DCs-primed CD4+ T cells were evaluated using flow cytometry. Precursor frequency (PF) was calculated to monitor the allostimulatory effects of DCs on CD4+ and CD8+ T cells. Results:AML cell supernatant-treated DCs showed significantly lower expression of co-stimulatory molecules CD80 and CD86, and reduced response to cytokines IL-1beta, IL-6, TNF-alpha,and PGE-2. The allostimulatory effects of AML cell supernatant-treated DCs on CD4+ and CD8+ T cells were significantly lower than those of normal mature DCs [PF: (1.8±0.5)% vs. (5.2±1.6)% for CD4+ T cells, (2.1±0.6)% vs. (6.5±2.0)%for CD8+ T cells, P<0.01]. These AML supematant-induced DCs could also induce allogeneic CD4+ T cells to differentiate into CD4+CD25high T cells, which had immunophenotyping characteristics of regulatory T cells, i.e. they expressed Foxp3 but not active maker CD69. Conclusion: This study demonstrates that soluble factors secreted by AML cells can inhibit development and functions of DCs. In addition, AML supematant-induced DCs can induce the generation of CD4+CD25high T cells from CD4+T cells, which may be a mechanism of increased prevalence of CD4+CD25high regulatory T cells and immune dysfunction in AML patients.

  14. Inhibitory effects of apoptotic cell ingestion upon endotoxin-driven myeloid dendritic cell maturation.

    Science.gov (United States)

    Stuart, Lynda M; Lucas, Mark; Simpson, Cathy; Lamb, Jonathan; Savill, John; Lacy-Hulbert, Adam

    2002-02-15

    Dendritic cells (DCs) are the sentinels of the immune system, able to interact with both naive and memory T cells. The recent observation that DCs can ingest cells dying by apoptosis has raised the possibility that DCs may, in fact, present self-derived Ags, initiating both autoimmunity and tumor-specific responses, especially if associated with appropriate danger signals. Although the process of ingestion of apoptotic cells has not been shown to induce DC maturation, the exact fate of these phagocytosing DCs remains unclear. In this paper we demonstrate that DCs that ingest apoptotic cells are able to produce TNF-alpha but have a diminished ability to produce IL-12 in response to external stimuli, a property that corresponds to a failure to up-regulate CD86. By single-cell analysis we demonstrate that these inhibitory effects are restricted to those DCs that have engulfed apoptotic cells, with bystander DCs remaining unaffected. These changes were independent of the production of anti-inflammatory cytokines TGF-beta1 and IL-10 and corresponded with a diminished capacity to stimulate naive T cells. Thus, the ingestion of apoptotic cells is not an immunologically null event but is capable of modulating DC maturation. These results have important implications for our understanding of the role of clearance of dying cells by DCs not only in the normal resolution of inflammation but also in control of subsequent immune responses to apoptotic cell-derived Ags.

  15. Mature adipocyte-derived dedifferentiated fat cells can transdifferentiate into skeletal myocytes in vitro.

    Science.gov (United States)

    Kazama, Tomohiko; Fujie, Masaki; Endo, Tuyoshi; Kano, Koichiro

    2008-12-19

    We have previously reported the establishment of preadipocyte cell lines, termed dedifferentiated fat (DFAT) cells, from mature adipocytes of various animals. DFAT cells possess long-term viability and can redifferentiate into adipocytes both in vivo and in vitro. Furthermore, DFAT cells can transdifferentiate into osteoblasts and chondrocytes under appropriate culture conditions. However, it is unclear whether DFAT cells are capable of transdifferentiating into skeletal myocytes, which is common in the mesodermal lineage. Here, we show that DFAT cells can be induced to transdifferentiate into skeletal myocytes in vitro. Myogenic induction of DFAT cells resulted in the expression of MyoD and myogenin, followed by cell fusion and formation of multinucleated cells expressing sarcomeric myosin heavy chain. These results indicate that DFAT cells derived from mature adipocytes can transdifferentiate into skeletal myocytes in vitro.

  16. Induction of mast cell proliferation, maturation, and heparin synthesis by the rat c-kit ligand, stem cell factor

    Energy Technology Data Exchange (ETDEWEB)

    Tsai, M.; Takeishi, Takashi; Geissler, E.N. (Beth Israel Hospital, Boston, MA (United States)); Thompson, H.; Metcalfe, D.D. (National Inst. of Health, Bethesda, MD (United States)); Langley, K.E.; Zsebo, K.M.; Galli, S.J. (Amgen, Inc., Thousand Oaks, CA (United States))

    1991-07-15

    The authors investigated the effects of a newly recognized multifunctional growth factor, the c-kit ligand stem cell factor (SCF), on mouse mast cell proliferation and phenotype. Recombinant rat SCF{sup 164} (rrSCF{sup 164}) induced the development of large numbers of dermal mast cells in normal mice in vivo. Many of these mast cells had features of connective tissue-type mast cells (CTMC), in that they were reactive both with the heparin-binding fluorescent dye berberine sulfate and with safranin. In vitro, rrSCF{sup 164} induced the proliferation of cloned interleukin 3 (IL-3)-dependent mouse mast cells and primary populations of IL-3-dependent, bone marrow-derived cultured mast cells (BMCMC), which represent immature mast cells, and purified peritoneal mast cells, which represent a type of mature CTMC> BMCMC maintained in rrSCF{sup 164} not only proliferated but also matured. These findings identify SCF as a single cytokine that can induce immature, IL-3-dependent mast cells to mature and to acquire multiple characteristics of CTMC. These findings also directly demonstrate that SCF can regulate the development of a cellular lineage expressing c-kit through effects on both proliferation and maturation.

  17. NKT cell-TCR expression activates conventional T cells in vivo, but is largely dispensable for mature NKT cell biology.

    Science.gov (United States)

    Vahl, J Christoph; Heger, Klaus; Knies, Nathalie; Hein, Marco Y; Boon, Louis; Yagita, Hideo; Polic, Bojan; Schmidt-Supprian, Marc

    2013-01-01

    Natural killer T (NKT) cell development depends on recognition of self-glycolipids via their semi-invariant Vα14i-TCR. However, to what extent TCR-mediated signals determine identity and function of mature NKT cells remains incompletely understood. To address this issue, we developed a mouse strain allowing conditional Vα14i-TCR expression from within the endogenous Tcrα locus. We demonstrate that naïve T cells are activated upon replacement of their endogenous TCR repertoire with Vα14i-restricted TCRs, but they do not differentiate into NKT cells. On the other hand, induced TCR ablation on mature NKT cells did not affect their lineage identity, homeostasis, or innate rapid cytokine secretion abilities. We therefore propose that peripheral NKT cells become unresponsive to and thus are independent of their autoreactive TCR.

  18. NKT cell-TCR expression activates conventional T cells in vivo, but is largely dispensable for mature NKT cell biology.

    Directory of Open Access Journals (Sweden)

    J Christoph Vahl

    Full Text Available Natural killer T (NKT cell development depends on recognition of self-glycolipids via their semi-invariant Vα14i-TCR. However, to what extent TCR-mediated signals determine identity and function of mature NKT cells remains incompletely understood. To address this issue, we developed a mouse strain allowing conditional Vα14i-TCR expression from within the endogenous Tcrα locus. We demonstrate that naïve T cells are activated upon replacement of their endogenous TCR repertoire with Vα14i-restricted TCRs, but they do not differentiate into NKT cells. On the other hand, induced TCR ablation on mature NKT cells did not affect their lineage identity, homeostasis, or innate rapid cytokine secretion abilities. We therefore propose that peripheral NKT cells become unresponsive to and thus are independent of their autoreactive TCR.

  19. A proliferation-inducing ligand sustains the proliferation of human naïve (CD27⁻) B cells and mediates their differentiation into long-lived plasma cells in vitro via transmembrane activator and calcium modulator and cyclophilin ligand interactor and B-cell mature antigen.

    Science.gov (United States)

    Matsuda, Yoshiko; Haneda, Masataka; Kadomatsu, Kenji; Kobayashi, Takaaki

    2015-06-01

    Long-lived plasma cells (PCs) contribute to humoral immunity through an undefined mechanism. Memory B cells, but not human naïve B cells, can be induced to differentiate into long-lived PCs in vitro. Because evidence links a proliferation-inducing ligand (APRIL), a tumor necrosis factor family member, to the ability of bone marrow to mediate long-term PC survival, we reasoned that APRIL influences the proliferation and differentiation of naïve B cells. We describe here the development of a simple cell culture system that allowed us to show that APRIL sustained the proliferation of naïve human B cells and induced them to differentiate into long-lived PCs. Blocking the transmembrane activator and calcium modulator and cyclophilin ligand interactor or B-cell mature antigen shows they were required for the differentiation of naïve B cells into long-lived PCs in vitro. Our in vitro culture system will reveal new insights into the biology of long-lived PCs.

  20. [Production of mature red blood cell by using peripheral blood mononuclear cells].

    Science.gov (United States)

    Jia, Yan-Jun; Liu, Jiang; Zhang, Ke-Ying; Shang, Xiao-Yan; Li, Wei; Wang, Li-Jun; Liu, Na; Wang, Lin; Cui, Shuang; Ni, Lei; Zhao, Bo-Tao; Wang, Dong-Mei; Gao, Song-Ming; Zhang, Zhi-Xin

    2014-10-01

    Most protocols for in vitro producing red blood cells (RBC) use the CD34(+) cells or embryonic stem cells from cord blood, bone marrow or peripheral blood as the start materials. This study was purposed to produce the mature RBC in vitro by using peripheral blood mononuclear cells as start material. The peripheral blood mononuclear cells (PBMNC) were isolated from buffy coat after blood leukapheresis, the mature red blood cells (RBC) were prepared by a 4-step culture protocol. The results showed that after culture by inducing with the different sets of cytokines and supporting by mouse MS-5 cell line, the expansion of PBMNC reached about 1000 folds at the end of the culture. About 90% of cultured RBC were enucleated mature cells which had the comparable morphological characteristics with normal RBC. Colony-forming assays showed that this culture system could stimulate the proliferation of progenitors in PBMNC and differentiate into erythroid cells. The structure and function analysis indicated that the mean cell volume of in vitro cultured RBC was 118 ± 4 fl, which was slight larger than that of normal RBC (80-100 fl); the mean cell hemoglobin was 36 ± 1.2 pg, which was slight higher than that of normal RBC (27-31 pg); the maximal deformation index was 0.46, which approachs level of normal RBC; the glucose-6-phosphate dehydrogenase and pyrurvate kinase levels was consistant with young RBC. It is concluded that PBMNC are feasble, convenient and low-cost source for producing cultured RBC and this culture system is suitable to generate the RBC from PBMNC.

  1. Wnt-β-Catenin Signaling Promotes the Maturation of Mast Cells

    Directory of Open Access Journals (Sweden)

    Tomoko Yamaguchi

    2016-01-01

    Full Text Available Mast cells play an important role in the pathogenesis of allergic diseases. Immature mast cells migrate into peripheral tissues from the bone marrow and undergo complete maturation. Interestingly, mast cells have characteristics similar to hematopoietic stem cells (HSCs, such as self-renewal and c-kit expression. In HSCs, Wnt signaling is involved in their maintenance and differentiation. On the other hand, the relation between Wnt signaling and mast cell differentiation is poorly understood. To study whether Wnt signals play a role in the maturation of mast cells, we studied the effect of Wnt proteins on mast cell maturation of bone marrow-derived mast cells (BMMCs. The expression levels of CD81 protein and histidine decarboxylase mRNA and activity of mast cell-specific protease were all elevated in BMMCs treated with Wnt5a. In addition, Wnt5a induced the expression of Axin2 and TCF mRNA in BMMCs. These results showed that Wnt5a could promote the maturation of mast cells via the canonical Wnt signaling pathway and provide important insights into the molecular mechanisms underlying the differentiation of mast cells.

  2. Transcriptional networks in developing and mature B cells.

    Science.gov (United States)

    Matthias, Patrick; Rolink, Antonius G

    2005-06-01

    The development of B cells from haematopoietic stem cells proceeds along a highly ordered, yet flexible, pathway. At multiple steps along this pathway, cells are instructed by transcription factors on how to further differentiate, and several check-points have been identified. These check-points are initial commitment to lymphocytic progenitors, specification of pre-B cells, entry to the peripheral B-cell pool, maturation of B cells and differentiation into plasma cells. At each of these regulatory nodes, there are transcriptional networks that control the outcome, and much progress has recently been made in dissecting these networks. This article reviews our current understanding of this exciting field.

  3. Mutation in Fas Ligand Impairs Maturation of Thymocytes Bearing Moderate Affinity T Cell Receptors

    OpenAIRE

    2003-01-01

    Fas ligand, best known as a death-inducer, is also a costimulatory molecule required for maximal proliferation of mature antigen-specific CD4+ and CD8+ T cells. We now extend the role of Fas ligand by showing that it can also influence thymocyte development. T cell maturation in some, but not all, strains of TCR transgenic mice is severely impaired in thymocytes expressing mutant Fas ligand incapable of interacting with Fas. Mutant Fas ligand inhibits neither negative selection nor death by n...

  4. Cord blood mesenchymal stem cells propel human dendritic cells to an intermediate maturation state and boost interleukin-12 production by mature dendritic cells.

    NARCIS (Netherlands)

    Berk, L.C.J. van den; Roelofs, H.; Huijs, T.; Siebers-Vermeulen, K.G.C.; Raymakers, R.A.P.; Kogler, G.; Figdor, C.G.; Torensma, R.

    2009-01-01

    Pathogen-derived entities force the tissue-resident dendritic cells (DCs) towards a mature state, followed by migration to the draining lymph node to present antigens to T cells. Bone marrow mesenchymal stem cells (MSCs) modulate the differentiation, maturation and function of DCs. In umbilical cord

  5. The fertilization—induced Ca2+ oscillation in mouse oocytes is cytoplasmic maturation dependent

    Institute of Scientific and Technical Information of China (English)

    DENGMANQI; FANGZHENSUN

    1996-01-01

    Mature eggs (at metaphase II stage) produce a series of Ca2+ oscillation at fertilization.To define whether the fertilization-induced Ca2+ oscillation is restrict to the metaphase II eggs and cell cycle dependent,mouse oocytes at prophase I (arrested at germinal vesicle stage),metaphase I,metaphase II,as well as the pronuclear embryos at interphase of the first mitotic division derived from fertilization of parthenogenetic activation were inseminated after removal of zona pellucida,The results show that the fertilization-induced Ca2+ oscillation is not specific to metaphase II eggs.This is supported by the fact that immature oocytes generated the Ca2+ oscillations at fertilization regardless of their nuclear progression from prophase I to metaphase I (in vitro matured) stage.More interestingly,it was first found that pronuclear embryos at interphase derived from parthenogenetic activation showed Ca2+ oscillations in response to fertilization while the zygotes at interphase did not after reinsemination or intracytoplasmic injection of sperm extracts which induce Ca2+ oscillations in MII eggs.This suggests that the ability of oocytes to generate Ca2+ oscillation in response to sperm penetration is not regulated in a cell cycle dependent manner but dependent on the cytoplasmic maturation.

  6. Weaning triggers a maturation step of pancreatic β cells

    DEFF Research Database (Denmark)

    Stolovich-Rain, Miri; Enk, Jonatan; Vikesa, Jonas;

    2015-01-01

    Because tissue regeneration deteriorates with age, it is generally assumed that the younger the animal, the better it compensates for tissue damage. We have examined the effect of young age on compensatory proliferation of pancreatic β cells in vivo. Surprisingly, β cells in suckling mice fail...... to enter the cell division cycle in response to a diabetogenic injury or increased glycolysis. The potential of β cells for compensatory proliferation is acquired following premature weaning to normal chow, but not to a diet mimicking maternal milk. In addition, weaning coincides with enhanced glucose...... a discrete maturation step of β cells, elevating both the mitogenic and secretory response to glucose....

  7. Cord blood mesenchymal stem cells propel human dendritic cells to an intermediate maturation state and boost interleukin-12 production by mature dendritic cells.

    Science.gov (United States)

    van den Berk, Lieke C J; Roelofs, Helene; Huijs, Tonnie; Siebers-Vermeulen, Kim G C; Raymakers, Reinier A; Kögler, Gesine; Figdor, Carl G; Torensma, Ruurd

    2009-12-01

    Pathogen-derived entities force the tissue-resident dendritic cells (DCs) towards a mature state, followed by migration to the draining lymph node to present antigens to T cells. Bone marrow mesenchymal stem cells (MSCs) modulate the differentiation, maturation and function of DCs. In umbilical cord blood an immature MSC population was identified. Remarkably, these immature stem cells modulated DCs in a different way. Marker expression was unchanged during the differentiation of monocytes towards immature DCs (iDCs) when cocultured with cord blood MSC [unrestricted somatic stem cells (USSCs)]. The maturation to mature DCs (mDCs) was enhanced when DCs were co-cultured with USSC, as evidenced by the up-regulation of costimulatory molecules. Endocytosis of dextran by iDCs was hampered in the presence of USSCs, which is indicative for the maturation of iDCs. Despite this maturation, the migration of iDCs cocultured with USSCs appeared to be identical to iDCs cultured alone. However, USSCs increased the migration of mDCs towards CCL21 and boosted interleukin-12 production. So, USSCs mature iDCs, thereby redirecting the antigen-uptake phenotype towards a mature phenotype. Furthermore, DC maturation by lipopolysaccharide (LPS) or USSCs reflects two distinct pathways because migration was unaffected when iDCs were matured by coculture with USSCs, while it was strongly enhanced in the presence of LPS. DCs are able to discriminate the different MSC subtypes, resulting in diverse differentiation programmes.

  8. Endogenous laminin is required for human airway smooth muscle cell maturation

    Directory of Open Access Journals (Sweden)

    Tran Thai

    2006-09-01

    Full Text Available Abstract Background Airway smooth muscle (ASM contraction underlies acute bronchospasm in asthma. ASM cells can switch between a synthetic-proliferative phenotype and a contractile phenotype. While the effects of extracellular matrix (ECM components on modulation of ASM cells to a synthetic phenotype have been reported, the role of ECM components on maturation of ASM cells to a contractile phenotype in adult lung is unclear. As both changes in ECM components and accumulation of contractile ASM are features of airway wall remodelling in asthma, we examined the role of the ECM protein, laminin, in the maturation of contractile phenotype in human ASM cells. Methods Human ASM cells were made senescence-resistant by stable expression of human telomerase reverse transcriptase. Maturation to a contractile phenotype was induced by 7-day serum deprivation, as assessed by immunoblotting for desmin and calponin. The role of laminin on ASM maturation was investigated by comparing the effects of exogenous laminin coated on culture plates, and of soluble laminin peptide competitors. Endogenous expression of laminin chains during ASM maturation was also measured. Results Myocyte binding to endogenously expressed laminin was required for ASM phenotype maturation, as laminin competing peptides (YIGSR or GRGDSP significantly reduced desmin and calponin protein accumulation that otherwise occurs with prolonged serum deprivation. Coating of plastic cell culture dishes with different purified laminin preparations was not sufficient to further promote accumulation of desmin or calponin during 7-day serum deprivation. Expression of α2, β1 and γ1 laminin chains by ASM cells was specifically up-regulated during myocyte maturation, suggesting a key role for laminin-2 in the development of the contractile phenotype. Conclusion While earlier reports suggest exogenously applied laminin slows the spontaneous modulation of ASM to a synthetic phenotype, we show for the

  9. Comparison of immature and mature bone marrow-derived dendritic cells by atomic force microscopy

    Science.gov (United States)

    Xing, Feiyue; Wang, Jiongkun; Hu, Mingqian; Yu, Yu; Chen, Guoliang; Liu, Jing

    2011-07-01

    A comparative study of immature and mature bone marrow-derived dendritic cells (BMDCs) was first performed through an atomic force microscope (AFM) to clarify differences of their nanostructure and adhesion force. AFM images revealed that the immature BMDCs treated by granulocyte macrophage-colony stimulating factor plus IL-4 mainly appeared round with smooth surface, whereas the mature BMDCs induced by lipopolysaccharide displayed an irregular shape with numerous pseudopodia or lamellapodia and ruffles on the cell membrane besides becoming larger, flatter, and longer. AFM quantitative analysis further showed that the surface roughness of the mature BMDCs greatly increased and that the adhesion force of them was fourfold more than that of the immature BMDCs. The nano-features of the mature BMDCs were supported by a high level of IL-12 produced from the mature BMDCs and high expression of MHC-II on the surface of them. These findings provide a new insight into the nanostructure of the immature and mature BMDCs.

  10. BMP2 induces osteoblast apoptosis in a maturation state and noggin-dependent manner.

    Science.gov (United States)

    Hyzy, Sharon L; Olivares-Navarrete, Rene; Schwartz, Zvi; Boyan, Barbara D

    2012-10-01

    Large doses of bone morphogenetic protein 2 (BMP2) are used clinically to induce bone formation in challenging bone defects. However, complications after treatment include swelling, ectopic bone formation, and adjacent bone resorption. While BMP2 can be effective, it is important to characterize the mechanism of the deleterious effects to optimize its use. The aim of this study was to determine the effect of BMP2 on apoptosis in osteoblast lineage cells and to determine the role of the BMP inhibitor Noggin in this process. Human mesenchymal stem cells (MSCs), immature osteoblast-like MG63 cells, and mature normal human osteoblasts (NHOst) were treated with BMP2. A model system of increased endogenous BMP signaling was created by silencing Noggin (shNOG-MG63). Finally, the BMP pathway regulating apoptosis in NHOst was examined using BMP signaling inhibitors (5Z-7-oxozeaenol, dorsomorphin, H-8). Apoptosis was characterized by caspase-3, BAX/BCL2, p53, and DNA fragmentation. BMP2 induced apoptosis in a cell-type dependent manner. While the effect was minor in MSCs, MG63 cells had modest increases and NHOst cells had robust increases apoptosis after BMP2 treatment. Apoptosis was significantly higher in shNOG-MG63 than MG63 cells. 5Z-7-oxozeaenol and dorsomorphin eliminated the BMP2-induced increase in DNA fragmentation in NHOst, suggesting roles for TAB/TAK1 and Smad signaling. These results indicate that the apoptotic effect of BMP2 is dependent on cell maturation state, inducing apoptosis in committed osteoblasts through Smad and TAB/TAK1 signaling, and is regulated by Noggin. Dose and delivery must be optimized in therapeutic applications of BMP2 to minimize complications.

  11. Maturation of spinal motor neurons derived from human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Tomonori Takazawa

    Full Text Available Our understanding of motor neuron biology in humans is derived mainly from investigation of human postmortem tissue and more indirectly from live animal models such as rodents. Thus generation of motor neurons from human embryonic stem cells and human induced pluripotent stem cells is an important new approach to model motor neuron function. To be useful models of human motor neuron function, cells generated in vitro should develop mature properties that are the hallmarks of motor neurons in vivo such as elaborated neuronal processes and mature electrophysiological characteristics. Here we have investigated changes in morphological and electrophysiological properties associated with maturation of neurons differentiated from human embryonic stem cells expressing GFP driven by a motor neuron specific reporter (Hb9::GFP in culture. We observed maturation in cellular morphology seen as more complex neurite outgrowth and increased soma area over time. Electrophysiological changes included decreasing input resistance and increasing action potential firing frequency over 13 days in vitro. Furthermore, these human embryonic stem cell derived motor neurons acquired two physiological characteristics that are thought to underpin motor neuron integrated function in motor circuits; spike frequency adaptation and rebound action potential firing. These findings show that human embryonic stem cell derived motor neurons develop functional characteristics typical of spinal motor neurons in vivo and suggest that they are a relevant and useful platform for studying motor neuron development and function and for modeling motor neuron diseases.

  12. The role of phosphatidylinositol signaling pathway in regulating serotonin-induced oocyte maturation in Mercenaria mercenaria

    Institute of Scientific and Technical Information of China (English)

    WANG Qing; ZHANG Tao

    2011-01-01

    Serotonin (5-HT) has been found to stimulate meiotic maturation of oocytes in many molluscs. During maturation, several signaling pathways are involved, especially the phosphatidylinositol and cAMP pathways. In order to examine the possible role of the phosphatidylinositol signaling pathway in regulating oocyte maturation in Mercenaria mercenaria, the effects of the activator/inhibitor of phospholipase (PLC) and protein kinase C (PKC) on serotonin-induced maturation were studied. Results show that high-concentrations of neomycin (inhibitor of PLC) blocked oocyte maturation, while 9, 10-dimethyl- 1, 2-benzanthracene (DMBA, activator of PLC) promoted oocyte maturation in the presence of serotonin. It was also found that in the presence of serotonin, phorbol 12-myristate 13-acetate (PMA,activator of PKC) inhibited oocyte maturation, while sphingosine (inhibitor of PKC) stimulated oocyte maturation. These results indicate that serotonin-induced oocyte maturation requires the activation of the phosphatidylinositol pathway. Decrease of PLC inhibited serotonin-induced oocyte maturation, whereas a decrease of PKC stimulated the maturation. Thus, our study indicates that serotonin promotes maturation of M. mercenaria oocytes through PLC stimulated increase in calcium ion concentration via inositol-1,4, 5-trisphosphate (IP3) but not PKC.

  13. Squamous Cell Carcinoma Arising in a Mature Cystic Teratoma

    Directory of Open Access Journals (Sweden)

    Suna Avcı

    2012-01-01

    Full Text Available Introduction. Malignant transformation in a mature cystic teratoma of the ovary is a rare complication. Squamous cell carcinoma is the most common transformation. We describe a new case of squamous cell carcinoma arising in a mature cystic teratoma. Case Report. A premenopausal 52-year-old female patient is diagnosed with vaginal bleeding. According to examination made on the women and the pelvic scanning, 7 cm mass is found on the right adnexa of the patient. Total abdominal hysterectomy, bilateral salpingo-oophorectomy, omentectomy, pelvic lymph node dissection, and debulking were the treatments completed on the patient. According to histopathological diagnosis, squamous cell carcinoma arising in a mature cystic teratoma is diagnosed as a reason for the mass in the right adnexa of the patient. Conclusion. The prognosis of the malign transformation of MCT depends on surgery stage; however it is extremely poor. The patient should receive chemotherapy regardless of stage. We have decided to administer second cycle carboplatin and paclitaxel treatments on the patient.

  14. Human dendritic cell maturation and cytokine secretion upon stimulation with Bordetella pertussis filamentous haemagglutinin.

    Science.gov (United States)

    Dirix, Violette; Mielcarek, Nathalie; Debrie, Anne-Sophie; Willery, Eve; Alonso, Sylvie; Versheure, Virginie; Mascart, Françoise; Locht, Camille

    2014-07-01

    In addition to antibodies, Th1-type T cell responses are also important for long-lasting protection against pertussis. However, upon immunization with the current acellular vaccines, many children fail to induce Th1-type responses, potentially due to immunomodulatory effects of some vaccine antigens, such as filamentous haemagglutinin (FHA). We therefore analysed the ability of FHA to modulate immune functions of human monocyte-derived dendritic cells (MDDC). FHA was purified from pertussis toxin (PTX)-deficient or from PTX- and adenylate cyclase-deficient Bordetella pertussis strains, and residual endotoxin was neutralized with polymyxin B. FHA from both strains induced phenotypic maturation of human MDDC and cytokine secretion (IL-10, IL-12p40, IL-12p70, IL-23 and IL-6). To identify the FHA domains responsible for MDDC immunomodulation, MDDC were stimulated with FHA containing a Gly→Ala substitution at its RGD site (FHA-RAD) or with an 80-kDa N-terminal moiety of FHA (Fha44), containing its heparin-binding site. Whereas FHA-RAD induced maturation and cytokine production comparable to those of FHA, Fha44 did not induce IL-10 production, but maturated MDDC at least partially. Nevertheless, Fha44 induced the secretion of IL-12p40, IL-12p70, IL-23 and IL-6 by MDDC, albeit at lower levels than FHA. Thus, FHA can modulate MDDC responses in multiple ways, and IL-10 induction can be dissociated from the induction of other cytokines.

  15. Mediation of transitional B cell maturation in the absence of functional Bruton’s tyrosine kinase

    Science.gov (United States)

    Tanwar, Shalini; Dhar, Atika; Varanasi, Vineeth; Mukherjee, Tapas; Boppana, Ramanamurthy; Basak, Soumen; Bal, Vineeta; George, Anna; Rath, Satyajit

    2017-01-01

    X-linked immune-deficient (Xid) mice, carrying a mutation in Bruton’s tyrosine kinase (Btk), have multiple B cell lineage differentiation defects. We now show that, while Xid mice showed only mild reduction in the frequency of the late transitional (T2) stage of peripheral B cells, the defect became severe when the Xid genotype was combined with either a CD40-null, a TCRbeta-null or an MHC class II (MHCII)-null genotype. Purified Xid T1 and T2 B cells survived poorly in vitro compared to wild-type (WT) cells. BAFF rescued WT but not Xid T1 and T2 B cells from death in culture, while CD40 ligation equivalently rescued both. Xid transitional B cells ex vivo showed low levels of the p100 protein substrate for non-canonical NF-kappaB signalling. In vitro, CD40 ligation induced equivalent activation of the canonical but not of the non-canonical NF-kappaB pathway in Xid and WT T1 and T2 B cells. CD40 ligation efficiently rescued p100-null T1 B cells from neglect-induced death in vitro. These data indicate that CD40-mediated signals, likely from CD4 T cells, can mediate peripheral transitional B cell maturation independent of Btk and the non-canonical NF-kappaB pathway, and thus contribute to the understanding of the complexities of peripheral B cell maturation. PMID:28378771

  16. Neural induction from ES cells portrays default commitment but instructive maturation.

    Directory of Open Access Journals (Sweden)

    Nibedita Lenka

    Full Text Available The neural induction has remained a debatable issue pertaining to whether it is a mere default process or it involves precise instructive cues. We have chosen the embryonic stem (ES cell model to address this issue. In a devised monoculture strategy, the cell-cell interaction availed through optimum cell plating density could define the niche for the attainment of efficient in vitro neurogenesis from the ES cells. The medium plating density was found ideal in generating optimum number of progenitors and also yielded about 80% mature neurons in a serum free culture set up barring any exogenous inducers. We could also demarcate and quantify the neural stem cells/progenitors among the heterogeneous cell population of differentiating ES cells using nestin intron II driven EGFP expression as a tool. The one week post-plating was determined to be the critical time window for optimum neural progenitor generation from ES cells that helped us further in purifying these cells and in demonstrating their proliferation and multipotent differentiation potential. Seeding cells at varying densities, we could decipher an interesting paradoxical scenario that interlinked both commitment and maturation with the initial plating density having a vital influence on neuronal maturation but not specification and the secretory factors were apparently playing a key role during this process. Thus it was comprehended that, the neural specification was a default process independent of exogenous factors and cellular interaction. Conversely, a defined number of cells at the specification stage itself seemed critical to provide an auto-/paracrine means of signaling threshold for the maturation process to materialize.

  17. Role for endogenous estrogen in prepubertal Sertoli cell maturation.

    Science.gov (United States)

    Kao, Eddy; Villalon, Rosalina; Ribeiro, Salustiano; Berger, Trish

    2012-11-01

    Reducing prepubertal endogenous estrogens led to increased numbers of Sertoli cells and the associated increased testicular size and testicular sperm production capacity in boars. The increased number of Sertoli cells might be due to a longer time for proliferation; delayed differentiation of Sertoli cells during suppressed endogenous estrogens would be consistent with this hypothesized, prolonged proliferation interval. This study used immunohistochemical detection of anti-Müllerian hormone (AMH), a marker of immature Sertoli cells, and of CDKN1B, a cell cycle inhibitor associated with more mature Sertoli cells, to determine if suppressing endogenous estrogens detectably delayed "differentiation" of porcine Sertoli cells. Testes were from littermate pairs of boars previously treated with Letrozole, an aromatase inhibitor, or vehicle, from the first week of age until tissue collection at 2, 3, 4, 5 or 6 months of age. Four animals were examined at each age following Letrozole treatment and their corresponding littermates evaluated following treatment with vehicle. Amount of AMH protein in Sertoli cells decreased with age of boar and could not be detected at 6 months of age. The AMH labeling was greater in the Letrozole-treated boars compared with littermate vehicle controls at 4 months of age (P=0.03). The percentage of CDKN1B-labeled Sertoli cells apparently increased with age through 5 months of age. At 4 and 5 months of age, the mean percentage of CDKN1B-labeled Sertoli cells was less in the Letrozole-treated animals than in the vehicle control animals (P = 0.03 and 0.04, respectively). These results are consistent with the hypothesis that continual inhibition of aromatase (and concomitatant reduced estrogen synthesis) causes a delay in Sertoli cell maturation in boars.

  18. Interaction of poxvirus intracellular mature virion proteins with the TPR domain of kinesin light chain in live infected cells revealed by two-photon-induced fluorescence resonance energy transfer fluorescence lifetime imaging microscopy.

    Science.gov (United States)

    Jeshtadi, Ananya; Burgos, Pierre; Stubbs, Christopher D; Parker, Anthony W; King, Linda A; Skinner, Michael A; Botchway, Stanley W

    2010-12-01

    Using two-photon-induced fluorescence lifetime imaging microscopy, we corroborate an interaction (previously demonstrated by yeast two-hybrid domain analysis) of full-length vaccinia virus (VACV; an orthopoxvirus) A36 protein with the cellular microtubule motor protein kinesin. Quenching of enhanced green fluorescent protein (EGFP), fused to the C terminus of VACV A36, by monomeric red fluorescent protein (mDsRed), fused to the tetratricopeptide repeat (TPR) domain of kinesin, was observed in live chicken embryo fibroblasts infected with either modified vaccinia virus Ankara (MVA) or wild-type fowlpox virus (FWPV; an avipoxvirus), and the excited-state fluorescence lifetime of EGFP was reduced from 2.5 ± 0.1 ns to 2.1 ± 0.1 ns due to resonance energy transfer to mDsRed. FWPV does not encode an equivalent of intracellular enveloped virion surface protein A36, yet it is likely that this virus too must interact with kinesin to facilitate intracellular virion transport. To investigate possible interactions between innate FWPV proteins and kinesin, recombinant FWPVs expressing EGFP fused to the N termini of FWPV structural proteins Fpv140, Fpv168, Fpv191, and Fpv198 (equivalent to VACV H3, A4, p4c, and A34, respectively) were generated. EGFP fusions of intracellular mature virion (IMV) surface protein Fpv140 and type II membrane protein Fpv198 were quenched by mDsRed-TPR in recombinant FWPV-infected cells, indicating that these virion proteins are found within 10 nm of mDsRed-TPR. In contrast, and as expected, EGFP fusions of the IMV core protein Fpv168 did not show any quenching. Interestingly, the p4c-like protein Fpv191, which demonstrates late association with preassembled IMV, also did not show any quenching.

  19. Effect of Different Titanium Surfaces on Maturation of Murine Bone Marrow-Derived Dendritic Cells

    Science.gov (United States)

    Zheng, Xiaofei; Zhou, Fengjuan; Gu, Yifei; Duan, Xiaobo; Mo, Anchun

    2017-01-01

    Dendritic cells (DCs) play a pivotal role in the host response to implanted biomaterials. Osseointegration of titanium (Ti) implant is an immunological and inflammatory-driven process. However, the role of DCs in this complex process is largely unknown. This study aimed to investigate the effect of different Ti surfaces on DC maturation, and evaluate its subsequent potential on osteogenic differentiation of preosteoblasts. Murine bone marrow-derived DCs were seeded on Ti disks with different surface treatments, including pretreatment (PT), sandblasted/acid-etched (SLA) and modified SLA (modSLA) surface. Compared with DCs cultured on PT and SLA surfaces, the cells seeded on modSLA surface demonstrated a more round morphology with lower expression of CD86 and MHC-II, the DC maturation markers. Those cells also secreted high levels of anti-inflammatory cytokine IL-10 and TGF-β. Notably, addition of conditioned medium (CM) from modSLA-induced DCs significantly increased the mRNA expression of Runx2 and ALP as well as ALP activity by murine preosteoblast MC3T3-E1 cells. Our data demonstrated that Ti disks with different surfaces lead to differential DCs responses. PT and SLA surfaces induce DCs mature, while DCs seeded on modSLA-Ti surface maintain an immature phenotype and exhibit a potential of promoting osteogenic differentiation of MC3T3-E1 cells. PMID:28157196

  20. Influence of low dose irradiation on differentiation, maturation and T-cell activation of human dendritic cells

    Energy Technology Data Exchange (ETDEWEB)

    Jahns, Jutta [Department of Radiotherapy and Radiation Oncology, University of Leipzig, Stephanstrasse 21, 04103 Leipzig (Germany); Anderegg, Ulf; Saalbach, Anja [Department for Dermatology, Venerology and Allergology, University of Leipzig, Johannisallee 30, 04103 Leipzig (Germany); Rosin, Britt; Patties, Ina; Glasow, Annegret [Department of Radiotherapy and Radiation Oncology, University of Leipzig, Stephanstrasse 21, 04103 Leipzig (Germany); Kamprad, Manja [Institute for Clinical Immunology and Transfusion Medicine, University of Leipzig, Johannisallee 30, 04103 Leipzig (Germany); Scholz, Markus [Institute for Medical Informatics, Statistics and Epidemiology, University of Leipzig, Haertelstr. 16-18, 04103 Leipzig (Germany); Hildebrandt, Guido, E-mail: Guido.Hildebrandt@uni-rostock.de [Department of Radiotherapy and Radiation Oncology, University of Rostock, Suedring 75, 18059 Rostock (Germany); Department of Radiotherapy and Radiation Oncology, University of Leipzig, Stephanstrasse 21, 04103 Leipzig (Germany)

    2011-05-10

    Ionizing irradiation could act directly on immune cells and may induce bystander effects mediated by soluble factors that are released by the irradiated cells. This is the first study analyzing both the direct effect of low dose ionizing radiation (LDIR) on the maturation and cytokine release of human dendritic cells (DCs) and the functional consequences for co-cultured T-cells. We showed that irradiation of DC-precursors in vitro does not influence surface marker expression or cytokine profile of immature DCs nor of mature DCs after LPS treatment. There was no difference of single dose irradiation versus fractionated irradiation protocols on the behavior of the mature DCs. Further, the low dose irradiation did not change the capacity of the DCs to stimulate T-cell proliferation. But the irradiation of the co-culture of DCs and T-cells revealed significantly lower proliferation of T-cells with higher doses. Summarizing the data from approx. 50 DC preparations there is no significant effect of low dose ionizing irradiation on the cytokine profile, surface marker expression and maturation of DCs in vitro although functional consequences cannot be excluded.

  1. Maturation, proliferation and apoptosis of seminal tubule cells at puberty after administration of estradiol, follicle stimulating hormone or both

    Institute of Scientific and Technical Information of China (English)

    Renata Walczak-Jedrzejowska; Jolanta Slowikowska-Hilczer; Katarzyna Marchlewska; Krzysztof Kula

    2008-01-01

    Aim: To assess proliferative and apoptotic potential of the seminiferous epithelium cells in relation to Sertoli cell maturation in newborn rats under the influence of estradiol, follicle stimulating hormone (FSH) or both agents given together. Methods: From postnatal day (PND) 5 to 15 male rats were daily injected with 12.5 μg of 17β-estradiol benzoate (EB) or 7.5 IU of human purified FSH (hFSH) or EB + hFSH or solvents (control). On postnatal day 16, autopsy was performed. Sertoli cell maturation/function was assessed by morphometry. Proliferation of the semini- ferous epithelium cells was quantitatively evaluated using immunohistochemical labeling against proliferating cell nuclear antigen and apoptosis using the TUNEL method. Results: Although EB inhibited Sertoli cell maturation and hFSH was not effective, a pronounced acceleration of Sertoli cell maturation occurred after EB + hFSH. Whereas hFSH stimulated Sertoli cell proliferation, EB or EB + hFSH inhibited Sertoli cell proliferation. All treatments signifi- cantly stimulated germ cell proliferation. Apoptosis of Sertoli cells increased 9-fold and germ cells 2-fold after EB, and was not affected by hFSH but was inhibited after EB + hFSH. Conclusion: At puberty, estradiol inhibits Sertoli cell maturation, increases Sertoli and germ cell apoptosis but stimulates germ cell proliferation. Estradiol in synergism with FSH, but neither of the hormones alone, accelerates Sertoli cell maturation associated with an increase in germ cell survival. Estradiol and FSH cooperate to induce seminal tubule maturation and trigger first spermatogenesis. (Asian J Androl 2008 Jul; 10: 585-592)

  2. A Key Role for Inhibins in Dendritic Cell Maturation and Function.

    Science.gov (United States)

    Olguín-Alor, Roxana; de la Fuente-Granada, Marisol; Bonifaz, Laura C; Antonio-Herrera, Laura; García-Zepeda, Eduardo A; Soldevila, Gloria

    2016-01-01

    Inhibins are members of the TGFβ superfamily, which regulate many cellular processes including differentiation, proliferation, survival and apoptosis. Although initially described as hormones regulating the hypothalamus-pituitary-gonadal axis, based on their ability to antagonize Activins, our group has recently reported that they play a role in thymocyte differentiation and survival, as well as in thymic stromal cell maturation and nTreg generation. Here, we used Inhibin knock out mice (Inhα-/-) to investigate the role of Inhibins in peripheral dendritic cell maturation and function. We first demonstrated that LPS treated Inhα+/+ bone marrow derived dendritic cells (BMDC) were capable to produce significant levels of Inhibin A. Interestingly, Inhα-/- BMDC showed reduced MHCII and CD86 upregulation and increased PD-L1 expression in response to LPS compared to Inhα+/+, which correlated with reduced ability to induce proliferation of allogeneic T cells. The "semi-mature" phenotype displayed by Inhα-/- mBMDC correlated with increased levels of IL-10 and slightly decreased IL-6 production after LPS stimulation. In addition, Inhα-/- mBMDC showed impaired migration towards CCL19 and CCL21, assessed by in vitro chemotaxis and in vivo competitive homing experiments, despite their normal CCR7 expression. Furthermore, in vivo LPS-induced DC maturation was also diminished in Inhα-/- mice, specially within the LC (CD207+ CD11b+ CD103-) subpopulation. Finally, analysis of delayed type hypersensitivity responses in Inhα-/- mice, showed reduced ear swelling as a result of reduced cellular infiltration in the skin, correlating with impaired homing of CD207+ DCs to the draining lymph nodes. In summary, our data demonstrate for the first time that Inhibins play a key role in peripheral DC maturation and function, regulating the balance between immunity and tolerance.

  3. TLR-independent induction of dendritic cell maturation and adaptive immunity by negative-strand RNA viruses.

    Science.gov (United States)

    López, Carolina B; Moltedo, Bruno; Alexopoulou, Lena; Bonifaz, Laura; Flavell, Richard A; Moran, Thomas M

    2004-12-01

    TLR signaling leads to dendritic cell (DC) maturation and immunity to diverse pathogens. The stimulation of TLRs by conserved viral structures is the only described mechanism leading to DC maturation after a virus infection. In this report, we demonstrate that mouse myeloid DCs mature normally after in vivo and in vitro infection with Sendai virus (SeV) in the absence of TLR3, 7, 8, or 9 signaling. DC maturation by SeV requires virus replication not necessary for TLR-mediated triggering. Moreover, DCs deficient in TLR signaling efficiently prime for Th1 immunity after infection with influenza or SeV, generating IFN-gamma-producing T cells, CTLs and antiviral Abs. We have previously demonstrated that SeV induces DC maturation independently of the presence of type I IFN, which has been reported to mature DCs in a TLR-independent manner. The data presented here provide evidence for the existence of a novel intracellular pathway independent of TLR-mediated signaling responsible for live virus triggering of DC maturation and demonstrate its critical role in the onset of antiviral immunity. The revelation of this pathway should stimulate invigorating research into the mechanism for virus-induced DC maturation and immunity.

  4. Tumour tissue microenvironment can inhibit dendritic cell maturation in colorectal cancer.

    LENUS (Irish Health Repository)

    Michielsen, Adriana J

    2011-01-01

    Inflammatory mediators in the tumour microenvironment promote tumour growth, vascular development and enable evasion of anti-tumour immune responses, by disabling infiltrating dendritic cells. However, the constituents of the tumour microenvironment that directly influence dendritic cell maturation and function are not well characterised. Our aim was to identify tumour-associated inflammatory mediators which influence the function of dendritic cells. Tumour conditioned media obtained from cultured colorectal tumour explant tissue contained high levels of the chemokines CCL2, CXCL1, CXCL5 in addition to VEGF. Pre-treatment of monocyte derived dendritic cells with this tumour conditioned media inhibited the up-regulation of CD86, CD83, CD54 and HLA-DR in response to LPS, enhancing IL-10 while reducing IL-12p70 secretion. We examined if specific individual components of the tumour conditioned media (CCL2, CXCL1, CXCL5) could modulate dendritic cell maturation or cytokine secretion in response to LPS. VEGF was also assessed as it has a suppressive effect on dendritic cell maturation. Pre-treatment of immature dendritic cells with VEGF inhibited LPS induced upregulation of CD80 and CD54, while CXCL1 inhibited HLA-DR. Interestingly, treatment of dendritic cells with CCL2, CXCL1, CXCL5 or VEGF significantly suppressed their ability to secrete IL-12p70 in response to LPS. In addition, dendritic cells treated with a combination of CXCL1 and VEGF secreted less IL-12p70 in response to LPS compared to pre-treatment with either cytokine alone. In conclusion, tumour conditioned media strongly influences dendritic cell maturation and function.

  5. Commonly used prophylactic vaccines as an alternative for synthetically produced TLR ligands to mature monocyte-derived dendritic cells.

    Science.gov (United States)

    Schreibelt, Gerty; Benitez-Ribas, Daniel; Schuurhuis, Danita; Lambeck, Annechien J A; van Hout-Kuijer, Maaike; Schaft, Niels; Punt, Cornelis J A; Figdor, Carl G; Adema, Gosse J; de Vries, I Jolanda M

    2010-07-29

    Currently dendritic cell (DC)-based vaccines are explored in clinical trials, predominantly in cancer patients. Murine studies showed that only maturation with Toll-like receptor (TLR) ligands generates mature DCs that produce interleukin-12 and promote optimal T-cell help. Unfortunately, the limited availability of clinical-grade TLR ligands significantly hampers the translation of these findings into DC-based vaccines. Therefore, we explored 15 commonly used preventive vaccines as a possible source of TLR ligands. We have identified a cocktail of the vaccines BCG-SSI, Influvac, and Typhim that contains TLR ligands and is capable of optimally maturing DCs. These DCs (vaccine DCs) showed high expression of CD80, CD86, and CD83 and secreted interleukin-12. Although vaccine DCs exhibited an impaired migratory capacity, this could be restored by addition of prostaglandin E(2) (PGE(2); vaccine PGE(2) DCs). Vaccine PGE(2) DCs are potent inducers of T-cell proliferation and induce Th1 polarization. In addition, vaccine PGE(2) DCs are potent inducers of tumor antigen-specific CD8(+) effector T cells. Finally, vaccine PGE(2)-induced DC maturation is compatible with different antigen-loading strategies, including RNA electroporation. These data thus identify a new clinical application for a mixture of commonly used preventive vaccines in the generation of Th1-inducing clinical-grade mature DCs.

  6. Expression of maturation-specific nuclear antigens in differentiating human myeloid leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Murao, S.; Epstein, A.L.; Clevenger, C.V.; Huberman, E.

    1985-02-01

    The expression of three myeloid-specific nuclear antigens was studied by indirect immunofluorescence with murine monoclonal antibodies in human myeloid (HL-60, ML-2, KG-1, and B-II) leukemia cells treated with chemical inducers of cell differentiation. Treatment of the promyelocytic HL-60 cells with dimethyl sulfoxide or 1,25-dihydroxyvitamin DT induced the cells to acquire a phenotype that resembled that of granulocytes and monocytesmacrophages, respectively. These phenotypes were characterized by changes in cell growth, cell morphology, expression of specific cell surface antigens, and activities of lysozyme and nonspecific esterase enzymes. Induction of these differentiation markers in the HL-60 cells was associated with induction of the myeloid-specific nuclear antigens. The ML-2 cells, which are arrested at the myeloblast-promyelocyte stage, were also susceptible to the induction of cell differentiation and to changes in the expression of the nuclear antigens, but the degree of susceptibility was less than in the HL-60 cells. The less-differentiated KG-1 and B-II myeloid cells were either not responsive or responded only in a limited degree to the induction of cell differentiation or to changes in the expression of the nuclear antigens. The authors suggest that the reactivity of cells with monoclonal antibodies to specific nuclear antigens can be used as a maturational marker in cell differentiation studies. Furthermore, nuclear antigens expressed early in cellular differentiation may provide information about changes in regulatory elements in normal and malignant cells. 40 references, 2 figures, 1 table.

  7. Rapid developmental maturation of neocortical FS cell intrinsic excitability.

    Science.gov (United States)

    Goldberg, Ethan M; Jeong, Hyo-Young; Kruglikov, Ilya; Tremblay, Robin; Lazarenko, Roman M; Rudy, Bernardo

    2011-03-01

    Fast-spiking (FS) cells are a prominent subtype of neocortical γ-aminobutyric acidergic interneurons that mediate feed-forward inhibition and the temporal sculpting of information transfer in neural circuits, maintain excitation/inhibition balance, and contribute to network oscillations. FS cell dysfunction may be involved in the pathogenesis of disorders such as epilepsy, autism, and schizophrenia. Mature FS cells exhibit coordinated molecular and cellular specializations that facilitate rapid responsiveness, including brief spikes and sustained high-frequency discharge. We show that these features appear during the second and third postnatal weeks driven by upregulation of K(+) channel subunits of the Kv3 subfamily. The low membrane resistance and fast time constant characteristic of FS cells also appears during this time, driven by expression of a K(+) leak current mediated by K(ir)2 subfamily inward rectifier K(+) channels and TASK subfamily 2-pore K(+) channels. Blockade of this leak produces dramatic depolarization of FS cells suggesting the possibility for potent neuromodulation. Finally, the frequency of FS cell membrane potential oscillations increases during development and is markedly slower in TASK-1/3 knockout mice, suggesting that TASK channels regulate FS cell rhythmogenesis. Our findings imply that some of the effects of acidosis and/or anesthetics on brain function may be due to blockade of TASK channels in FS cells.

  8. Inhibition of phenotypic and functional maturation of dendritic cells by manassantin a.

    Science.gov (United States)

    Kim, Jee Youn; Kang, Jong Soon; Kim, Hwan Mook; Kim, Young Kook; Lee, Hong Kyung; Song, Sukgil; Hong, Jin Tae; Kim, Youngsoo; Han, Sang-Bae

    2009-04-01

    Manassantin A (MSA) inhibits nitric oxide production by macrophages through the inhibition of NF-kappaB activation, but the effect of MSA on dendritic cells has not been elucidated yet. Here we investigated the inhibitory effects of MSA on immune functions of dendritic cells (DCs). MSA inhibited lipopolysaccharide (LPS)-induced phenotypic maturation of DCs, which was proved by the decreased expression of CD40, CD80, CD86, MHC-I, and MHC-II. MSA also inhibited functional maturation of DCs, that is, decreased the gene expression of IL-12, IL-1beta, TNF-alpha, and IFN-alpha/beta; enhanced antigen capture capacity of DCs; and impaired induction of allogenic T cell activation. As a mechanism of action, MSA inhibited LPS-induced activation of NF-kappaB, ERK, p38, and JNK, which played pivotal roles in toll-like receptor 4-mediated DC maturation. Collectively, these results suggested that MSA might be used for the treatment of DC-related immune diseases.

  9. Effects of maturation-inducing hormone on heterologous gap junctional coupling in ovarian follicles of Atlantic croaker

    Science.gov (United States)

    Yoshizaki, G.; Patino, R.; Thomas, P.; Bolamba, D.; Chang, Xiaotian

    2001-01-01

    A previous ultrastructural study of heterologous (granulosa cell-oocyte) gap junction (GJ) contacts in ovarian follicles of Atlantic croaker suggested that these contacts disappear late during the process of resumption of oocyte meiosis. This observation suggested that, unlike scenarios proposed for a number of other species, uncoupling of GJ is not necessary for the onset of meiotic resumption in croaker follicles. However, the functionality of heterologous GJ contacts and the temporal association between maturation-inducing hormone (MIH)-induced changes in heterologous coupling and resumption of oocyte meiosis have not been examined in Atlantic croaker. These questions were addressed with a cell-cell coupling assay that is based on the transfer of a GJ marker, Lucifer Yellow, from oocytes to granulosa cells. Follicle-enclosed oocytes injected with Lucifer Yellow allowed transfer of the dye into the follicle cell layer, thus confirming that there is functional heterologous coupling between the oocyte and the granulosa cells. Dye transfer was observed in vitellogenic, full-grown/maturation-incompetent, and full-grown /maturation-competent follicles. Treatment of maturation-competent follicles with MIH caused a time-dependent decline in the number of follicles transferring dye. However, although GJ uncoupling in some of the follicles was observed before germinal vesicle breakdown (GVBD, index of meiotic resumption), about 50% of the follicles maintained the ability to transfer dye even after GVBD had occurred. Further, a known GJ inhibitor (phorbol 12-myristate 13-acetate) blocked heterologous GJ within a time frame similar to that seen with MIH but without inducing any of the morphological changes (including GVBD) associated with follicular maturation. In conclusion, uncoupling of heterologous GJ seems insufficient and unnecessary for the onset of meiotic resumption in ovarian follicles of Atlantic croaker. ?? 2001 Elsevier Science.

  10. Composition of MHC class II-enriched lipid microdomains is modified during maturation of primary dendritic cells.

    Science.gov (United States)

    Setterblad, Niclas; Roucard, Corinne; Bocaccio, Claire; Abastado, Jean-Pierre; Charron, Dominique; Mooney, Nuala

    2003-07-01

    Dendritic cells (DCs) are the most potent antigen presenting cells. Major histocompatibility complex (MHC) class II molecule expression changes with maturation; immature DCs concentrate MHC class II molecules intracellularly, whereas maturation increases surface expression of MHC class II and costimulatory molecules to optimize antigen presentation. Signal transduction via MHC class II molecules localized in lipid microdomains has been described in B lymphocytes and in the THP-1 monocyte cell line. We have characterized MHC class II molecules throughout human DC maturation with particular attention to their localization in lipid-rich microdomains. Only immature DCs expressed empty MHC class II molecules, and maturation increased the level of peptide-bound heterodimers. Ligand binding to surface human leukocyte antigen (HLA)-DR induced rapid internalization in immature DCs. The proportion of cell-surface detergent-insoluble glycosphingolipid-enriched microdomain-clustered HLA-DR was higher in immature DCs despite the higher surface expression of HLA-DR in mature DCs. Constituents of HLA-DR containing microdomains included the src kinase Lyn and the cytoskeletal protein tubulin in immature DCs. Maturation modified the composition of the HLA-DR-containing microdomains to include protein kinase C (PKC)-delta, Lyn, and the cytoskeletal protein actin, accompanied by the loss of tubulin. Signaling via HLA-DR redistributed HLA-DR and -DM and PKC-delta as well as enriching the actin content of mature DC microdomains. The increased expression of HLA-DR as a result of DC maturation was therefore accompanied by modification of the spatial organization of HLA-DR. Such regulation could contribute to the distinct responses induced by ligand binding to MHC class II molecules in immature versus mature DCs.

  11. XIAP inhibits mature Smac-induced apoptosis by degrading it through ubiquitination in NSCLC

    Science.gov (United States)

    Qin, Sida; Yang, Chengcheng; Zhang, Boxiang; Li, Xiang; Sun, Xin; Li, Gang; Zhang, Jing; Xiao, Guodong; Gao, Xiao; Huang, Guanghong; Wang, Peili; Ren, Hong

    2016-01-01

    X-linked inhibitor of apoptosis protein (XIAP) and second mitochondrial-derived activator of caspase (Smac) are two important prognostic biomarkers for cancer. They are negatively correlated in many types of cancer. However, their relationship is still unknown in lung cancer. In the present study, we found that there was a negative correlation between Smac and XIAP at the level of protein but not mRNA in NSCLC patients. However, XIAP overexpression had no effect on degrading endogenous Smac in lung cancer cell lines. Therefore, we constructed plasmids with full length of Smac (fSmac) and mature Smac (mSmac) which located in cytoplasm instead of original mitochondrial location, and was confirmed by immunofluorescence. Subsequently, we found that mSmac rather than fSmac was degraded by XIAP and inhibited cell viability. CHX chase assay and ubiquitin assay were performed to illustrate XIAP degraded mSmac through ubiquitin pathway. Overexpression of XIAP partially reverted apoptotic induction and cell viability inhibition by mSmac, which was due to inhibiting caspase-3 activation. In nude mouse xenograft experiments, mSmac inhibited Ki-67 expression and slowed down lung cancer growth, while XIAP partially reversed the effect of mSmac by degrading it. In conclusion, XIAP inhibits mature Smac-induced apoptosis by degrading it through ubiquitination in NSCLC. PMID:27498621

  12. Rab8a vesicles regulate Wnt ligand delivery and Paneth cell maturation at the intestinal stem cell niche.

    Science.gov (United States)

    Das, Soumyashree; Yu, Shiyan; Sakamori, Ryotaro; Vedula, Pavan; Feng, Qiang; Flores, Juan; Hoffman, Andrew; Fu, Jiang; Stypulkowski, Ewa; Rodriguez, Alexis; Dobrowolski, Radek; Harada, Akihiro; Hsu, Wei; Bonder, Edward M; Verzi, Michael P; Gao, Nan

    2015-06-15

    Communication between stem and niche supporting cells maintains the homeostasis of adult tissues. Wnt signaling is a crucial regulator of the stem cell niche, but the mechanism that governs Wnt ligand delivery in this compartment has not been fully investigated. We identified that Wnt secretion is partly dependent on Rab8a-mediated anterograde transport of Gpr177 (wntless), a Wnt-specific transmembrane transporter. Gpr177 binds to Rab8a, depletion of which compromises Gpr177 traffic, thereby weakening the secretion of multiple Wnts. Analyses of generic Wnt/β-catenin targets in Rab8a knockout mouse intestinal crypts indicate reduced signaling activities; maturation of Paneth cells - a Wnt-dependent cell type - is severely affected. Rab8a knockout crypts show an expansion of Lgr5(+) and Hopx(+) cells in vivo. However, in vitro, the knockout enteroids exhibit significantly weakened growth that can be partly restored by exogenous Wnts or Gsk3β inhibitors. Immunogold labeling and surface protein isolation identified decreased plasma membrane localization of Gpr177 in Rab8a knockout Paneth cells and fibroblasts. Upon stimulation by exogenous Wnts, Rab8a-deficient cells show ligand-induced Lrp6 phosphorylation and transcriptional reporter activation. Rab8a thus controls Wnt delivery in producing cells and is crucial for Paneth cell maturation. Our data highlight the profound tissue plasticity that occurs in response to stress induced by depletion of a stem cell niche signal.

  13. A Key Role for Inhibins in Dendritic Cell Maturation and Function

    Science.gov (United States)

    Olguín-Alor, Roxana; de la Fuente-Granada, Marisol; Bonifaz, Laura C.; Antonio-Herrera, Laura; García-Zepeda, Eduardo A.; Soldevila, Gloria

    2016-01-01

    Inhibins are members of the TGFβ superfamily, which regulate many cellular processes including differentiation, proliferation, survival and apoptosis. Although initially described as hormones regulating the hypothalamus-pituitary-gonadal axis, based on their ability to antagonize Activins, our group has recently reported that they play a role in thymocyte differentiation and survival, as well as in thymic stromal cell maturation and nTreg generation. Here, we used Inhibin knock out mice (Inhα-/-) to investigate the role of Inhibins in peripheral dendritic cell maturation and function. We first demonstrated that LPS treated Inhα+/+ bone marrow derived dendritic cells (BMDC) were capable to produce significant levels of Inhibin A. Interestingly, Inhα-/- BMDC showed reduced MHCII and CD86 upregulation and increased PD-L1 expression in response to LPS compared to Inhα+/+, which correlated with reduced ability to induce proliferation of allogeneic T cells. The “semi-mature” phenotype displayed by Inhα-/- mBMDC correlated with increased levels of IL-10 and slightly decreased IL-6 production after LPS stimulation. In addition, Inhα-/- mBMDC showed impaired migration towards CCL19 and CCL21, assessed by in vitro chemotaxis and in vivo competitive homing experiments, despite their normal CCR7 expression. Furthermore, in vivo LPS-induced DC maturation was also diminished in Inhα-/- mice, specially within the LC (CD207+ CD11b+ CD103-) subpopulation. Finally, analysis of delayed type hypersensitivity responses in Inhα-/- mice, showed reduced ear swelling as a result of reduced cellular infiltration in the skin, correlating with impaired homing of CD207+ DCs to the draining lymph nodes. In summary, our data demonstrate for the first time that Inhibins play a key role in peripheral DC maturation and function, regulating the balance between immunity and tolerance. PMID:27936218

  14. Chronic alcohol consumption enhances iNKT cell maturation and activation

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Hui, E-mail: hzhang@wsu.edu; Zhang, Faya; Zhu, Zhaohui; Luong, Dung; Meadows, Gary G.

    2015-01-15

    Alcohol consumption exhibits diverse effects on different types of immune cells. NKT cells are a unique T cell population and play important immunoregulatory roles in different types of immune responses. The effects of chronic alcohol consumption on NKT cells remain to be elucidated. Using a mouse model of chronic alcohol consumption, we found that alcohol increases the percentage of NKT cells, especially iNKT cells in the thymus and liver, but not in the spleen or blood. Alcohol consumption decreases the percentage of NK1.1{sup −} iNKT cells in the total iNKT cell population in all of the tissues and organs examined. In the thymus, alcohol consumption increases the number of NK1.1{sup +}CD44{sup hi} mature iNKT cells but does not alter the number of NK1.1{sup −} immature iNKT cells. A BrdU incorporation assay shows that alcohol consumption increases the proliferation of thymic NK1.1{sup −} iNKT cells, especially the NK1.1{sup −}CD44{sup lo} Stage I iNKT cells. The percentage of NKG2A{sup +} iNKT cells increases in all of the tissues and organs examined; whereas CXCR3{sup +} iNKT cells only increases in the thymus of alcohol-consuming mice. Chronic alcohol consumption increases the percentage of IFN-γ-producing iNKT cells and increases the blood concentration of IFN-γ and IL-12 after in vivo α-galactosylceramide (αGalCer) stimulation. Consistent with the increased cytokine production, the in vivo activation of iNKT cells also enhances the activation of dendritic cells (DC) and NK, B, and T cells in the alcohol-consuming mice. Taken together the data indicate that chronic alcohol consumption enhances iNKT cell maturation and activation, which favors the Th1 immune response. - Highlights: • Chronic alcohol consumption increases iNKT cells in the thymus and liver • Chronic alcohol consumption enhances thymic Stage I iNKT cell proliferation • Chronic alcohol consumption enhances iNKT cell maturation in thymus and periphery • Chronic alcohol

  15. Harvest-induced maturation evolution under different life-history trade-offs and harvesting regimes

    NARCIS (Netherlands)

    Poos, J.J.; Brannstrom, A.; Dieckman, U.

    2011-01-01

    The potential of harvesting to induce adaptive changes in exploited populations is now increasingly recognized. While early studies predicted that elevated mortalities among larger individuals select for reduced maturation size, recent theoretical studies have shown conditions under which other, mor

  16. Acquisition of a Quantitative, Stoichiometrically Conserved Ratiometric Marker of Maturation Status in Stem Cell-Derived Cardiac Myocytes

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    Fikru B. Bedada

    2014-10-01

    Full Text Available There is no consensus in the stem cell field as to what constitutes the mature cardiac myocyte. Thus, helping formalize a molecular signature for cardiac myocyte maturation would advance the field. In the mammalian heart, inactivation of the “fetal” TNNI gene, TNNI1 (ssTnI, together in temporal concert with its stoichiometric replacement by the adult TNNI gene product, TNNI3 (cTnI, represents a quantifiable ratiometric maturation signature. We examined the TNNI isoform transition in human induced pluripotent stem cell (iPSC cardiac myocytes (hiPSC-CMs and found the fetal TNNI signature, even during long-term culture. Rodent stem cell-derived and primary myocytes, however, transitioned to the adult TnI profile. Acute genetic engineering of hiPSC-CMs enabled a rapid conversion toward the mature TnI profile. While there is no single marker to denote the mature cardiac myocyte, we propose that tracking the cTnI:ssTnI protein isoform ratio provides a valuable maturation signature to quantify myocyte maturation status across laboratories.

  17. Influence of Skin Epithelial cells and Human Umbilical VEIN CELLS Conditioned Media on Maturation of Type 1 Dendritic Cells(DC1

    Directory of Open Access Journals (Sweden)

    M Ganjybakhsh

    2011-06-01

    Full Text Available Introduction: Dendritic cells have a high potential in presentation of antigens and can be generated and manipulated in invitro culture conditions. Dendritic cells(DC are therefore used in cancer immunotherapy, in prevention of graft rejection, treatment of allergy, autoimmune diseases and certain infectious diseases. Methods: Dendritic cell was generated in two stages. IN the first stage, monocyte cells were converted to immature DC affected GM-CSF and IL-4 .In the second stage, dendritic cells were maturated in the presence of supernatant skin epithelial cells(A375 and human umbilical vein endothelial cells(HUVEC and maturation factors. The ability of phagocytosis, expression phenotype, stimulation of T lymphocytes and cytokines was studied. Results: Mature Dendritic cells decreased their power of phagocytosis and increased expression of their surface markers. The ability of T cells stimulation and cytokine production(IL-12 increased . Conclusion: Mixture condition medium of epithelial cells and human skin umbilical vein endothelium cells induces maturation of monocyte-derived DCs. This condition medium improves their phenotype and their functions. The mentioned condition medium generates DC1 and Th1 in vitro.

  18. HIV and mature dendritic cells: Trojan exosomes riding the Trojan horse?

    Directory of Open Access Journals (Sweden)

    Nuria Izquierdo-Useros

    2010-03-01

    Full Text Available Exosomes are secreted cellular vesicles that can induce specific CD4(+ T cell responses in vivo when they interact with competent antigen-presenting cells like mature dendritic cells (mDCs. The Trojan exosome hypothesis proposes that retroviruses can take advantage of the cell-encoded intercellular vesicle traffic and exosome exchange pathway, moving between cells in the absence of fusion events in search of adequate target cells. Here, we discuss recent data supporting this hypothesis, which further explains how DCs can capture and internalize retroviruses like HIV-1 in the absence of fusion events, leading to the productive infection of interacting CD4(+ T cells and contributing to viral spread through a mechanism known as trans-infection. We suggest that HIV-1 can exploit an exosome antigen-dissemination pathway intrinsic to mDCs, allowing viral internalization and final trans-infection of CD4(+ T cells. In contrast to previous reports that focus on the ability of immature DCs to capture HIV in the mucosa, this review emphasizes the outstanding role that mature DCs could have promoting trans-infection in the lymph node, underscoring a new potential viral dissemination pathway.

  19. Coordinate reduction in cell proliferation and cell death in mouse olfactory epithelium from birth to maturity

    NARCIS (Netherlands)

    Fung, KM; Peringa, J; Venkatachalam, S; Lee, VMY; Trojanowski, JQ

    1997-01-01

    We investigated cell proliferation and cell death in the olfactory epithelium (OE) of mice from birth to maturity using bromodeoxyuridine and terminal deoxynucleotidyl transferase nick end labeling. We show that cell death events and proliferative activity diminish concomitantly with age in the OE.

  20. The effect of Propionibacterium acnes on maturation of dendritic cells derived from acne patients' peripherial blood mononuclear cells.

    Science.gov (United States)

    Michalak-Stoma, Anna; Tabarkiewicz, Jacek; Olender, Alina; Juszkiewicz-Borowiec, Maria; Stoma, Filip; Pietrzak, Aldona; Pozarowski, Piotr; Bartkowiak-Emeryk, Małgorzata

    2008-01-01

    Propionibacterium acnes (P. acnes) has been implicated in the pathogenesis of acne vulgaris which is the most common cutaneous disorder. It has a proinflammatory activity and takes part in immune reactions modulating the Th1/Th2 cellular response. The exposure of dendritic cells (DCs) to whole bacteria, their components, cytokines or other inflammatory stimuli and infectious agents induces differentiation from immature DCs into antigen-presenting mature DCs. The aim of the study was to evaluate the capability of P. acnes to induce the maturation of DCs. We stimulated monocyte derived dendritic cells (Mo-DCs) from acne patients with various concetrations of heat-killed P. acnes (10(6)-10(8) bacteria/ml) cultured from acne lesions. The results showed an increase in CD80+/CD86+/DR+ and CD83+/CD1a+/DR+ cells percentage depending on the concetration of P. acnes. The expression of CD83 and CD80 (shown as the mean fluorescence intensity - MFI) increased with higher concetrations of P. acnes. There were also significant correlations between MFI of CD83, CD80, CD86 and concetration of P. acnes. The study showed that P. acnes in the concetration of 10(8) bacteria/ml is most effective in the induction of Mo-DCs maturation. Futher studies concerning the influence on the function of T cells are needed.

  1. The effect of Propionibacterium acnes on maturation of dendritic cells derived from acne patients' peripherial blood mononuclear cells.

    Directory of Open Access Journals (Sweden)

    Maria Juszkiewicz-Borowiec

    2009-01-01

    Full Text Available Propionibacterium acnes (P. acnes has been implicated in the pathogenesis of acne vulgaris which is the most common cutaneous disorder. It has a proinflammatory activity and takes part in immune reactions modulating the Th1/Th2 cellular response. The exposure of dendritic cells (DCs to whole bacteria, their components, cytokines or other inflammatory stimuli and infectious agents induces differentiation from immature DCs into antigen-presenting mature DCs. The aim of the study was to evaluate the capability of P. acnes to induce the maturation of DCs. We stimulated monocyte derived dendritic cells (Mo-DCs from acne patients with various concetrations of heat-killed P. acnes (10(6-10(8 bacteria/ml cultured from acne lesions. The results showed an increase in CD80+/CD86+/DR+ and CD83+/CD1a+/DR+ cells percentage depending on the concetration of P. acnes. The expression of CD83 and CD80 (shown as the mean fluorescence intensity - MFI increased with higher concetrations of P. acnes. There were also significant correlations between MFI of CD83, CD80, CD86 and concetration of P. acnes. The study showed that P. acnes in the concetration of 10(8 bacteria/ml is most effective in the induction of Mo-DCs maturation. Futher studies concerning the influence on the function of T cells are needed.

  2. Maturation of dendritic cells by pullulan promotes anti-cancer effect

    Science.gov (United States)

    Xu, Li; Zhang, LiJun; Yu, Qing; Jin, Jun-O

    2016-01-01

    Previous studies have demonstrated that pullulan, a polysaccharide purified from Aureobasidium pullulans, has immune-stimulatory effects on T and B cells. Moreover, pullulan has been used as a carrier in the delivery of the antigen (Ag) peptide to lymphoid tissues. However, the in vivo effect of pullulan on dendritic cells (DC) has not been well characterized. In this study, we assessed the effect of pullulan on DC activation and anti-cancer immunity. The results showed that the pullulan treatment up-regulated co-stimulatory molecule expression and enhanced pro-inflammatory cytokine production in bone marrow-derived DCs (BMDC) in vitro and in spleen DCs in vivo. Moreover, the combination of ovalbumin (OVA) and pullulan induced OVA antigen-specific T cell activations in vivo. In tumor-bearing mice, pullulan induced the maturation of DCs in spleen and tumor draining lymph node (drLN), and promoted the OVA-specific T cell activation and migration of the T cells into the tumor. In addition, the combination of OVA and pullulan inhibited B16-OVA tumor growth and liver metastasis. The combination of tyrosinase-related protein 2 (TRP2) peptide and pullulan treatment also suppressed B16 melanoma growth. Thus, the results demonstrated that pullulan enhanced DC maturation and function, and it acted as an adjuvant in promoting Ag-specific immune responses in mice. Thus, pullulan could be a new and useful adjuvant for use in therapeutic cancer vaccines. PMID:27341129

  3. Piperlongumine Suppresses Dendritic Cell Maturation by Reducing Production of Reactive Oxygen Species and Has Therapeutic Potential for Rheumatoid Arthritis.

    Science.gov (United States)

    Xiao, Youjun; Shi, Maohua; Qiu, Qian; Huang, Mingcheng; Zeng, Shan; Zou, Yaoyao; Zhan, Zhongping; Liang, Liuqin; Yang, Xiuyan; Xu, Hanshi

    2016-06-15

    Piperlongumine (PLM) is a natural product from the plant Piper longum that inhibits platelet aggregation, atherosclerosis plaque formation, and tumor cell growth. It has potential value in immunomodulation and the management of autoimmune diseases. In this study, we investigated the role of PLM in regulating the differentiation and maturation of dendritic cells (DCs), a critical regulator of immune tolerance, and evaluated its clinical effects in a rheumatoid arthritis mouse model. We found that PLM treatment reduced LPS-induced murine bone marrow-derived DC maturation, characterized by reduced expression of CD80/86, secretion of MCP-1, IL-12p70, IL-6, TNFα, IFN-γ, and IL-23, and reduced alloproliferation of T cells; however, PLM does not affect cell differentiation. Furthermore, PLM reduced intracellular reactive oxygen species (ROS) production by DCs and inhibited the activation of p38, JNK, NF-κB, and PI3K/Akt signaling pathways. Conversely, PLM increased the expression of GSTP1 and carbonyl reductase 1, two enzymes that counteract ROS effects. ROS inhibition by exogenous N-acetyl-l-cysteine suppressed DC maturation. PLM treatment improved the severity of arthritis and reduced in vivo splenic DC maturation, collagen-specific CD4(+) T cell responses, and ROS production in mice with collagen-induced arthritis. Taken together, these results suggest that PLM inhibits DC maturation by reducing intracellular ROS production and has potential as a therapeutic agent for rheumatoid arthritis.

  4. HIV-1-infected monocyte-derived dendritic cells do not undergo maturation but can elicit IL-10 production and T cell regulation

    Science.gov (United States)

    Granelli-Piperno, Angela; Golebiowska, Angelika; Trumpfheller, Christine; Siegal, Frederick P.; Steinman, Ralph M.

    2004-05-01

    Dendritic cells (DCs) undergo maturation during virus infection and thereby become potent stimulators of cell-mediated immunity. HIV-1 replicates in immature DCs, but we now find that infection is not accompanied by many components of maturation in either infected cells or uninfected bystanders. The infected cultures do not develop potent stimulating activity for the mixed leukocyte reaction (MLR), and the DCs producing HIV-1 gag p24 do not express CD83 and DC-lysosome-associated membrane protein maturation markers. If different maturation stimuli are applied to DCs infected with HIV-1, the infected cells selectively fail to mature. When DCs from HIV-1-infected patients are infected and cultured with autologous T cells, IL-10 was produced in 6 of 10 patients. These DC-T cell cocultures could suppress another immune response, the MLR. The regulation was partially IL-10-dependent and correlated in extent with the level of IL-10 produced. Suppressor cells only developed from infected patients, rather than healthy controls, and the DCs had to be exposed to live virus rather than HIV-1 gag peptides or protein. These results indicate that HIV-1-infected DCs have two previously unrecognized means to evade immune responses: maturation can be blocked reducing the efficacy of antigen presentation from infected cells, and T cell-dependent suppression can be induced.

  5. Caspase-2-dependent dendritic cell death, maturation, and priming of T cells in response to Brucella abortus infection.

    Directory of Open Access Journals (Sweden)

    Xinna Li

    Full Text Available Smooth virulent Brucella abortus strain 2308 (S2308 causes zoonotic brucellosis in cattle and humans. Rough B. abortus strain RB51, derived from S2308, is a live attenuated cattle vaccine strain licensed in the USA and many other countries. Our previous report indicated that RB51, but not S2308, induces a caspase-2-dependent apoptotic and necrotic macrophage cell death. Dendritic cells (DCs are professional antigen presenting cells critical for bridging innate and adaptive immune responses. In contrast to Brucella-infected macrophages, here we report that S2308 induced higher levels of apoptotic and necrotic cell death in wild type bone marrow-derived DCs (WT BMDCs than RB51. The RB51 and S2308-induced BMDC cell death was regulated by caspase-2, indicated by the minimal cell death in RB51 and S2308-infected BMDCs isolated from caspase-2 knockout mice (Casp2KO BMDCs. More S2308 bacteria were taken up by Casp2KO BMDCs than wild type BMDCs. Higher levels of S2308 and RB51 cells were found in infected Casp2KO BMDCs compared to infected WT BMDCs at different time points. RB51-infected wild type BMDCs were mature and activated as shown by significantly up-regulated expression of CD40, CD80, CD86, MHC-I, and MHC-II. RB51 induced the production of cytokines TNF-α, IL-6, IFN-γ and IL12/IL23p40 in infected BMDCs. RB51-infected WT BMDCs also stimulated the proliferation of CD4(+ and CD8(+ T cells compared to uninfected WT BMDCs. However, the maturation, activation, and cytokine secretion are significantly impaired in Casp2KO BMDCs infected with RB51 or Salmonella (control. S2308-infected WT and Casp2KO BMDCs were not activated and could not induce cytokine production. These results demonstrated that virulent smooth strain S2308 induced more apoptotic and necrotic dendritic cell death than live attenuated rough vaccine strain RB51; however, RB51, but not its parent strain S2308, induced caspase-2-mediated DC maturation, cytokine production, antigen

  6. Glial cell line-derived neurotrophic factor promotes barrier maturation and wound healing in intestinal epithelial cells in vitro.

    Science.gov (United States)

    Meir, Michael; Flemming, Sven; Burkard, Natalie; Bergauer, Lisa; Metzger, Marco; Germer, Christoph-Thomas; Schlegel, Nicolas

    2015-10-15

    Recent data suggest that neurotrophic factors from the enteric nervous system are involved in intestinal epithelial barrier regulation. In this context the glial cell line-derived neurotrophic factor (GDNF) was shown to affect gut barrier properties in vivo directly or indirectly by largely undefined processes in a model of inflammatory bowel disease (IBD). We further investigated the potential role and mechanisms of GDNF in the regulation of intestinal barrier functions. Immunostaining of human gut specimen showed positive GDNF staining in enteric neuronal plexus and in enterocytes. In Western blots of the intestinal epithelial cell lines Caco2 and HT29B6, significant amounts of GDNF were detected, suggesting that enterocytes represent an additional source of GDNF. Application of recombinant GDNF on Caco2 and HT29B6 cells for 24 h resulted in significant epithelial barrier stabilization in monolayers with immature barrier functions. Wound-healing assays showed a significantly faster closure of the wounded areas after GDNF application. GDNF augmented cAMP levels and led to significant inactivation of p38 MAPK in immature cells. Activation of p38 MAPK signaling by SB-202190 mimicked GDNF-induced barrier maturation, whereas the p38 MAPK activator anisomycin blocked GDNF-induced effects. Increasing cAMP levels had adverse effects on barrier maturation, as revealed by permeability measurements. However, increased cAMP augmented the proliferation rate in Caco2 cells, and GDNF-induced proliferation of epithelial cells was abrogated by the PKA inhibitor H89. Our data show that enterocytes represent an additional source of GDNF synthesis. GDNF contributes to wound healing in a cAMP/PKA-dependent manner and promotes barrier maturation in immature enterocytes cells by inactivation of p38 MAPK signaling.

  7. Dynamic imaging of cell-free and cell-associated viral capture in mature dendritic cells.

    Science.gov (United States)

    Izquierdo-Useros, Nuria; Esteban, Olga; Rodriguez-Plata, Maria T; Erkizia, Itziar; Prado, Julia G; Blanco, Julià; García-Parajo, Maria F; Martinez-Picado, Javier

    2011-12-01

    Dendritic cells (DCs) capture human immunodeficiency virus (HIV) through a non-fusogenic mechanism that enables viral transmission to CD4(+) T cells, contributing to in vivo viral dissemination. Although previous studies have provided important clues to cell-free viral capture by mature DCs (mDCs), dynamic and kinetic insight on this process is still missing. Here, we used three-dimensional video microscopy and single-particle tracking approaches to dynamically dissect both cell-free and cell-associated viral capture by living mDCs. We show that cell-free virus capture by mDCs operates through three sequential phases: virus binding through specific determinants expressed in the viral particle, polarized or directional movements toward concrete regions of the cell membrane and virus accumulation in a sac-like structure where trapped viral particles display a hindered diffusive behavior. Moreover, real-time imaging of cell-associated viral transfer to mDCs showed a similar dynamics to that exhibited by cell-free virus endocytosis leading to viral accumulation in compartments. However, cell-associated HIV type 1 transfer to mDCs was the most effective pathway, boosted throughout enhanced cellular contacts with infected CD4(+) T cells. Our results suggest that in lymphoid tissues, mDC viral uptake could occur either by encountering cell-free or cell-associated virus produced by infected cells generating the perfect scenario to promote HIV pathogenesis and impact disease progression.

  8. Comparison of two in vitro dendritic cell maturation models for screening contact sensitizers using a panel of methacrylates.

    NARCIS (Netherlands)

    Rustemeyer, T.; Preuss, M; Blomberg - van der Flier, von B.M.E.; Das, PK; Scheper, R.J.

    2003-01-01

    Allergen-induced emigration and maturation of dendritic cells (DC) are pivotal steps in sparking off allergic contact dermatitis. In vitro models, reflecting these steps, may provide tools for assessment of sensitizing capacities of putative contact allergens. Here, we evaluated the applicability of

  9. Hydrostatic Pressure Affects In Vitro Maturation of Oocytes and Follicles and Increases Granulosa Cell Death

    Directory of Open Access Journals (Sweden)

    Isac Karimi

    2013-01-01

    Full Text Available Objective: This study examines the effects of hydrostatic pressure on in vitro maturation (IVM of oocytes derived from in vitro grown follicles.Materials and Methods: In this experimental study, preantral follicles were isolated from 12-day-old female NMRI mice. Each follicle was cultured individually in Alpha Minimal Essential Medium (α-MEM under mineral oil for 12 days. Then, follicles were induced for IVM and divided into two groups, control and experiment. In the experiment group follicles were subjected to 20 mmHg pressure for 30 minutes and cultured for 24-48 hours. We assessed for viability and IVM of the oocytes. The percentage of apoptosis in cumulus cells was determined by the TUNEL assay. A comparison between groups was made using the student’s t test.Results: The percentage of metaphase II oocytes (MII increased in hydrostatic pressure-treated follicles compared to controls (p<0.05. Cumulus cell viability reduced in hydrostatic pressure-treated follicles compared to controls (p<0.05. Exposure of follicles to pressure increased apoptosis in cumulus cells compared to controls (p<0.05.Conclusion: Hydrostatic pressure, by inducing apoptosis in cumulus cells, participates in the cumulus oocyte coupled relationship with oocyte maturation.

  10. Lipid body formation during maturation of human mast cells.

    Science.gov (United States)

    Dichlberger, Andrea; Schlager, Stefanie; Lappalainen, Jani; Käkelä, Reijo; Hattula, Katarina; Butcher, Sarah J; Schneider, Wolfgang J; Kovanen, Petri T

    2011-12-01

    Lipid droplets, also called lipid bodies (LB) in inflammatory cells, are important cytoplasmic organelles. However, little is known about the molecular characteristics and functions of LBs in human mast cells (MC). Here, we have analyzed the genesis and components of LBs during differentiation of human peripheral blood-derived CD34(+) progenitors into connective tissue-type MCs. In our serum-free culture system, the maturing MCs, derived from 18 different donors, invariably developed triacylglycerol (TG)-rich LBs. Not known heretofore, the MCs transcribe the genes for perilipins (PLIN)1-4, but not PLIN5, and PLIN2 and PLIN3 display different degrees of LB association. Upon MC activation and ensuing degranulation, the LBs were not cosecreted with the cytoplasmic secretory granules. Exogenous arachidonic acid (AA) enhanced LB genesis in Triacsin C-sensitive fashion, and it was found to be preferentially incorporated into the TGs of LBs. The large TG-associated pool of AA in LBs likely is a major precursor for eicosanoid production by MCs. In summary, we demonstrate that cultured human MCs derived from CD34(+) progenitors in peripheral blood provide a new tool to study regulatory mechanisms involving LB functions, with particular emphasis on AA metabolism, eicosanoid biosynthesis, and subsequent release of proinflammatory lipid mediators from these cells.

  11. Mature dendritic cells derived from human monocytes within 48 hours: a novel strategy for dendritic cell differentiation from blood precursors.

    Science.gov (United States)

    Dauer, Marc; Obermaier, Bianca; Herten, Jan; Haerle, Carola; Pohl, Katrin; Rothenfusser, Simon; Schnurr, Max; Endres, Stefan; Eigler, Andreas

    2003-04-15

    It is widely believed that generation of mature dendritic cells (DCs) with full T cell stimulatory capacity from human monocytes in vitro requires 5-7 days of differentiation with GM-CSF and IL-4, followed by 2-3 days of activation. Here, we report a new strategy for differentiation and maturation of monocyte-derived DCs within only 48 h of in vitro culture. Monocytes acquire immature DC characteristics by day 2 of culture with GM-CSF and IL-4; they down-regulate CD14, increase dextran uptake, and respond to the inflammatory chemokine macrophage inflammatory protein-1alpha. To accelerate DC development and maturation, monocytes were incubated for 24 h with GM-CSF and IL-4, followed by activation with proinflammatory mediators for another 24 h (FastDC). FastDC expressed mature DC surface markers as well as chemokine receptor 7 and secreted IL-12 (p70) upon CD40 ligation in the presence of IFN-gamma. The increase in intracellular calcium in response to 6Ckine showed that chemokine receptor 7 expression was functional. When FastDC were compared with mature monocyte-derived DCs generated by a standard 7-day protocol, they were equally potent in inducing Ag-specific T cell proliferation and IFN-gamma production as well as in priming autologous naive T cells using tetanus toxoid as a model Ag. These findings indicate that FastDC are as effective as monocyte-derived DCs in stimulating primary, Ag-specific, Th 1-type immune responses. Generation of FastDC not only reduces labor, cost, and time required for in vitro DC development, but may also represent a model more closely resembling DC differentiation from monocytes in vivo.

  12. Studies on the physiological function of spermine in the process of progesterone induced toad oocyte maturation

    Institute of Scientific and Technical Information of China (English)

    LIRUNSHENG; JIAKETSO

    1992-01-01

    Spermidine or spermine but not putrescine inhibited progesterone induced Bufo bufo gargarizans oocyte maturation.The ID50 for spermine inhibition via intra -oocyte microinjection on maturation induced by progesterone was 6.8mM(100nl).Spermine could inhibit MPF induced toad oocyte maturation with a much higher ID50.A 55 kD protein was dephosphorylated during the process of progesterone induced oocyte maturation .Spermine selectively promoted the level of phosphorylation of this protein in both progesterone-stimulated and hormone-untreated oocytes.The extent of its dephosphorylation was fairly Correlated with the percentage of GVBD in the hormone stimulated oocytes.The level of endogenous spermine was reduced by 28% between the perod of 0.40 GVBD50 and 0.60 GVBD50,at which 55 kD protein was dephosphorylated.Spermine inhibited progesterone-stimulated protein synthesis in almost the same dose dependent manner as its inhititory effect on the hormone-induced maturation,The endogenous spermine regulated 55 kD protein dephosphorylation which may trigger the increase of protein dephosphorylation which may trigger the increase of protein synthesis and in turn promote the activation of MPF,It is possible that 55 kD protein may be one of the components of messenger ribonucleoprotein(mRNP) particles.

  13. Induced Maturation and Spawning by Sex Pheromones in Female Mudskipper(Boleophthalmus pectinirostris)

    Institute of Scientific and Technical Information of China (English)

    Zhao Weihong; Hong Wanshu; Zhang Qiyong; Jiang Xinxin; Wu Dingxun

    2003-01-01

    Maturation of the 4th phase oocytes of mudskipper is induced in vitro by sex pheromones, the extract of ovary, testis and seminal vesicle, oxytocini and deoxycorticosterone ( DOC ). Maturation rates ofoocytes are related to pheromones and their dosages. The single use of prostaglandin E1(PGE 1) is not effective in the induction of maturation, but the combination of PGE1 with HCG promotes oocyte maturation rates.Maturation is induced by injections of the extract of the ovary at dosages of 0.01 cm3/a fish,seminal vesicle extract at dosages of 0.1 cm3/a fish, testis extract at dosage of 0.1 cm3/a fish, or PGE1 at dosage of 10μ g/fish, respectively. Among them, seminal vesicle extract is the most effective in the induction of maturation. The artificial nests, with the extract of the ovary,testis or seminal vesicle inside, attract more heterosexual mudskippers than homosexual mudskippers to enter. Especially, the extract of seminal vesicle is the most effective in attracting females. Female mudskippers could be induced to spawn by the extracts of ovary,testis and seminal vesicle placed inside both the ceramic and sponge nests. More spawned eggs and higher fertilization rates are observed in the ceramic nests than in the sponge ones.

  14. Human CD8 T cells generated in vitro from hematopoietic stem cells are functionally mature

    Directory of Open Access Journals (Sweden)

    Zúñiga-Pflücker Juan

    2011-03-01

    Full Text Available Abstract Background T cell development occurs within the highly specialized thymus. Cytotoxic CD8 T cells are critical in adaptive immunity by targeting virally infected or tumor cells. In this study, we addressed whether functional CD8 T cells can be generated fully in vitro using human umbilical cord blood (UCB hematopoietic stem cells (HSCs in coculture with OP9-DL1 cells. Results HSC/OP9-DL1 cocultures supported the differentiation of CD8 T cells, which were TCR/CD3hi CD27hi CD1aneg and thus phenotypically resembled mature functional CD8 single positive thymocytes. These in vitro-generated T cells also appeared to be conventional CD8 cells, as they expressed high levels of Eomes and low levels of Plzf, albeit not identical to ex vivo UCB CD8 T cells. Consistent with the phenotypic and molecular characterization, upon TCR-stimulation, in vitro-generated CD8 T cells proliferated, expressed activation markers (MHC-II, CD25, CD38, secreted IFN-γ and expressed Granzyme B, a cytotoxic T-cell effector molecule. Conclusion Taken together, the ability to direct human hematopoietic stem cell or T-progenitor cells towards a mature functional phenotype raises the possibility of establishing cell-based treatments for T-immunodeficiencies by rapidly restoring CD8 effector function, thereby mitigating the risks associated with opportunistic infections.

  15. Rab3D is critical for secretory granule maturation in PC12 cells.

    Directory of Open Access Journals (Sweden)

    Tanja Kögel

    Full Text Available Neuropeptide- and hormone-containing secretory granules (SGs are synthesized at the trans-Golgi network (TGN as immature secretory granules (ISGs and complete their maturation in the F-actin-rich cell cortex. This maturation process is characterized by acidification-dependent processing of cargo proteins, condensation of the SG matrix and removal of membrane and proteins not destined to mature secretory granules (MSGs. Here we addressed a potential role of Rab3 isoforms in these maturation steps by expressing their nucleotide-binding deficient mutants in PC12 cells. Our data show that the presence of Rab3D(N135I decreases the restriction of maturing SGs to the F-actin-rich cell cortex, blocks the removal of the endoprotease furin from SGs and impedes the processing of the luminal SG protein secretogranin II. This strongly suggests that Rab3D is implicated in the subcellular localization and maturation of ISGs.

  16. Antigen-specific IL-23/17 pathway activation by murine semi-mature DC-like cells

    Energy Technology Data Exchange (ETDEWEB)

    Nagasaka, Shinya; Iwasaki, Takumi; Okano, Tomoko [Department of Biological Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510 (Japan); Chiba, Joe, E-mail: chibaj@rs.noda.tus.ac.jp [Department of Biological Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510 (Japan)

    2009-09-11

    We analyzed the phenotype and function of bone marrow-derived dendritic cells (DCs) induced in vitro without using any serum during the late stage of cultivation. These 'serum-free' DCs (SF-DCs) possessed the ability to induce T cell proliferation as well as antibody responses, indicating that they were functional DCs. Surprisingly, the SF-DCs akin to semi-mature DCs in terms of both phenotypic and functional characteristics. The SF-DCs did not produce IL-12 but produced large amounts of IL-23 following lipopolysaccharide stimulation. The antigen-specific production of IL-17 by CD4{sup +} T cells co-cultured with OVA-loaded SF-DCs was significantly higher than that with OVA-loaded conventional DCs. These results suggest that SF-DCs tend to produce IL-23 and can consequently induce the IL-17 producing CD4{sup +} T cells. The semi-mature DC-like cells reported here will be useful vehicles for DC immunization and might contribute to studies on the possible involvement of semi-mature DCs in Th17 cell differentiation.

  17. The methodological approach for the generation of human dendritic cells from monocytes affects the maturation state of the resultant dendritic cells.

    Science.gov (United States)

    Mucci, Ilaria; Legitimo, Annalisa; Compagnino, Marta; Consolini, Rita; Migliaccio, Pasquale; Metelli, Maria Rita; Scatena, Fabrizio

    2009-10-01

    Dendritic cells (DCs) are effective as antigen-presenting cells in the immune system and are present at two functional stages depending on their maturation state. For experimental investigation of this concept, CD14(+) monocytes from blood are isolated and cultured to generate in vitro the DCs needed for functional analysis. For positive selection of CD14(+) monocytes we compared two immunomagnetic bead technologies: MACS Separation, created by Miltenyi Biotec, and EasySep Selection, created by StemCell Technologies. The monocytes provided dendritic cells for their functional analysis. Lipopolysaccharide was added to cultured DCs to induce maturation. Although both systems generated DCs from the positively selected CD14(+) cells, there were certain differences between them. Morphological, phenotypic, and functional analysis showed that MACS-selection provided DCs that have typical features corresponding to day 6 or 7 of maturation. EasySep-DCs exist in a partially-mature state from day 6 onward, even without the addition of a maturation stimulus. The reason behind this partial maturation is possibly based on the dextran-coated beads that are associated with the EasySep product. Both methods provide pure and viable DCs, but we would recommend using the MACS system for obtaining DCs suitable for functional studies.

  18. Glycyrrhiza uralensis water extract enhances dendritic cell maturation and antitumor efficacy of HPV dendritic cell-based vaccine

    Science.gov (United States)

    Aipire, Adila; Li, Jinyu; Yuan, Pengfei; He, Jiang; Hu, Yelang; Liu, Lu; Feng, Xiaoli; Li, Yijie; Zhang, Fuchun; Yang, Jianhua; Li, Jinyao

    2017-01-01

    Licorice has been used as herbal medicine and natural sweetener. Here, we prepared Glycyrrhiza uralensis water extract (GUWE) and investigated the effect of GUWE on the maturation and function of dendritic cells (DCs) and its adjuvant effect on DC-based vaccine. We observed that GUWE dose-dependently promoted DC maturation and cytokine secretion through TLR4 signaling pathway. The capacity of DC to stimulate allogenic splenocyte proliferation was also enhanced by GUWE treatment. Compared with control group, GUWE treated DCs pulsed with human papillomavirus (HPV)-16 E6/E7 peptides significantly inhibited the tumor growth in both early and late therapeutic groups. In early therapeutic group, the frequencies of induced regulatory T cells (iTregs: CD4+CD25−Fopx3+) and CD4+ and CD8+ T cells were significantly decreased and increased, respectively. HPV-16-specific CD8+ T cell responses were significantly induced and negatively correlated with iTreg frequencies and tumor weight. These results indicated the immunoregulatory activities of licorice. PMID:28272545

  19. MUC5B silencing reduces chemo-resistance of MCF-7 breast tumor cells and impairs maturation of dendritic cells.

    Science.gov (United States)

    García, Enrique P; Tiscornia, Inés; Libisch, Gabriela; Trajtenberg, Felipe; Bollati-Fogolín, Mariela; Rodríguez, Ernesto; Noya, Verónica; Chiale, Carolina; Brossard, Natalie; Robello, Carlos; Santiñaque, Federico; Folle, Gustavo; Osinaga, Eduardo; Freire, Teresa

    2016-05-01

    Mucins participate in cancer progression by regulating cell growth, adhesion, signaling, apoptosis or chemo-resistance to drugs. The secreted mucin MUC5B, the major component of the respiratory tract mucus, is aberrantly expressed in breast cancer, where it could constitute a cancer biomarker. In this study we evaluated the role of MUC5B in breast cancer by gene silencing the MUC5B expression with short hairpin RNA on MCF-7 cells. We found that MUC5B-silenced MCF-7 cells have a reduced capacity to grow, adhere and form cell colonies. Interestingly, MUC5B knock-down increased the sensitivity to death induced by chemotherapeutic drugs. We also show that MUC5B silencing impaired LPS-maturation of DCs, and production of cytokines. Furthermore, MUC5B knock-down also influenced DC-differentiation and activation since it resulted in an upregulation of IL-1β, IL-6 and IL-10, cytokines that might be involved in cancer progression. Thus, MUC5B could enhance the production of LPS-induced cytokines, suggesting that the use of MUC5B-based cancer vaccines combined with DC-maturation stimuli, could favor the induction of an antitumor immune response.

  20. Growth dynamics and cytoskeleton organization during stem maturation and gravity-induced stem bending in Zea mays L

    Science.gov (United States)

    Collings, D. A.; Winter, H.; Wyatt, S. E.; Allen, N. S.; Davies, E. (Principal Investigator)

    1998-01-01

    Characterization of gravitropic bending in the maize stem pulvinus, a tissue that functions specifically in gravity responses, demonstrates that the pulvinus is an ideal system for studying gravitropism. Gravistimulation during the second of three developmental phases of the pulvinus induces a gradient of cell elongation across the non-growing cells of the pulvinus, with the most elongation occurring on the lower side. This cell elongation is spatially and temporally separated from normal internodal cell elongation. The three characterized growth phases in the pulvinus correspond closely to a specialized developmental sequence in which structural features typical of cells not fully matured are retained while cell maturation occurs in surrounding internodal and nodal tissue. For example, the lignification of supporting tissue and rearrangement of transverse microtubules to oblique that occur in the internode when cell elongation ceases are delayed for up to 10 d in the adjacent cells of the pulvinus, and only occurs as a pulvinus loses its capacity to respond to gravistimulation. Gravistimulation does not modify this developmental sequence. Neither wall lignification nor rearrangement of transverse microtubules occurs in the rapidly elongating lower side or non-responsive upper side of the pulvinus until the pulvinus loses the capacity to bend further. Gravistimulation does, however, lead to the formation of putative pit fields within the expanding cells of the pulvinus.

  1. On/off TLR segnaling decides immunogenic or tolerogenic dendritic cell maturation upon NKT cell contact

    OpenAIRE

    Caielli,

    2009-01-01

    Invariant Natural Killer (iNK)T cells play opposite immune functions. They participate in the innate immune response to promote anti-microbial and anti-tumor immunity and they are crucial to maintain T cell tolerance and prevent autoimmune diseases. While it is well known that the adjuvant function of iNKT cells is mediated through maturation of dendritic cells (DC), the mechanism underlying the tolerogenic function of iNKT cells remains unclear. We performed co-culture experiments with immat...

  2. The Specification and Maturation of Nociceptive Neurons from Human Embryonic Stem Cells.

    Science.gov (United States)

    Boisvert, Erin M; Engle, Sandra J; Hallowell, Shawn E; Liu, Ping; Wang, Zhao-Wen; Li, Xue-Jun

    2015-11-19

    Nociceptive neurons play an essential role in pain sensation by transmitting painful stimuli to the central nervous system. However, investigations of nociceptive neuron biology have been hampered by the lack of accessibility of human nociceptive neurons. Here, we describe a system for efficiently guiding human embryonic stem cells into nociceptive neurons by first inducing these cells to the neural lineage. Subsequent addition of retinoic acid and BMP4 at specific time points and concentrations yielded a high population of neural crest progenitor cells (AP2α(+), P75(+)), which further differentiated into nociceptive neurons (TRKA(+), Nav1.7(+), P2X3(+)). The overexpression of Neurogenin 1 (Neurog1) promoted the neurons to express genes related to sensory neurons (Peripherin, TrkA) and to further mature into TRPV1(+) nociceptive neurons. Importantly, the overexpression of Neurog1 increased the response of these neurons to capsaicin stimulation, a hallmark of mature functional nociceptive neurons. Taken together, this study reveals the important role that Neurog1 plays in generating functional human nociceptive neurons.

  3. Size-dependent effects of gold nanoparticles uptake on maturation and antitumor functions of human dendritic cells in vitro.

    Science.gov (United States)

    Tomić, Sergej; Ðokić, Jelena; Vasilijić, Saša; Ogrinc, Nina; Rudolf, Rebeka; Pelicon, Primož; Vučević, Dragana; Milosavljević, Petar; Janković, Srđa; Anžel, Ivan; Rajković, Jelena; Rupnik, Marjan Slak; Friedrich, Bernd; Colić, Miodrag

    2014-01-01

    Gold nanoparticles (GNPs) are claimed as outstanding biomedical tools for cancer diagnostics and photo-thermal therapy, but without enough evidence on their potentially adverse immunological effects. Using a model of human dendritic cells (DCs), we showed that 10 nm- and 50 nm-sized GNPs (GNP10 and GNP50, respectively) were internalized predominantly via dynamin-dependent mechanisms, and they both impaired LPS-induced maturation and allostimulatory capacity of DCs, although the effect of GNP10 was more prominent. However, GNP10 inhibited LPS-induced production of IL-12p70 by DCs, and potentiated their Th2 polarization capacity, while GNP50 promoted Th17 polarization. Such effects of GNP10 correlated with a stronger inhibition of LPS-induced changes in Ca2+ oscillations, their higher number per DC, and more frequent extra-endosomal localization, as judged by live-cell imaging, proton, and electron microscopy, respectively. Even when released from heat-killed necrotic HEp-2 cells, GNP10 inhibited the necrotic tumor cell-induced maturation and functions of DCs, potentiated their Th2/Th17 polarization capacity, and thus, impaired the DCs' capacity to induce T cell-mediated anti-tumor cytotoxicity in vitro. Therefore, GNP10 could potentially induce more adverse DC-mediated immunological effects, compared to GNP50.

  4. The glycoprotein-hormones activin A and inhibin A interfere with dendritic cell maturation

    Directory of Open Access Journals (Sweden)

    Reichardt Holger M

    2008-05-01

    Full Text Available Abstract Background Pregnancy represents an exclusive situation in which the immune and the endocrine system cooperate to prevent rejection of the embryo by the maternal immune system. While immature dendritic cells (iDC in the early pregnancy decidua presumably contribute to the establishment of peripheral tolerance, glycoprotein-hormones of the transforming growth factor beta (TGF-beta family including activin A (ActA and inhibin A (InA are candidates that could direct the differentiation of DCs into a tolerance-inducing phenotype. Methods To test this hypothesis we generated iDCs from peripheral-blood-monocytes and exposed them to TGF-beta1, ActA, as well as InA and Dexamethasone (Dex as controls. Results Both glycoprotein-hormones prevented up-regulation of HLA-DR during cytokine-induced DC maturation similar to Dex but did not influence the expression of CD 40, CD 83 and CD 86. Visualization of the F-actin cytoskeleton confirmed that the DCs retained a partially immature phenotype under these conditions. The T-cell stimulatory capacity of DCs was reduced after ActA and InA exposure while the secretion of cytokines and chemokines was unaffected. Conclusion These findings suggest that ActA and InA interfere with selected aspects of DC maturation and may thereby help preventing activation of allogenic T-cells by the embryo. Thus, we have identified two novel members of the TGF-beta superfamily that could promote the generation of tolerance-inducing DCs.

  5. Carbon black nanoparticles promote the maturation and function of mouse bone marrow-derived dendritic cells.

    Science.gov (United States)

    Koike, Eiko; Takano, Hirohisa; Inoue, Ken-Ichiro; Yanagisawa, Rie; Kobayashi, Takahiro

    2008-09-01

    Particulate matter including carbon black (CB) nanoparticles can enhance antigen-related inflammation and immunoglobulin production in vivo. Dendritic cells (DC) as antigen-presenting cells (APC) are the most capable inducers of immune responses. The present study was designed to determine whether CB nanoparticles affect the maturation/activation and function of DC in vitro. DC were differentiated from bone marrow (BM) cells of BALB/c mice by culture with granulocyte macrophage colony stimulating factor (GM-CSF). At day 8 of culture, BM-derived DC (BMDC) were exposed to CB nanoparticles with a diameter of 14nm or 56nm for 24h. The expression of major histocompatibility complex (MHC) class II, DEC205, CD80, and CD86 (maturation/activation markers of BMDC) was measured by flow cytometry. BMDC function was evaluated by an allogeneic mixed lymphocyte reaction (MLR) assay. CB nanoparticles significantly increased the expression of DEC205 and CD86 in BMDC and tended to increase MHC class II and CD80 expression; however, a size-dependent effect was not observed. On the other hand, BMDC-mediated MLR was significantly enhanced by the CB nanoparticles and the enhancement was greater by 14nm CB nanoparticles than by 56nm CB nanoparticles. Taken together, CB nanoparticles can promote the maturation/activation and function of BMDC, which could be related to their effects on allergic diseases and/or responses. In addition, BMDC-mediated MLR might be useful assay for in vitro screening for adjuvant activity of environmental toxicants.

  6. Characterization of monocyte-derived dendritic cells maturated with IFN-alpha

    DEFF Research Database (Denmark)

    Svane, I M; Nikolajsen, K; Walter, M R;

    2006-01-01

    Dendritic cells (DC) are promising candidates for cancer immunotherapy. These cells can be generated from peripheral blood monocytes cultured with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). In order to obtain full functional capacity, maturation is required......, maturation with IFN-alpha has only a small effect on induction of autologous T-cell stimulatory capacity of the DC. However, an increase in DC allogeneic T-cell stimulatory capacity was observed. These data suggest that IFN-alpha has a potential as a maturation agent used in DC-based cancer vaccine trials...

  7. Sertoli cell only syndrome: Status of sertoli cell maturation and function

    Directory of Open Access Journals (Sweden)

    Manish Jain

    2012-01-01

    Full Text Available Background of the study: Mature and functional Sertoli cells are essential for the survival of germ cells in testes. In Sertoli cell only syndrome (SCOS, there is no germ cells. Then, question arises whether absence of germ cells in SCOS secondary to Sertoli cells immaturity or mal function. Sertoli cells maturational and functional status is unclear in SCOS. This study investigated status of maturation and function of Sertoli cells in patients with SCOS. Materials and Methods: The present study was comprised of 37 cases of SCOS and 50 normal control males. Detailed clinical examination and investigation were carried out as per pre-determined proforma. Semen analysis, hormonal analysis (FSH, LH, testosterone, etc., and fine needle aspiration cytology (FNAC of testes (bilateral were performed. Fluorescence in situ hybridization (FISH with XY probes was carried out in addition to conventional chromosome analysis to find out chromosomal abnormalities, in particular sex chromosome aneuploidy, including mosaicism. Yq microdeletion status was also investigated. The anti-mullerian hormone (AMH, inhibin B, and seminal lactate were estimated by ELISA methods. Results: The study did not find any case of high AMH. About 78% cases had low inhibin B, and 60% had low AMH. FSH was high in about 78% cases. Low level of lactate was found in 49% cases. There was one case of high level of inhibin B. There were 6 (16.2% cases of chromosomal abnormality (2 mosaic Klinefelter and 4 Klinefelter syndrome and 4 (10.8% cases of Yq microdeletion. Conclusion: We conclude that Sertoli cell immaturity does not play any role in SCOS (no case of high AMH. It seems, in majority cases, Sertoli cells are functionally- and/or numerically-deficient (low inhibin B, AMH and lactate. However, in about 22% cases, Sertoli cell function and/or number remains normal (normal inhibin B, AMH. Inhibin B and FSH seems best predictor/marker of Sertoli cell function.

  8. Direct Induction and Functional Maturation of Forebrain GABAergic Neurons from Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Alfred Xuyang Sun

    2016-08-01

    Full Text Available Gamma-aminobutyric acid (GABA-releasing interneurons play an important modulatory role in the cortex and have been implicated in multiple neurological disorders. Patient-derived interneurons could provide a foundation for studying the pathogenesis of these diseases as well as for identifying potential therapeutic targets. Here, we identified a set of genetic factors that could robustly induce human pluripotent stem cells (hPSCs into GABAergic neurons (iGNs with high efficiency. We demonstrated that the human iGNs express neurochemical markers and exhibit mature electrophysiological properties within 6–8 weeks. Furthermore, in vitro, iGNs could form functional synapses with other iGNs or with human-induced glutamatergic neurons (iENs. Upon transplantation into immunodeficient mice, human iGNs underwent synaptic maturation and integration into host neural circuits. Taken together, our rapid and highly efficient single-step protocol to generate iGNs may be useful to both mechanistic and translational studies of human interneurons.

  9. Yttrium Y 90 Basiliximab and Combination Chemotherapy Before Stem Cell Transplant in Treating Patients With Mature T-cell Non-Hodgkin Lymphoma

    Science.gov (United States)

    2016-10-11

    Mature T-Cell and NK-Cell Non-Hodgkin Lymphoma; Recurrent Mature T- and NK-Cell Non-Hodgkin Lymphoma; Refractory Mature T-Cell and NK-Cell Non-Hodgkin Lymphoma; Recurrent Cutaneous T-Cell Non-Hodgkin Lymphoma; Refractory Cutaneous T-Cell Non-Hodgkin Lymphoma

  10. Enhancer mutations of Akv murine leukemia virus inhibit the induction of mature B-cell lymphomas and shift disease specificity towards the more differentiated plasma cell stage

    DEFF Research Database (Denmark)

    Sørensen, Karina Dalsgaard; Kunder, Sandra; Quintanilla-Martinez, Leticia;

    2007-01-01

    This study investigates the role of the proviral transcriptional enhancer for B-lymphoma induction by exogenous Akv murine leukemia virus. Infection of newborn inbred NMRI mice with Akv induced 35% plasma cell proliferations (PCPs) (consistent with plasmacytoma), 33% diffuse large B-cell lymphomas...... showed that many of the tumors/cell proliferations induced by each virus were polyclonal. Our results indicate that enhancer mutations weaken the ability of Akv to induce mature B-cell lymphomas prior to the plasma cell stage, whereas development of plasma cell proliferations is less dependent of viral......, 25% follicular B-cell lymphomas and few splenic marginal zone and small B-cell lymphomas. Deleting one copy of the 99-bp proviral enhancer sequence still allowed induction of multiple B-cell tumor types, although PCPs dominated (77%). Additional mutation of binding sites for the glucocorticoid...

  11. Simian virus 40 inhibits differentiation and maturation of rhesus macaque DC-SIGN+-dendritic cells

    Directory of Open Access Journals (Sweden)

    Changyong G

    2010-09-01

    Full Text Available Abstract Dendritic cells (DC are the initiators and modulators of the immune responses. Some species of pathogenic microorganisms have developed immune evasion strategies by controlling antigen presentation function of DC. Simian virus 40 (SV40 is a DNA tumor virus of rhesus monkey origin. It can induce cell transformation and tumorigenesis in many vertebrate species, but often causes no visible effects and persists as a latent infection in rhesus monkeys under natural conditions. To investigate the interaction between SV40 and rhesus monkey DC, rhesus monkey peripheral blood monocyte-derived DC were induced using recombinant human Interleukin-4 (rhIL-4 and infective SV40, the phenotype and function of DC-specific intracellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN+ DC were analyzed by flow cytometry (FCM and mixed lymphocyte reaction (MLR. Results showed that SV40 can down-regulate the expression of CD83 and CD86 on DC and impair DC-induced activation of T cell proliferation. These findings suggest that SV40 might also cause immune suppression by influencing differentiation and maturation of DC.

  12. Soluble forms of VEGF receptor-1 and -2 promote vascular maturation via mural cell recruitment.

    Science.gov (United States)

    Lorquet, Sophie; Berndt, Sarah; Blacher, Silvia; Gengoux, Emily; Peulen, Olivier; Maquoi, Erik; Noël, Agnès; Foidart, Jean-Michel; Munaut, Carine; Péqueux, Christel

    2010-10-01

    Two soluble forms of vascular endothelial growth factor (VEGF) receptors, sVEGFR-1 and sVEGFR-2, are physiologically released and overproduced in some pathologies. They are known to act as anti-VEGF agents. Here we report that these soluble receptors contribute to vessel maturation by mediating a dialogue between endothelial cells (ECs) and mural cells that leads to blood vessel stabilization. Through a multidisciplinary approach, we provide evidence that these soluble VEGF receptors promote mural cell migration through a paracrine mechanism involving interplay in ECs between VEGF/VEGFR-2 and sphingosine-1-phosphate type-1 (S1P)/S1P1 pathways that leads to endothelial nitric oxyde synthase (eNOS) activation. This new paradigm is supported by the finding that sVEGFR-1 and -2 perform the following actions: 1) induce an eNOS-dependent outgrowth of a mural cell network in an ex vivo model of angiogenesis, 2) increase the mural cell coverage of neovessels in vitro and in vivo, 3) promote mural cell migration toward ECs, and 4) stimulate endothelial S1P1 overproduction and eNOS activation that promote the migration and the recruitment of neighboring mural cells. These findings provide new insights into mechanisms regulating physiological and pathological angiogenesis and vessel stabilization.

  13. Transcriptional determinants of tolerogenic and immunogenic states during dendritic cell maturation

    Science.gov (United States)

    Vander Lugt, Bryan; Riddell, Jeremy; Khan, Aly A.; Lesch, Justin; DeVoss, Jason; Weirauch, Matthew T.

    2017-01-01

    Dendritic cells (DCs) promote either tolerogenic or immunogenic T cell responses, the latter upon sensing microbes. Using an in vitro system, we analyzed transcriptional determinants that enable mature DCs to direct these opposing T cell outcomes. In the absence of microbial products, the transcription factor interferon regulatory factor 4 (IRF4) promotes regulatory T cell (Treg) generation by enhancing expression of genes required for antigen presentation along with those for T cell tolerance. IRF4-deficient DCs were impaired for Treg generation in vivo. When exposed to microbial stimuli, DCs activated nuclear factor (NF)-κB, which induced expression of a proinflammatory cytokine module that, along with the antigen presentation module, promoted the generation of effector T cells. NF-κB was, however, dispensable for Treg development. Chromatin profiling revealed transcriptional motifs associated with the divergent DC programs. Thus, DCs modulate their ability to prime tolerogenic or immunogenic T cells by expressing a core antigen presentation module that is overlaid by distinctive regulatory modules to promote either tolerance or immunity. PMID:28130292

  14. Casein kinase G may be the target of spermine during progesterone-induced oocyte maturation

    Institute of Scientific and Technical Information of China (English)

    LIRUNSHENG; JIAKETSO

    1993-01-01

    Casein kinase G (CKG) with more than 2500-fold enrichraent was purified from Bufo bufo gargarizans ovaries. The catalytic activity of the enzyme was found to be associated with its 42 kD subunit, and its 26 kD subunit was found to be the major tsrget for the enzyme autophos phorylation. Each fuU-grown oocyte contained 1.9 units of CKG corresponding to an intracellular concentration of 93 nM. After injecting an amount of 0,38 units of the enzyme into the oocyte, approximately 50% of the progesterone-induoed maturation was inhibited. The inhibitory effect was enhanced in oocytes pretreated with spermine, which was consistent with the results that the enzyme was activated in vitro in the presence of spermine, The MPF-induced oocyte maturation was delayed and even prohibited in the kinase-microinjected oocytes. A 55 kD oocyte protein was identified as an suhstrate of CKG both in vivo and in vitro, and the enhancement of the 55 kD protein phosphory[ation was associated with kinase inhibition on maturation and on protein synthesis in kinase-microinjected oocytes. As the endogenous spermine level decreased in the course of progesteroneinduced oocyte maturation. 55 kD protein was dephosphorylated, Heparin, a specific inhibitor of CKG, potentiated the progesterone-induced oocyte maturation. Altogether the experimental reSults indicated Strongly that CKG may be the physiological target of spermine.

  15. Salvianolic acid B suppresses maturation of human monocyte-derived dendritic cells by activating PPARγ

    Science.gov (United States)

    Sun, Aijun; Liu, Hongying; Wang, Shijun; Shi, Dazhuo; Xu, Lei; Cheng, Yong; Wang, Keqiang; Chen, Keji; Zou, Yunzeng; Ge, Junbo

    2011-01-01

    BACKGROUND AND PURPOSE Salvianolic acid B (Sal B), a water-soluble antioxidant derived from a Chinese medicinal herb, is known to be effective in the prevention of atherosclerosis. Here, we tested the hypothesis that the anti-atherosclerotic effect of Sal B might be mediated by suppressing maturation of human monocyte-derived dendritic cells (h-monDC). EXPERIMENTAL APPROACH h-monDC were derived by incubating purified human monocytes with GM-CSF and IL-4. h-monDC were pre-incubated with or without Sal B and stimulated by oxidized low-density lipoprotein (ox-LDL) in the presence or absence of PPARγ siRNA. Expression of h-monDC membrane molecules (CD40, CD86, CD1a, HLA-DR) were analysed by FACS, cytokines were measured by elisa and the TLR4-associated signalling pathway was determined by Western blotting. KEY RESULTS Ox-LDL promoted h-monDC maturation, stimulated CD40, CD86, CD1a, HLA-DR expression and IL-12, IL-10, TNF-α production; and up-regulated TLR4 signalling. These effects were inhibited by Sal B. Sal B also triggered PPARγ activation and promoted PPARγ nuclear translocation, attenuated ox-LDL-induced up-regulation of TLR4 and myeloid differentiation primary-response protein 88 and inhibited the downstream p38-MAPK signalling cascade. Knocking down PPARγ with the corresponding siRNA blocked these effects of Sal B. CONCLUSIONS AND IMPLICATIONS Our data suggested that Sal B effectively suppressed maturation of h-monDC induced by ox-LDL through PPARγ activation. PMID:21649636

  16. Inhibition of Progenitor Dendritic Cell Maturation by Plasma from Patients with Peripartum Cardiomyopathy: Role in Pregnancy-associated Heart Disease

    Directory of Open Access Journals (Sweden)

    Jane E. Ellis

    2005-01-01

    Full Text Available Dendritic cells (DCs play dual roles in innate and adaptive immunity based on their functional maturity, and both innate and adaptive immune responses have been implicated in myocardial tissue remodeling associated with cardiomyopathies. Peripartum cardiomyopathy (PPCM is a rare disorder which affects women within one month antepartum to five months postpartum. A high occurrence of PPCM in central Haiti (1 in 300 live births provided the unique opportunity to study the relationship of immune activation and DC maturation to the etiology of this disorder. Plasma samples from two groups (n = 12 of age- and parity-matched Haitian women with or without evidence of PPCM were tested for levels of biomarkers of cardiac tissue remodeling and immune activation. Significantly elevated levels of GM-CSF, endothelin-1, proBNP and CRP and decreased levels of TGF- were measured in PPCM subjects relative to controls. Yet despite these findings, in vitro maturation of normal human cord blood derived progenitor dendritic cells (CBDCs was significantly reduced (p < 0.001 in the presence of plasma from PPCM patients relative to plasma from post-partum control subjects as determined by expression of CD80, CD86, CD83, CCR7, MHC class II and the ability of these matured CBDCs to induce allo-responses in PBMCs. These results represent the first findings linking inhibition of DC maturation to the dysregulation of normal physiologic cardiac tissue remodeling during pregnancy and the pathogenesis of PPCM.

  17. Nitric oxide acts through different signaling pathways in maturation of cumulus cell-enclosed mouse oocytes

    Directory of Open Access Journals (Sweden)

    M Abbasi

    2009-03-01

    Full Text Available ABSTRACT Background: Nitric oxide (NO have a dual action in mouse oocyte meiotic maturation which depends on its concentration, but the mechanisms by which it influences oocyte maturation has not been exactly clarified. In this study different signaling mechanisms which exist for in vitro maturation of meiosis was examined in cumulus cell-enclosed oocytes (CEOs after injection of pregnant mare's serum gonadotropin (PMSG to immature female mice. Methods: The CEOs were cultured in spontaneous maturation and hypoxanthine (HX arrested model. Results: Sodium nitroprusside (SNP, an NO donor, 10mM delayed germinal vesicle breakdown (GVBD significantly during the first 5 hrs of incubation and inhibited the formation of first polar body (PB1 at the end of 24 hrs of incubation. SNP (10-5M stimulated the meiotic maturation of oocytes significantly by overcoming the inhibition of HX. Sildenafil (a cGMP stimulator, 100 nM, had a significant inhibitory effects on both spontaneous meiotic maturation and HX-arrested meiotic maturation. Forskolin (an adenylate cyclase stimulator, 6µM and SNP (10mM had the same effects on GVBD. Forskolin reversed the SNP (10-5M stimulated meiotic maturation. Conclusion: These results suggest that differences in pathways are present between SNP-inhibited spontaneous meiotic maturation and SNP-stimulated meiotic maturation in mouse oocytes

  18. Status epilepticus increases mature granule cells in the molecular layer of the dentate gyrus in rats

    Institute of Scientific and Technical Information of China (English)

    Zhaoliang Liang; Fei Gao; Fajun Wang; Xiaochen Wang; Xinyu Song; Kejing Liu; Ren-Zhi Zhan

    2013-01-01

    Enhanced neurogenesis in the dentate gyrus of the hippocampus following seizure activity, especially status epilepticus, is associated with ectopic residence and aberrant integration of newborn granule cells. Hilar ectopic granule cells may be detrimental to the stability of dentate circuitry by means of their electrophysiological properties and synaptic connectivity. We hypothesized that status epilepticus also increases ectopic granule cells in the molecular layer. Status epilepticus was induced in male Sprague-Dawley rats by intraperitoneal injection of pilocarpine. Immunostaining showed that many doublecortin-positive cells were present in the molecular layer and the hilus 7 days after the induction of status epilepticus. At least 10 weeks after status epilepticus, the estimated number of cells positive for both prospero homeobox protein 1 and neuron-specific nuclear protein in the hilus was significantly increased. A similar trend was also found in the molecular layer. These findings indicate that status epilepticus can increase the numbers of mature and ectopic newborn granule cells in the molecular layer.

  19. The Hematopoietic Differentiation and Production of Mature Myeloid Cells from Human Pluripotent Stem Cells

    OpenAIRE

    Choi, Kyung-Dal; Vodyanik, Maxim; Slukvin, Igor I.

    2011-01-01

    Here we describe a protocol for hematopoietic differentiation of human pluripotent stem cells (hPSCs) and generation of mature myeloid cells from hPSCs through expansion and differentiation of hPSC-derived lin-CD34+CD43+CD45+ multipotent progenitors. The protocol is comprised of three major steps: (i) induction of hematopoietic differentiation by coculture of hPSCs with OP9 bone marrow stromal cells, (ii) short-term expansion of multipotent myeloid progenitors with a high dose of GM-CSF, and ...

  20. IFN-alpha promotes definitive maturation of dendritic cells generated by short-term culture of monocytes with GM-CSF and IL-4.

    Science.gov (United States)

    Dauer, Marc; Schad, Katharina; Junkmann, Jana; Bauer, Christian; Herten, Jan; Kiefl, Rosemarie; Schnurr, Max; Endres, Stefan; Eigler, Andreas

    2006-08-01

    Dendritic cells (DC) generated in vitro have to be viable and phenotypically mature to be capable of inducing T cell-mediated immunity after in vivo administration. To facilitate optimization of DC-based vaccination protocols, we investigated whether the cytokine environment and the mode of activation affect maturation and survival of DC derived from monocytes by a short-term protocol. Monocytes cultured for 24 h with granulocyte macrophage-colony stimulating factor and interleukin-4 were stimulated with proinflammatory mediators for another 36 h to generate mature DC. Additional activation with CD40 ligand and interferon (IFN)-gamma increased viability of DC and promoted definitive maturation as defined by maintenance of a mature phenotype after withdrawal of cytokines. Addition of IFN-alpha to DC cultures prior to stimulation further enhanced definitive maturation: IFN-alpha-primed DC expressed high levels of costimulatory molecules and CC chemokine receptor 7 (CCR7) up to 5 days after cytokine withdrawal. Compared with unprimed DC, IFN-alpha-primed DC displayed equal capacity to migrate upon CCR7 ligation and to prime antigen-specific T helper cell as well as cytolytic T cell responses. In conclusion, we show that optimal maturation and survival of monocyte-derived DC require multiple activation signals. Furthermore, we identified a novel role for IFN-alpha in DC development: IFN-alpha priming of monocytes promotes definitive maturation of DC upon activation.

  1. ROS-induced autophagy in cancer cells assists in evasion from determinants of immunogenic cell death

    NARCIS (Netherlands)

    Garg, A.D.; Dudek, A.M.D.; Ferreira, G.B.; Verfaillie, T.; Vandenabeele, P.; Krysko, D.V.; Mathieu, C.; Agostinis, P.

    2013-01-01

    Calreticulin surface exposure (ecto-CALR), ATP secretion, maturation of dendritic cells (DCs) and stimulation of T cells are prerequisites for anticancer therapy-induced immunogenic cell death (ICD). Recent evidence suggests that chemotherapy-induced autophagy may positively regulate ICD by favoring

  2. Human cytomegalovirus alters localization of MHC class II and dendrite morphology in mature Langerhans cells.

    Science.gov (United States)

    Lee, Andrew W; Hertel, Laura; Louie, Ryan K; Burster, Timo; Lacaille, Vashti; Pashine, Achal; Abate, Davide A; Mocarski, Edward S; Mellins, Elizabeth D

    2006-09-15

    Hemopoietic stem cell-derived mature Langerhans-type dendritic cells (LC) are susceptible to productive infection by human CMV (HCMV). To investigate the impact of infection on this cell type, we examined HLA-DR biosynthesis and trafficking in mature LC cultures exposed to HCMV. We found decreased surface HLA-DR levels in viral Ag-positive as well as in Ag-negative mature LC. Inhibition of HLA-DR was independent of expression of unique short US2-US11 region gene products by HCMV. Indeed, exposure to UV-inactivated virus, but not to conditioned medium from infected cells, was sufficient to reduce HLA-DR on mature LC, implicating particle binding/penetration in this effect. Reduced surface levels reflected an altered distribution of HLA-DR because total cellular HLA-DR was not diminished. Accumulation of HLA-DR was not explained by altered cathepsin S activity. Mature, peptide-loaded HLA-DR molecules were retained within cells, as assessed by the proportion of SDS-stable HLA-DR dimers. A block in egress was implicated, as endocytosis of surface HLA-DR was not increased. Immunofluorescence microscopy corroborated the intracellular retention of HLA-DR and revealed markedly fewer HLA-DR-positive dendritic projections in infected mature LC. Unexpectedly, light microscopic analyses showed a dramatic loss of the dendrites themselves and immunofluorescence revealed that cytoskeletal elements crucial for the formation and maintenance of dendrites are disrupted in viral Ag-positive cells. Consistent with these dendrite effects, HCMV-infected mature LC exhibit markedly reduced chemotaxis in response to lymphoid chemokines. Thus, HCMV impedes MHC class II molecule trafficking, dendritic projections, and migration of mature LC. These changes likely contribute to the reduced activation of CD4+ T cells by HCMV-infected mature LC.

  3. Subtoxic concentrations of allergenic haptens induce LC migration and maturation in a human organotypic skin explant culture model: A novel method for identifying potential contact allergens

    NARCIS (Netherlands)

    Lehé, C.L.; Jacobs, J.J.L.; Hua, C.M.; Courtellemont, P.; Elliott, G.R.; Das, P.K.

    2006-01-01

    The accelerated migration of Langerhans cells (LCs) out of the epidermis and up-regulation of maturation markers, upon treatment with subtoxic concentrations of chemicals, were used as the criteria to determine the potential of allergenic chemicals capable of inducing a hapten-specific delayed-type

  4. Dendritic cell maturation results in pronounced changes in glycan expression affecting recognition by siglecs and galectins

    NARCIS (Netherlands)

    Bax, Marieke; Garcia-Vallejo, Juan J.; Jang-Lee, Jihye; North, Simon J.; Gilmartin, Tim J.; Hernandez, Gilberto; Crocker, Paul R.; Leffler, Hakon; Head, Steven R.; Haslam, Stuart M.; Dell, Anne; van Kooyk, Yvette

    2007-01-01

    Dendritic cells (DC) are the most potent APC in the organism. Immature dendritic cells (iDC) reside in the tissue where they capture pathogens whereas mature dendritic cells (mDC) are able to activate T cells in the lymph node. This dramatic functional change is mediated by an important genetic repr

  5. Molecular signatures of maturing dendritic cells: implications for testing the quality of dendritic cell therapies

    Directory of Open Access Journals (Sweden)

    Wang Ena

    2010-01-01

    Full Text Available Abstract Background Dendritic cells (DCs are often produced by granulocyte-macrophage colony-stimulating factor (GM-CSF and interleukin-4 (IL-4 stimulation of monocytes. To improve the effectiveness of DC adoptive immune cancer therapy, many different agents have been used to mature DCs. We analyzed the kinetics of DC maturation by lipopolysaccharide (LPS and interferon-γ (IFN-γ induction in order to characterize the usefulness of mature DCs (mDCs for immune therapy and to identify biomarkers for assessing the quality of mDCs. Methods Peripheral blood mononuclear cells were collected from 6 healthy subjects by apheresis, monocytes were isolated by elutriation, and immature DCs (iDCs were produced by 3 days of culture with GM-CSF and IL-4. The iDCs were sampled after 4, 8 and 24 hours in culture with LPS and IFN-γ and were then assessed by flow cytometry, ELISA, and global gene and microRNA (miRNA expression analysis. Results After 24 hours of LPS and IFN-γ stimulation, DC surface expression of CD80, CD83, CD86, and HLA Class II antigens were up-regulated. Th1 attractant genes such as CXCL9, CXCL10, CXCL11 and CCL5 were up-regulated during maturation but not Treg attractants such as CCL22 and CXCL12. The expression of classical mDC biomarker genes CD83, CCR7, CCL5, CCL8, SOD2, MT2A, OASL, GBP1 and HES4 were up-regulated throughout maturation while MTIB, MTIE, MTIG, MTIH, GADD45A and LAMP3 were only up-regulated late in maturation. The expression of miR-155 was up-regulated 8-fold in mDCs. Conclusion DCs, matured with LPS and IFN-γ, were characterized by increased levels of Th1 attractants as opposed to Treg attractants and may be particularly effective for adoptive immune cancer therapy.

  6. Antigen receptors on immature, but not mature, B and T cells are coupled to cytosolic phospholipase A2 activation: expression and activation of cytosolic phospholipase A2 correlate with lymphocyte maturation.

    Science.gov (United States)

    Gilbert, J J; Stewart, A; Courtney, C A; Fleming, M C; Reid, P; Jackson, C G; Wise, A; Wakelam, M J; Harnett, M M

    1996-03-15

    The Ag receptors on mature B and T cells are not coupled to the activation of cytosolic phospholipase A2 (cPLA2) and arachidonic acid release. Moreover, phorbol esters such as PMA, which can activate cPLA2 via mitogen-activated protein (MAP) kinase in most cell types, also failed to induce the release of arachidonate from mature cells, suggesting that the cPLA2 pathway may not be functional in mature lymphocytes. Interestingly, Western blot analysis revealed that cPLA2, which had previously been thought to be expressed ubiquitously, is not expressed in mature B or T cells and that cytosolic phospholipase A2 expression could not be up-regulated in lymphocytes following culture with a range of cytokines most likely to be involved in an immune response such as IL-1 alpha, IL-3, or TNF-alpha. In contrast, cPLA2 was shown to be expressed and activated in thymocytes and immature B cells under conditions in which ligation of the Ag receptors led to growth arrest and/or apoptosis. Taken together, these data suggest that cPLA2 does not play a role in Ag receptor-mediated lymphocyte activation, but may be involved in the molecular mechanisms underlying lymphocyte maturation and/or self tolerance by clonal deletion.

  7. α-fetoprotein involvement during glucocorticoid-induced precocious maturation in rat colon

    Institute of Scientific and Technical Information of China (English)

    Min Chen; Peng Sun; Xiao-Yan Liu; Dan Dong; Jun Du; Luo Gu; Ying-Bin Ge

    2011-01-01

    AIM: To investigate the role of α-fetoprotein (AFP), a cancer-associated fetal glycoprotein, in glucocorticoidinduced precocious maturation in rat colon. METHODS: Colons from suckling Sprague-Dawley rats were used in this study. Corticosterone acetate at a dose of 100 μg/g body weight was given to normal pups on days 7, 9 and 11 after birth to induce hypercorticoidism. Control animals were injected with identical volumes of normal saline. Some rats receiving corticosterone 7 d after birth were also treated with mifepristone (RU38486), a glucocorticoid cytoplasm receptor antagonist to investigate the effects of glucocorticoids (GCs). The morphological changes of the crypt depth and villous height of the villous zone in colon were observed as indices of colon maturation. Expression levels of AFP in colons were detected by reverse transcriptase polymerase chain reaction and Western blotting. To identify the cellular localization of AFP in developing rat colons, double-immunofluorescent staining was performed using antibodies to specific mesenchymal cell marker and AFP. RESULTS: Corticosterone increased the crypt depth and villous height in the colon of 8- and 10-d-old rats with hypercorticoidism compared with that in the control animals (120% in 8-d-old rats and 118% in 10-d-old rats in villous height, P = 0.021; 145% in 8-d-old rats and 124% in 10-d-old rats in crypt depth, P = 0.017). These increases were accompanied by an increase of AFP expression in both mRNA and protein (2.5-folds in 8-dold and 2.5-folds in 10-d-old rats higher than in control animals, P = 0.035; 1.8-folds in 8-d-old and 1.3-folds in 10-d-old rats higher than in control animals, P = 0.023). Increased crypt depth and villous height and increased expression of AFP in the colon of rats with hypercorticoidism were blocked by mifepristone. Both had positive staining for AFP or vimentin, and overlapped in mesenchymal cells at each tested colon.

  8. The presence of centrioles and centrosomes in ovarian mature cystic teratoma cells suggests human parthenotes developed in vitro can differentiate into mature cells without a sperm centriole

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Bo Yon, E-mail: boyonlee@gmail.com [Department of Obstetrics and Gynecology, Kyung Hee University Hospital, Kyung Hee University, School of Medicine, Seoul (Korea, Republic of); Shim, Sang Woo; Kim, Young Sun; Kim, Seung Bo [Department of Obstetrics and Gynecology, Kyung Hee University Hospital, Kyung Hee University, School of Medicine, Seoul (Korea, Republic of)

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer The sperm centriole is the progenitor of centrosomes in all somatic cells. Black-Right-Pointing-Pointer Centrioles and centrosomes exist in parthenogenetic ovarian teratoma cells. Black-Right-Pointing-Pointer Without a sperm centriole, parthenogenetic oocytes produce centrioles and centrosomes. Black-Right-Pointing-Pointer Parthenogenetic human oocytes can develop and differentiate into mature cells. -- Abstract: In most animals, somatic cell centrosomes are inherited from the centriole of the fertilizing spermatozoa. The oocyte centriole degenerates during oogenesis, and completely disappears in metaphase II. Therefore, the embryos generated by in vitro parthenogenesis are supposed to develop without any centrioles. Exceptional acentriolar and/or acentrosomal developments are possible in mice and in some experimental cells; however, in most animals, the full developmental potential of parthenogenetic cells in vitro and the fate of their centrioles/centrosomes are not clearly understood. To predict the future of in vitro human parthenogenesis, we explored the centrioles/centrosomes in ovarian mature cystic teratoma cells by immunofluorescent staining and transmission electron microscopy. We confirmed the presence of centrioles and centrosomes in these well-known parthenogenetic ovarian tumor cells. Our findings clearly demonstrate that, even without a sperm centriole, parthenotes that develop from activated oocytes can produce their own centrioles/centrosomes, and can even develop into the well-differentiated mature tissue.

  9. Priming by Chemokines Restricts Lateral Mobility of the Adhesion Receptor LFA-1 and Restores Adhesion to ICAM-1 Nano-Aggregates on Human Mature Dendritic Cells

    NARCIS (Netherlands)

    Borgman, K.J.; Zanten, T.S. van; Manzo, C.; Cabezon, R.; Cambi, A.; Benitez-Ribas, D.; Garcia-Parajo, M.F.

    2014-01-01

    LFA-1 is a leukocyte specific beta2 integrin that plays a major role in regulating adhesion and migration of different immune cells. Recent data suggest that LFA-1 on mature dendritic cells (mDCs) may function as a chemokine-inducible anchor during homing of DCs through the afferent lymphatics into

  10. Priming by Chemokines Restricts Lateral Mobility of the Adhesion Receptor LFA-1 and Restores Adhesion to ICAM-1 Nano-Aggregates on Human Mature Dendritic Cells

    NARCIS (Netherlands)

    Borgman, K.J.; Zanten, van T.S.; Manzo, C.; Cabezon, R.; Cambi, A.; Benitez-Ribas, D.; Garcia Parajo, M.F.

    2014-01-01

    LFA-1 is a leukocyte specific β2 integrin that plays a major role in regulating adhesion and migration of different immune cells. Recent data suggest that LFA-1 on mature dendritic cells (mDCs) may function as a chemokine-inducible anchor during homing of DCs through the afferent lymphatics into the

  11. Autophagic vesicles on mature human reticulocytes explain phosphatidylserine-positive red cells in sickle cell disease.

    Science.gov (United States)

    Mankelow, Tosti J; Griffiths, Rebecca E; Trompeter, Sara; Flatt, Joanna F; Cogan, Nicola M; Massey, Edwin J; Anstee, David J

    2015-10-08

    During maturation to an erythrocyte, a reticulocyte must eliminate any residual organelles and reduce its surface area and volume. Here we show this involves a novel process whereby large, intact, inside-out phosphatidylserine (PS)-exposed autophagic vesicles are extruded. Cell surface PS is a well-characterized apoptotic signal initiating phagocytosis. In peripheral blood from patients after splenectomy or in patients with sickle cell disease (SCD), the number of circulating red cells exposing PS on their surface is elevated. We show that in these patients PS is present on the cell surface of red cells in large (∼1.4 µm) discrete areas corresponding to autophagic vesicles. The autophagic vesicles found on reticulocytes are identical to those observed on red cells from splenectomized individuals and patients with SCD. Our data suggest the increased thrombotic risk associated with splenectomy, and patients with hemoglobinopathies is a possible consequence of increased levels of circulating mature reticulocytes expressing inside-out PS-exposed autophagic vesicles because of asplenia.

  12. Semi-mature MyD88-silenced bone marrow dendritic cells prolong the allograft survival in a rat model of intestinal transplantation

    Institute of Scientific and Technical Information of China (English)

    YANG Xiao-jun; MENG Song; Zhu Chun-fu; JIANG Hong; WU Wen-xi

    2011-01-01

    Background Semi-mature dendritic cells (DCs) may induce tolerance rather than immunity.However,little is known about the regulatory mechanism by which these DCs induce transplant tolerance.Myeloid differentiation factor 88 (MyD88) is a key adaptor of Toil-like receptor signaling,which plays a critical role in DC maturation.Activation of MyD88-silenced immature DCs results in the generation of semi-mature DCs.We explored the possibility of using these DCs to induce intestinal transplant tolerance in rats.Methods MyD88 expression was silenced in bone marrow DCs (F344 rats) using small interfering RNAs for 24 hours,at which point,lipopolysaccharide (LPS) was added to the culture for another 48 hours.These cells were analyzed for their in vitro and in vivo tolerizing capacities.Results Semi-mature DCs expressing moderate levels of MHC class Ⅱ and low levels of co-stimulatory molecules were found to produce interleukin (IL)-10,while IL-12 production was decreased.In vitro co-culture with completely allogeneic T cells from Wistar rats led to a significant decrease in alloreactive T-cell responses.In vivo,the transfer of semi-mature DCs (1×106 ceils) followed by the transplantation of fully mismatched intestinal grafts (F344 rats) led to significantly prolonged survival compared to rats receiving immature and mature DCs.Serum from semi-mature DC-treated rats contained lower concentrations of the pro-inflammatory cytokines IL-2 and interferon-Y 5 days after transplantation.Conclusion Semi-mature DCs may promote inducible allograft tolerance and this study suggests a new strategy by which to facilitate the induction of transplant tolerance.

  13. Induced pluripotent stem cells

    Institute of Scientific and Technical Information of China (English)

    Siddhartha Bhowmik; LI Yong

    2011-01-01

    Induced pluripotent stem (iPS) cells are a recent development which has brought a promise of great therapeutic values. The previous technique of somatic cell nuclear transfer (SCNT) has been ineffective in humans. Recent discoveries show that human fibroblasts can be reprogrammed by a transient over expression of a small number of genes; they can undergo induced pluripotency. iPS were first produced in 2006. By 2008, work was underway to remove the potential oncogenes from their structure. In 2009, protein iPS (piPS) cells were discovered. Surface markers and reporter genes play an important role in stem cell research. Clinical applications include generation of self renewing stem cells, tissue replacement and many more. Stem cell therapy has the ability to dramatically change the treatment of human diseases.

  14. Maturation of dendritic cells by recombinant human CD40L-trimer leads to a homogeneous cell population with enhanced surface marker expression and increased cytokine production

    DEFF Research Database (Denmark)

    Würtzen, P A; Nissen, Mogens Holst; Claesson, M H

    2001-01-01

    Dendritic cells (DC) have been shown to be potent inducers of specific cytotoxic T-cell responses both in vivo and in vitro. Furthermore, exposure to cytokines such as tumour necrosis factor (TNF)-alpha or CD40 triggering changes DC phenotype and cytokine production and may enhance the T...... enhanced by exposure to huCD40LT even compared to TNF-alpha exposure. Only a moderate cytokine production was observed initially, while TNF-alpha addition or CD40 triggering, especially, induced enhanced production of IL-6 and IL-12 p40. Surprisingly, comparable induction of T-cell proliferation by a DC...... marker expression and high production of pro-inflammatory cytokines. In addition, the induction of responses to allo or recall antigens presented by huCD40LT maturated DC was comparable to the responses obtained with the DC maturated through TNF-alpha exposure....

  15. Osteopontin promotes dendritic cell maturation and function in response to HBV antigens

    Directory of Open Access Journals (Sweden)

    Cui GY

    2015-06-01

    HBV antigens. In addition, OPN deficiency in DCs reduced the HBV antigen-induced inflammatory response in the liver of mice. Importantly, OPN administration significantly promoted the maturation of DCs from CHB patients in vitro.Conclusion: These findings suggested that OPN could improve the maturation and functioning of DCs in the immune response to HBV antigens, which might be useful to further improve the effect of DC vaccine. Keywords: osteopontin, dendritic cells, hepatitis B virus

  16. Electroporated Antigen-Encoding mRNA Is Not a Danger Signal to Human Mature Monocyte-Derived Dendritic Cells

    Directory of Open Access Journals (Sweden)

    Stefanie Hoyer

    2015-01-01

    Full Text Available For therapeutic cancer vaccination, the adoptive transfer of mRNA-electroporated dendritic cells (DCs is frequently performed, usually with monocyte-derived, cytokine-matured DCs (moDCs. However, DCs are rich in danger-sensing receptors which could recognize the exogenously delivered mRNA and induce DC activation, hence influencing the DCs’ immunogenicity. Therefore, we examined whether electroporation of mRNA with a proper cap and a poly-A tail of at least 64 adenosines had any influence on cocktail-matured moDCs. We used 16 different RNAs, encoding tumor antigens (MelanA, NRAS, BRAF, GNAQ, GNA11, and WT1, and variants thereof. None of those RNAs induced changes in the expression of CD25, CD40, CD83, CD86, and CD70 or the secretion of the cytokines IL-8, IL-6, and TNFα of more than 1.5-fold compared to the control condition, while an mRNA encoding an NF-κB-activation protein as positive control induced massive secretion of the cytokines. To determine whether mRNA electroporation had any effect on the whole transcriptome of the DCs, we performed microarray analyses of DCs of 6 different donors. None of 60,000 probes was significantly different between mock-electroporated DCs and MelanA-transfected DCs. Hence, we conclude that no transcriptional programs were induced within cocktail-matured DCs by electroporation of single tumor-antigen-encoding mRNAs.

  17. Stimulated mast cells promote maturation of myocardial microvascular endothelial cell neovessels by modulating the angiopoietin-Tie-2 signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Z.H. [Division of Cardiology, Shanghai Sixth People' s Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China, Division of Cardiology, Shanghai Sixth People’s Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai (China); Yancheng People' s First Hospital, Division of Cardiology, Yancheng, Jiangsu, China, Division of Cardiology, Yancheng People’s First Hospital, Yancheng, Jiangsu (China); Zhu, W.; Tao, J.P.; Zhang, Q.Y.; Wei, M. [Division of Cardiology, Shanghai Sixth People' s Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China, Division of Cardiology, Shanghai Sixth People’s Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai (China)

    2013-10-22

    Angiopoietin (Ang)-1 and Ang-2 interact in angiogenesis to activate the Tie-2 receptor, which may be involved in new vessel maturation and regression. Mast cells (MCs) are also involved in formation of new blood vessels and angiogenesis. The present study was designed to test whether MCs can mediate angiogenesis in myocardial microvascular endothelial cells (MMVECs). Using a rat MMVEC and MC co-culture system, we observed that Ang-1 protein levels were very low even though its mRNA levels were increased by MCs. Interestingly, MCs were able to enhance migration, proliferation, and capillary-like tube formation, which were associated with suppressed Ang-2 protein expression, but not Tie-2 expression levels. These MCs induced effects that could be reversed by either tryptase inhibitor [N-tosyl-L-lysine chloromethyl ketone (TLCK)] or chymase inhibitor (N-tosyl-L-phenylalanyl chloromethyl ketone), with TLCK showing greater effects. In conclusion, our data indicated that MCs can interrupt neovessel maturation via suppression of the Ang-2/Tie-2 signaling pathway.

  18. Energy metabolism in intestinal epithelial cells during maturation along the crypt-villus axis

    Science.gov (United States)

    Yang, Huansheng; Wang, Xiaocheng; Xiong, Xia; Yin, Yulong

    2016-01-01

    Intestinal epithelial cells continuously migrate and mature along crypt-villus axis (CVA), while the changes in energy metabolism during maturation are unclear in neonates. The present study was conducted to test the hypothesis that the energy metabolism in intestinal epithelial cells would be changed during maturation along CVA in neonates. Eight 21-day-old suckling piglets were used. Intestinal epithelial cells were isolated sequentially along CVA, and proteomics was used to analyze the changes in proteins expression in epithelial cells along CVA. The identified differentially expressed proteins were mainly involved in cellular process, metabolic process, biological regulation, pigmentation, multicellular organizational process and so on. The energy metabolism in intestinal epithelial cells of piglets was increased from the bottom of crypt to the top of villi. Moreover, the expression of proteins related to the metabolism of glucose, most of amino acids, and fatty acids was increased in intestinal epithelial cells during maturation along CVA, while the expression of proteins related to glutamine metabolism was decreased from crypt to villus tip. The expression of proteins involved in citrate cycle was also increased intestinal epithelial cells during maturation along CVA. Moreover, dietary supplementation with different energy sources had different effects on intestinal structure of weaned piglets. PMID:27558220

  19. Cdk5 regulates accurate maturation of newborn granule cells in the adult hippocampus.

    Directory of Open Access Journals (Sweden)

    Sebastian Jessberger

    2008-11-01

    Full Text Available Newborn granule cells become functionally integrated into the synaptic circuitry of the adult dentate gyrus after a morphological and electrophysiological maturation process. The molecular mechanisms by which immature neurons and the neurites extending from them find their appropriate position and target area remain largely unknown. Here we show that single-cell-specific knockdown of cyclin-dependent kinase 5 (cdk5 activity in newborn cells using a retrovirus-based strategy leads to aberrant growth of dendritic processes, which is associated with an altered migration pattern of newborn cells. Even though spine formation and maturation are reduced in cdk5-deficient cells, aberrant dendrites form ectopic synapses onto hilar neurons. These observations identify cdk5 to be critically involved in the maturation and dendrite extension of newborn neurons in the course of adult neurogenesis. The data presented here also suggest a mechanistic dissociation between accurate dendritic targeting and subsequent synapse formation.

  20. c-Rel is dispensable for the differentiation and functional maturation of M cells in the follicle-associated epithelium.

    Science.gov (United States)

    Sehgal, Anuj; Kobayashi, Atsushi; Donaldson, David S; Mabbott, Neil A

    2017-02-01

    M cells reside within the follicle-associated epithelium (FAE) overlying the gut-associated lymphoid tissues. These unique phagocytic epithelial cells enable the mucosal immune system to sample antigens within the lumen of the intestine. The differentiation of M cells from uncommitted precursors in the FAE is dependent on the production of receptor activator of nuclear factor-κB ligand (RANKL) by subepithelial stromal cells. The ligation of a variety of cell surface receptors activates the nuclear factor-κB (NF-κB) family of transcription factors which in-turn induce the transcription of multiple target genes. RANKL-stimulation can stimulate the nuclear translocation of the NF-κB subunit c-Rel. We therefore used c-Rel-deficient mice to determine whether the differentiation and functional maturation of M cells in the Peyer's patches was dependent on c-Rel. Our data show that c-Rel-deficiency does not influence the expression of RANKL or RANK in Peyer's patches, or the induction of M-cell differentiation in the FAE. RANKL-stimulation in the differentiating M cells induces the expression of SpiB which is essential for their subsequent maturation. However, SpiB expression in the FAE was also unaffected in the absence of c-Rel. As a consequence, the functional maturation of M cells was not impaired in the Peyer's patches of c-Rel-deficient mice. Although our data showed that the specific expression of CCL20 and ubiquitin D in the FAE was not impeded in the absence of c-Rel, the expression of ubiquitin D was dramatically reduced in the B cell-follicles of c-Rel-deficient mice. Coincident with this, we also observed that the status of follicular dendritic cells in the B cell-follicles was dramatically reduced in Peyer's patches from c-Rel-deficient mice. Taken together, our data show that c-Rel is dispensable for the RANKL-mediated differentiation and functional maturation of M cells.

  1. Mouse Oocytes Enable LH-Induced Maturation of the Cumulus-Oocyte Complex via Promoting EGF Receptor-Dependent Signaling

    Science.gov (United States)

    Su, You-Qiang; Sugiura, Koji; Li, Qinglei; Wigglesworth, Karen; Matzuk, Martin M.; Eppig, John J.

    2010-01-01

    LH triggers the maturation of the cumulus-oocyte complex (COC), which is followed by ovulation. These ovarian follicular responses to LH are mediated by epidermal growth factor (EGF)-like growth factors produced by granulosa cells and require the participation of oocyte-derived paracrine factors. However, it is not clear how oocytes coordinate with the EGF receptor (EGFR) signaling to achieve COC maturation. The aim of the present study was to test the hypothesis that oocytes promote the expression of EGFR by cumulus cells, thus enabling them to respond to the LH-induced EGF-like peptides. Egfr mRNA and protein expression were dramatically reduced in cumulus cells of mutant mice deficient in the production of the oocyte-derived paracrine factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15). Moreover, microsurgical removal of oocytes from wild-type COCs dramatically reduced expression of Egfr mRNA and protein, and these levels were restored by either coculture with oocytes or treatment with recombinant GDF9 or GDF9 plus recombinant BMP15. Blocking Sma- and Mad-related protein (SMAD)2/3 phosphorylation in vitro inhibited Egfr expression in wild-type COCs and in GDF9-treated wild-type cumulus cells, and conditional deletion of Smad2 and Smad3 genes in granulosa cells in vivo resulted in the reduction of Egfr mRNA in cumulus cells. These results indicate that oocytes promote expression of Egfr in cumulus cells, and a SMAD2/3-dependent pathway is involved in this process. At least two oocyte-derived growth factors, GDF9 and BMP15, are required for EGFR expression by cumulus cells. PMID:20382892

  2. A key role for PTP1B in dendritic cell maturation, migration, and T cell activation.

    Science.gov (United States)

    Martin-Granados, Cristina; Prescott, Alan R; Le Sommer, Samantha; Klaska, Izabela P; Yu, Tian; Muckersie, Elizabeth; Giuraniuc, Claudiu V; Grant, Louise; Delibegovic, Mirela; Forrester, John V

    2015-12-01

    Dendritic cells (DC) are the major antigen-presenting cells bridging innate and adaptive immunity, a function they perform by converting quiescent DC to active, mature DC with the capacity to activate naïve T cells. They do this by migrating from the tissues to the T cell area of the secondary lymphoid tissues. Here, we demonstrate that myeloid cell-specific genetic deletion of PTP1B (LysM PTP1B) leads to defects in lipopolysaccharide-driven bone marrow-derived DC (BMDC) activation associated with increased levels of phosphorylated Stat3. We show that myeloid cell-specific PTP1B deletion also causes decreased migratory capacity of epidermal DC, as well as reduced CCR7 expression and chemotaxis to CCL19 by BMDC. PTP1B deficiency in BMDC also impairs their migration in vivo. Further, immature LysM PTP1B BMDC display fewer podosomes, increased levels of phosphorylated Src at tyrosine 527, and loss of Src localization to podosome puncta. In co-culture with T cells, LysM PTP1B BMDC establish fewer and shorter contacts than control BMDC. Finally, LysM PTP1B BMDC fail to present antigen to T cells as efficiently as control BMDC. These data provide first evidence for a key regulatory role for PTP1B in mediating a central DC function of initiating adaptive immune responses in response to innate immune cell activation.

  3. The phenotype and tissue-specific nature of multipotent cells derived from human mature adipocytes.

    Science.gov (United States)

    Kou, Liang; Lu, Xiao-Wen; Wu, Min-Ke; Wang, Hang; Zhang, Yu-Jiao; Sato, Soh; Shen, Jie-Fei

    2014-02-21

    Dedifferentiated fat (DFAT) cells derived from mature adipocytes have been considered to be a homogeneous group of multipotent cells, which present to be an alternative source of adult stem cells for regenerative medicine. However, many aspects of the cellular nature about DFAT cells remained unclarified. This study aimed to elucidate the basic characteristics of DFAT cells underlying their functions and differentiation potentials. By modified ceiling culture technique, DFAT cells were converted from human mature adipocytes from the human buccal fat pads. Flow cytometry analysis revealed that those derived cells were a homogeneous population of CD13(+) CD29(+) CD105(+) CD44(+) CD31(-) CD34(-) CD309(-) α-SMA(-) cells. DFAT cells in this study demonstrated tissue-specific differentiation properties with strong adipogenic but much weaker osteogenic capacity. Neither did they express endothelial markers under angiogenic induction.

  4. Determination of Mother Centriole Maturation in CPAP-Depleted Cells Using the Ninein Antibody

    OpenAIRE

    Miseon Lee; Kunsoo Rhee

    2015-01-01

    Background Mutations in centrosomal protein genes have been identified in a number of genetic diseases in brain development, including microcephaly. Centrosomal P4.1-associated protein (CPAP) is one of the causal genes implicated in primary microcephaly. We previously proposed that CPAP is essential for mother centriole maturation during mitosis. Methods We immunostained CPAP-depleted cells using the ninein antibody, which selectively detects subdistal appendages in mature mother centrioles. ...

  5. Effects of TLR agonists on maturation and function of 3-day dendritic cells from AML patients in complete remission

    Directory of Open Access Journals (Sweden)

    Merk Martina

    2011-09-01

    Full Text Available Abstract Background Active dendritic cell (DC immunization protocols are rapidly gaining interest as therapeutic options in patients with acute myeloid leukemia (AML. Here we present for the first time a GMP-compliant 3-day protocol for generation of monocyte-derived DCs using different synthetic Toll-like receptor (TLR agonists in intensively pretreated patients with AML. Methods Four different maturation cocktails were compared for their impact on cell recovery, phenotype, cytokine secretion, migration, and lymphocyte activation in 20 AML patients and 25 healthy controls. Results Maturation cocktails containing the TLR7/8 agonists R848 or CL075, with and without the addition of the TLR3 agonist poly(I:C, induced DCs that had a positive costimulatory profile, secreted high levels of IL-12(p70, showed chemotaxis to CCR7 ligands, had the ability to activate NK cells, and efficiently stimulated antigen-specific CD8+ T cells. Conclusions Our results demonstrate that this approach translates into biologically improved DCs, not only in healthy controls but also in AML patients. This data supports the clinical application of TLR-matured DCs in patients with AML for activation of innate and adaptive immune responses.

  6. Evaluating the Effects of Cytomegalovirus Glycoprotein B on the Maturation and Function of Monocyte-derived dendritic cells

    Directory of Open Access Journals (Sweden)

    Afsson shariat

    2015-11-01

    Full Text Available Background & Objectives: Interaction of cytomegalovirus glycoprotein B with toll-like receptors of dendritic cells leads to early signaling and innate immune responses. The aim of this study is to evaluate the effects of cytomegalovirus glycoprotein B on the maturation and function of monocyte-derived dendritic cells in treated groups in comparison with control groups. Materials & Methods: Blood samples were taken from 5 healthy volunteers. Following the generation of monocyte-derived dendritic cells on the fifth day of cell culture, half of the immature dendritic cells were treated with cytomegalovirus glycoprotein B, and the rest of them were induced to mature dendritic untreated cells and were used as the control group. The maturation and function of dendritic cells were evaluated in these two groups. Results: The gene expression level of toll-like receptor-4 significantly increased in the group treated with glycoprotein B (p < 0.05, whereas there were no significant differences in the expression rates of CD83, CD86, CD1a, and HLA-DR and the secretion of IL-23 from monocyte-derived dendritic cells between the treated groups and the controls. Conclusion: The increase in the gene expression of toll-like receptor-4 in monocyte-derived dendritic cells treated with cytomegalovirus glycoprotein B showed that cell contact is required to elicit cellular antiviral response and toll-like receptor activation. Thus, it is critical to recognize the viral and cellular determinants of the immune system in order to develop new therapeutic strategies against cytomegalovirus.

  7. Adipose progenitor cells reside among the mature adipocytes: morphological research using an organotypic culture system.

    Science.gov (United States)

    Anayama, Hisashi; Fukuda, Ryo; Yamate, Jyoji

    2015-11-01

    The precise localization and biological characteristics of the adipose progenitor cells are still a focus of debate. In this study, the localization of the adipose progenitor cells was determined using an organotypic culture system of adipose tissue slices. The tissue slices of subcutaneous white adipose tissue from rats were placed on a porous membrane and cultured at the interface between air and the culture medium for up to 5 days with or without adipogenic stimulation. The structure of adipose tissue components was sufficiently preserved during the culture and, following adipogenic stimulation with insulin, dexamethasone, and 3-isobutyl-1-methylxanthine, numerous multilocular adipocytes appeared in the interstitium among the mature adipocytes. Histomorphological 3-D observation using confocal laser microscopy revealed the presence of small mesenchymal cells containing little or no fat residing in the perivascular region and on the mature adipocytes and differentiation from the pre-existing mesenchymal cells to multilocular adipocytes. Immunohistochemistry demonstrated that these cells were initially present within the fibronectin-positive extracellular matrix (ECM). The adipose differentiation of the mesenchymal cells was confirmed by the enhanced expression of C/EBP-β suggesting adipose differentiation and the concurrent advent of CD105-expressing mesenchymal cells within the interstitium of the mature adipocytes. Based on the above, the mesenchymal cells embedded in the ECM around the mature adipocytes were confirmed to be responsible for adipogenesis because the transition of the mesenchymal cells to the stem state contributed to the increase in the number of adipocytes in rat adipose tissue.

  8. Use of rat mature adipocyte-derived dedifferentiated fat cells as a cell source for periodontal tissue regeneration

    OpenAIRE

    Daisuke eAkita; Koichiro eKano; Yoko eSaito-Tamura; Takayuki eMashimo; Momoko eSato-Shionome; Niina eTsurumachi; Katsuyuki eYamanaka; Tadashi eKaneko; Taku eToriumi; Yoshinori eArai; Naoki eTsukimura; Taro eMatsumoto; Tomohiko eIshigami; Keitaro eIsokawa; Masaki eHonda

    2016-01-01

    Lipid-free fibroblast-like cells, known as dedifferentiated fat (DFAT) cells, can be generated from mature adipocytes with a large single lipid droplet. DFAT cells can re-establish their active proliferation ability and can transdifferentiate into various cell types under appropriate culture conditions. The first objective of this study was to compare the multilineage differentiation potential of DFAT cells with that of adipose-derived stem cells (ASCs) on mesenchymal stem cellsWe obtained DF...

  9. Cbln1 regulates rapid formation and maintenance of excitatory synapses in mature cerebellar Purkinje cells in vitro and in vivo.

    Science.gov (United States)

    Ito-Ishida, Aya; Miura, Eriko; Emi, Kyoichi; Matsuda, Keiko; Iijima, Takatoshi; Kondo, Tetsuro; Kohda, Kazuhisa; Watanabe, Masahiko; Yuzaki, Michisuke

    2008-06-04

    Although many synapse-organizing molecules have been identified in vitro, their functions in mature neurons in vivo have been mostly unexplored. Cbln1, which belongs to the C1q/tumor necrosis factor superfamily, is the most recently identified protein involved in synapse formation in the mammalian CNS. In the cerebellum, Cbln1 is predominantly produced and secreted from granule cells; cbln1-null mice show ataxia and a severe reduction in the number of synapses between Purkinje cells and parallel fibers (PFs), the axon bundle of granule cells. Here, we show that application of recombinant Cbln1 specifically and reversibly induced PF synapse formation in dissociated cbln1-null Purkinje cells in culture. Cbln1 also rapidly induced electrophysiologically functional and ultrastructurally normal PF synapses in acutely prepared cbln1-null cerebellar slices. Furthermore, a single injection of recombinant Cbln1 rescued severe ataxia in adult cbln1-null mice in vivo by completely, but transiently, restoring PF synapses. Therefore, Cbln1 is a unique synapse organizer that is required not only for the normal development of PF-Purkinje cell synapses but also for their maintenance in the mature cerebellum both in vitro and in vivo. Furthermore, our results indicate that Cbln1 can also rapidly organize new synapses in adult cerebellum, implying its therapeutic potential for cerebellar ataxic disorders.

  10. Insights into the osteoblast precursor differentiation towards mature osteoblasts induced by continuous BMP-2 signaling

    Directory of Open Access Journals (Sweden)

    Omar F. Zouani

    2013-07-01

    Mature osteoblasts are the cells responsible for bone formation and are derived from precursor osteoblasts. However, the mechanisms that control this differentiation are poorly understood. In fact, unlike the majority of organs in the body, which are composed of “soft” tissue from which cells can easily be isolated and studied, the “hard” mineralized tissue of bone has made it difficult to study the function of bone cells. Here, we established an in vitro model that mimics this differentiation under physiological conditions. We obtained mature osteoblasts and characterized them on the basis of the following parameters: the strong expression of osteoblastic markers, such as Runx2 and Col-I; the achievement of specific dimensions (the cell volume increases 26-fold compared to the osteoblast precursors; and the production of an abundant extracellular matrix also called osteoid. We demonstrated that the differentiation of osteoblast precursors into mature osteoblasts requires the continuous activation of Bone Morphogenetic Protein (BMP receptors, which we established with the immobilization of a BMP-2mimetic peptide on a synthetic matrix mimicking in vivo microenvironment. Importantly, we demonstrated that the organization of the F-actin network and acetylated microtubules of the cells were modified during the differentiation process. We showed that the perturbation of the F-actin cytoskeleton organization abolished the differentiation process. In addition, we demonstrated that expression of the Runx2 gene is required for this differentiation. These findings demonstrate the retro-regulation of cytoplasmic and genic components due to the continuous induction of BMP-2 and also provide more detailed insights into the correct signaling of BMPs for cell differentiation in bone tissue.

  11. Plasma cholesterol-induced lesion networks activated before regression of early, mature, and advanced atherosclerosis.

    Science.gov (United States)

    Björkegren, Johan L M; Hägg, Sara; Talukdar, Husain A; Foroughi Asl, Hassan; Jain, Rajeev K; Cedergren, Cecilia; Shang, Ming-Mei; Rossignoli, Aránzazu; Takolander, Rabbe; Melander, Olle; Hamsten, Anders; Michoel, Tom; Skogsberg, Josefin

    2014-02-01

    Plasma cholesterol lowering (PCL) slows and sometimes prevents progression of atherosclerosis and may even lead to regression. Little is known about how molecular processes in the atherosclerotic arterial wall respond to PCL and modify responses to atherosclerosis regression. We studied atherosclerosis regression and global gene expression responses to PCL (≥80%) and to atherosclerosis regression itself in early, mature, and advanced lesions. In atherosclerotic aortic wall from Ldlr(-/-)Apob (100/100) Mttp (flox/flox)Mx1-Cre mice, atherosclerosis regressed after PCL regardless of lesion stage. However, near-complete regression was observed only in mice with early lesions; mice with mature and advanced lesions were left with regression-resistant, relatively unstable plaque remnants. Atherosclerosis genes responding to PCL before regression, unlike those responding to the regression itself, were enriched in inherited risk for coronary artery disease and myocardial infarction, indicating causality. Inference of transcription factor (TF) regulatory networks of these PCL-responsive gene sets revealed largely different networks in early, mature, and advanced lesions. In early lesions, PPARG was identified as a specific master regulator of the PCL-responsive atherosclerosis TF-regulatory network, whereas in mature and advanced lesions, the specific master regulators were MLL5 and SRSF10/XRN2, respectively. In a THP-1 foam cell model of atherosclerosis regression, siRNA targeting of these master regulators activated the time-point-specific TF-regulatory networks and altered the accumulation of cholesterol esters. We conclude that PCL leads to complete atherosclerosis regression only in mice with early lesions. Identified master regulators and related PCL-responsive TF-regulatory networks will be interesting targets to enhance PCL-mediated regression of mature and advanced atherosclerotic lesions.

  12. Plasma Cholesterol–Induced Lesion Networks Activated before Regression of Early, Mature, and Advanced Atherosclerosis

    Science.gov (United States)

    Björkegren, Johan L. M.; Hägg, Sara; Jain, Rajeev K.; Cedergren, Cecilia; Shang, Ming-Mei; Rossignoli, Aránzazu; Takolander, Rabbe; Melander, Olle; Hamsten, Anders; Michoel, Tom; Skogsberg, Josefin

    2014-01-01

    Plasma cholesterol lowering (PCL) slows and sometimes prevents progression of atherosclerosis and may even lead to regression. Little is known about how molecular processes in the atherosclerotic arterial wall respond to PCL and modify responses to atherosclerosis regression. We studied atherosclerosis regression and global gene expression responses to PCL (≥80%) and to atherosclerosis regression itself in early, mature, and advanced lesions. In atherosclerotic aortic wall from Ldlr−/−Apob 100/100 Mttp flox/floxMx1-Cre mice, atherosclerosis regressed after PCL regardless of lesion stage. However, near-complete regression was observed only in mice with early lesions; mice with mature and advanced lesions were left with regression-resistant, relatively unstable plaque remnants. Atherosclerosis genes responding to PCL before regression, unlike those responding to the regression itself, were enriched in inherited risk for coronary artery disease and myocardial infarction, indicating causality. Inference of transcription factor (TF) regulatory networks of these PCL-responsive gene sets revealed largely different networks in early, mature, and advanced lesions. In early lesions, PPARG was identified as a specific master regulator of the PCL-responsive atherosclerosis TF-regulatory network, whereas in mature and advanced lesions, the specific master regulators were MLL5 and SRSF10/XRN2, respectively. In a THP-1 foam cell model of atherosclerosis regression, siRNA targeting of these master regulators activated the time-point-specific TF-regulatory networks and altered the accumulation of cholesterol esters. We conclude that PCL leads to complete atherosclerosis regression only in mice with early lesions. Identified master regulators and related PCL-responsive TF-regulatory networks will be interesting targets to enhance PCL-mediated regression of mature and advanced atherosclerotic lesions. PMID:24586211

  13. Plasma cholesterol-induced lesion networks activated before regression of early, mature, and advanced atherosclerosis.

    Directory of Open Access Journals (Sweden)

    Johan L M Björkegren

    2014-02-01

    Full Text Available Plasma cholesterol lowering (PCL slows and sometimes prevents progression of atherosclerosis and may even lead to regression. Little is known about how molecular processes in the atherosclerotic arterial wall respond to PCL and modify responses to atherosclerosis regression. We studied atherosclerosis regression and global gene expression responses to PCL (≥80% and to atherosclerosis regression itself in early, mature, and advanced lesions. In atherosclerotic aortic wall from Ldlr(-/-Apob (100/100 Mttp (flox/floxMx1-Cre mice, atherosclerosis regressed after PCL regardless of lesion stage. However, near-complete regression was observed only in mice with early lesions; mice with mature and advanced lesions were left with regression-resistant, relatively unstable plaque remnants. Atherosclerosis genes responding to PCL before regression, unlike those responding to the regression itself, were enriched in inherited risk for coronary artery disease and myocardial infarction, indicating causality. Inference of transcription factor (TF regulatory networks of these PCL-responsive gene sets revealed largely different networks in early, mature, and advanced lesions. In early lesions, PPARG was identified as a specific master regulator of the PCL-responsive atherosclerosis TF-regulatory network, whereas in mature and advanced lesions, the specific master regulators were MLL5 and SRSF10/XRN2, respectively. In a THP-1 foam cell model of atherosclerosis regression, siRNA targeting of these master regulators activated the time-point-specific TF-regulatory networks and altered the accumulation of cholesterol esters. We conclude that PCL leads to complete atherosclerosis regression only in mice with early lesions. Identified master regulators and related PCL-responsive TF-regulatory networks will be interesting targets to enhance PCL-mediated regression of mature and advanced atherosclerotic lesions.

  14. Analysis of maturation states of rat bone marrow-derived dendritic cells using an improved culture technique.

    Science.gov (United States)

    Grauer, Oliver; Wohlleben, Gisela; Seubert, Silvia; Weishaupt, Andreas; Kämpgen, Eckhart; Gold, Ralf

    2002-04-01

    In this study, we examined in more detail the development of rat bone marrow-derived dendritic cells (BMDC). A two-stage culture system was used to propagate BMDC from rat bone marrow precursors. BMDC developed within clusters of proliferating cells after repetitive addition of rat granulocyte/macrophage colony-stimulating factor and rat interleukin (IL)-4 at a concentration of 5 ng/ml to the cultures. Fluorescence-activated cell sorter analysis performed at an early stage of development (day 6) revealed an immature phenotype with intermediate levels of major histocompatibility complex (MHC) class II expression and low levels of the costimulator molecules CD80 and CD86. Upon further culture, a strong upregulation of MHC class II, costimulatory and adhesion molecules could be observed, whereas macrophage marker antigens were downregulated. Late-stage BMDC (day 10) showed a high expression of MHC class I and II, ICAM-1, Ox62 and CD11c, and revealed a split pattern of B7-1 and B7-2. The cell yield was about 40% of the initially plated bone marrow cells with 80% MHC class II-high and less than 20% MHC class II-low positive cells. Full maturation of rat BMDC (day 12) with an almost uniform expression of B7 was achieved by subsequent subculture and further stimulation with rat tumour necrosis factor alpha (TNF-alpha), lipopolysaccharide (LPS) or soluble CD40 ligand (CD40L). Analysis of the cell supernatant revealed a strong IL-12 production after LPS or CD40L, and to a lesser extent after TNF-alpha stimulation. Additionally, LPS-treated, but not CD40L-treated BMDC secreted TNF-alpha into the supernatant. Early-stage BMDC sufficiently triggered a T cell receptor (TCR) downregulation, but did not stimulate naive T cells in an allogeneic mixed leukocyte reaction (MLR) and revealed a low stimulatory capacity in an antigen-specific T cell assay. In contrast, late-stage BMDC and especially fully mature BMDC strongly induced TCR internalisation, elicited high T cell responses

  15. A temporal role of type I interferon signaling in CD8+ T cell maturation during acute West Nile virus infection.

    Directory of Open Access Journals (Sweden)

    Amelia K Pinto

    2011-12-01

    Full Text Available A genetic absence of the common IFN-α/β signaling receptor (IFNAR in mice is associated with enhanced viral replication and altered adaptive immune responses. However, analysis of IFNAR(-/- mice is limited for studying the functions of type I IFN at discrete stages of viral infection. To define the temporal functions of type I IFN signaling in the context of infection by West Nile virus (WNV, we treated mice with MAR1-5A3, a neutralizing, non cell-depleting anti-IFNAR antibody. Inhibition of type I IFN signaling at or before day 2 after infection was associated with markedly enhanced viral burden, whereas treatment at day 4 had substantially less effect on WNV dissemination. While antibody treatment prior to infection resulted in massive expansion of virus-specific CD8(+ T cells, blockade of type I IFN signaling starting at day 4 induced dysfunctional CD8(+ T cells with depressed cytokine responses and expression of phenotypic markers suggesting exhaustion. Thus, only the later maturation phase of anti-WNV CD8(+ T cell development requires type I IFN signaling. WNV infection experiments in BATF3(-/- mice, which lack CD8-α dendritic cells and have impaired priming due to inefficient antigen cross-presentation, revealed a similar effect of blocking IFN signaling on CD8(+ T cell maturation. Collectively, our results suggest that cell non-autonomous type I IFN signaling shapes maturation of antiviral CD8(+ T cell response at a stage distinct from the initial priming event.

  16. Loss of sensitivity to ACTH of adrenocortical cells isolated from maturing domestic fowl.

    Science.gov (United States)

    Carsia, R V; Scanes, C G; Malamed, S

    1985-07-01

    Maturation of domestic fowl corticosteroidogenesis was evaluated using purified adrenocortical cells. Basal corticosterone production decreased steadily from 2 days to 26 weeks after hatching. However, maximally stimulated corticosterone production was not changed. In contrast, the half-maximal steroidogenic concentrations (ED50 values or effective doses for 50% maximal effect) of ACTH analogs increased approximately 40 times by 26 weeks, but the ED50 values of 8-bromo-cyclic AMP and pregnenolone were not changed. This suggests that adrenocortical cell sensitivity to ACTH decreases with maturation of the domestic fowl.

  17. Lactobacilli differentially modulate expression of cytokines and maturation surface markers in murine dendritic cells

    DEFF Research Database (Denmark)

    Christensen, Hanne Risager; Frøkiær, Hanne; Pestka, J.J.

    2002-01-01

    with greatest capacity to induce IL-12 were most effective. Remarkably, Lactobacillus reuteri DSM12246, a poor IL-12 inducer, inhibited IL-12, IL-6, and TNF-alpha induction by the otherwise strong cytokine inducer L. casei CHCC3139, while IL-10 production remained unaltered. In analogous fashion, L. reuteri...... in the capacity to induce IL-12 and TNF-a production in the DC. Similar but less pronounced differences were observed among lactobacilli in the induction of IL-6 and IL-10. Although all strains up-regulated surface MHC class II and B7-2 (CD86), which is indicative of DC maturation, those lactobacilli...

  18. MUTZ-3 Langerhans cell maturation and CXCL12 independent migration in reconstructed human gingiva.

    Science.gov (United States)

    Kosten, Ilona J; Spiekstra, Sander W; de Gruijl, Tanja D; Gibbs, Susan

    2016-01-01

    Here we describe a reconstructed full thickness human oral mucosa (gingiva) equivalent with integrated Langerhans Cells (GE-LC) and use it to compare LC activation and migration from oral versus skin epithelium. The physiologically representative models consist of differentiated reconstructed epithelium (keratinocytes and Langerhans-like cells derived from the MUTZ-3 cell line) on a fibroblast-populated collagen hydrogel which serves as a lamina propria for gingiva and dermis for skin. Topical exposure of GE-LC and the skin equivalent (SE-LC) to sub-toxic concentrations of the allergens cinnamaldehyde, resorcinol and nickel sulphate, resulted in LC migration out of the epithelia. Neutralizing antibody to CXCL12 blocked allergen-induced LC migration in SE-LC but not in GE-LC. Also, gingival fibroblasts secreted very low amounts of CXCL12 compared to skin fibroblasts even when stimulated with rhTNFα or rhIL-1α. Surprisingly, cinnamaldehyde exposure of GE-LC resulted in an increase in MUTZ-3 LC and CD83 mRNA in the hydrogel but did not result in an increase in CD1a+ cells in the collagen hydrogel (as was observed for SE-LC. These results indicate that in gingiva, upon allergen exposure, MUTZ-3 LC migrate in a CXCL12 independent manner from epithelium-to-lamina propria and in doing so mature become CD1a- and increase CD83+ mRNA. These physiologically relevant in vitro models which not only are human but which also resemble specific tissues, may aid in the identification of factors regulating immune stimulation which in turn will aid the development of therapeutic interventions for allergy and inflammation, anti-cancer vaccines as well as improving diagnostics for skin and oral allergy.

  19. Induction of initial steps of angiogenic differentiation and maturation of endothelial cells by pericytes in vitro and the role of collagen IV.

    Science.gov (United States)

    Zhou, Zhigang; Pausch, Friederike; Schlötzer-Schrehardt, Ursula; Brachvogel, Bent; Pöschl, Ernst

    2016-05-01

    Activation of endothelial cells and recruitment of mural cells define critical steps during the formation of stable vascular elements. Both events are reflected by cocultures of endothelial cells and isolated murine pericyte-like cells and define a versatile platform for the analysis of distinct steps during the angiogenic process in vitro. Isolated pericyte-like cells promote the survival of endothelial cells, induce the assembly of endothelial cells as well as establish direct contacts with forming endothelial alignments. More importantly, they also induce characteristic steps of maturation including the assembly of stable cell-cell junctions, deposition of basement membrane-like matrices and local formation of a central lumen. The presence of pericyte-like cells induces the secretion of extracellular matrices enriched in collagen IV by endothelial cells, which improves endothelial tube formation and provides the adhesive substrate for mural cell recruitment. Collagen-binding integrins contribute differentially to the process, with α1β1 involved in the adhesion of pericyte-like cells to collagen IV and α2β1 mainly involved in endothelial cord formation. These data indicate that pericyte-like cells are essential for the survival of endothelial cells, the efficient formation of endothelial alignments as well as initial steps of maturation of capillary-like structures.

  20. Immature hematopoietic stem cells undergo maturation in the fetal liver.

    Science.gov (United States)

    Kieusseian, Aurelie; Brunet de la Grange, Philippe; Burlen-Defranoux, Odile; Godin, Isabelle; Cumano, Ana

    2012-10-01

    Hematopoietic stem cells (HSCs), which are defined by their capacity to reconstitute adult conventional mice, are first found in the dorsal aorta after 10.5 days post coitus (dpc) and in the fetal liver at 11 dpc. However, lympho-myeloid hematopoietic progenitors are detected in the dorsal aorta from 9 dpc, raising the issue of their role in establishing adult hematopoiesis. Here, we show that these progenitors are endowed with long-term reconstitution capacity, but only engraft natural killer (NK)-deficient Rag2γc(-/-) mice. This novel population, called here immature HSCs, evolves in culture with thrombopoietin and stromal cells, into HSCs, defined by acquisition of CD45 and MHC-1 expression and by the capacity to reconstitute NK-competent mice. This evolution occurs during ontogeny, as early colonization of fetal liver by immature HSCs precedes that of HSCs. Moreover, organ culture experiments show that immature HSCs acquire, in this environment, the features of HSCs.

  1. Dendritic cell maturation and cross-presentation: timing matters!

    Science.gov (United States)

    Alloatti, Andrés; Kotsias, Fiorella; Magalhaes, Joao Gamelas; Amigorena, Sebastian

    2016-07-01

    As a population, dendritic cells (DCs) appear to be the best cross-presenters of internalized antigens on major histocompatibility complex class I molecules in the mouse. To do this, DCs have developed a number of unique and dedicated means to control their endocytic and phagocytic pathways: among them, the capacity to limit acidification of their phagosomes, to prevent proteolytic degradation, to delay fusion of phagosomes to lysosomes, to recruit ER proteins to phagosomes, and to export phagocytosed antigens to the cytosol. The regulation of phagocytic functions, and thereby of antigen processing and presentation by innate signaling, represents a critical level of integration of adaptive and innate immune responses. Understanding how innate signals control antigen cross-presentation is critical to define effective vaccination strategies for CD8(+) T-cell responses.

  2. IL-12 directs further maturation of ex vivo differentiated NK cells with improved therapeutic potential.

    Directory of Open Access Journals (Sweden)

    Dorit Lehmann

    Full Text Available The possibility to modulate ex vivo human NK cell differentiation towards specific phenotypes will contribute to a better understanding of NK cell differentiation and facilitate tailored production of NK cells for immunotherapy. In this study, we show that addition of a specific low dose of IL-12 to an ex vivo NK cell differentiation system from cord blood CD34(+ stem cells will result in significantly increased proportions of cells with expression of CD62L as well as KIRs and CD16 which are preferentially expressed on mature CD56(dim peripheral blood NK cells. In addition, the cells displayed decreased expression of receptors such as CCR6 and CXCR3, which are typically expressed to a lower extent by CD56(dim than CD56(bright peripheral blood NK cells. The increased number of CD62L and KIR positive cells prevailed in a population of CD33(+NKG2A(+ NK cells, supporting that maturation occurs via this subtype. Among a series of transcription factors tested we found Gata3 and TOX to be significantly downregulated, whereas ID3 was upregulated in the IL-12-modulated ex vivo NK cells, implicating these factors in the observed changes. Importantly, the cells differentiated in the presence of IL-12 showed enhanced cytokine production and cytolytic activity against MHC class I negative and positive targets. Moreover, in line with the enhanced CD16 expression, these cells exhibited improved antibody-dependent cellular cytotoxicity for B-cell leukemia target cells in the presence of the clinically applied antibody rituximab. Altogether, these data provide evidence that IL-12 directs human ex vivo NK cell differentiation towards more mature NK cells with improved properties for potential cancer therapies.

  3. An HDAC2-TET1 switch at distinct chromatin regions significantly promotes the maturation of pre-iPS to iPS cells.

    Science.gov (United States)

    Wei, Tingyi; Chen, Wen; Wang, Xiukun; Zhang, Man; Chen, Jiayu; Zhu, Songcheng; Chen, Long; Yang, Dandan; Wang, Guiying; Jia, Wenwen; Yu, Yangyang; Duan, Tao; Wu, Minjuan; Liu, Houqi; Gao, Shaorong; Kang, Jiuhong

    2015-06-23

    The maturation of induced pluripotent stem cells (iPS) is one of the limiting steps of somatic cell reprogramming, but the underlying mechanism is largely unknown. Here, we reported that knockdown of histone deacetylase 2 (HDAC2) specifically promoted the maturation of iPS cells. Further studies showed that HDAC2 knockdown significantly increased histone acetylation, facilitated TET1 binding and DNA demethylation at the promoters of iPS cell maturation-related genes during the transition of pre-iPS cells to a fully reprogrammed state. We also found that HDAC2 competed with TET1 in the binding of the RbAp46 protein at the promoters of maturation genes and knockdown of TET1 markedly prevented the activation of these genes. Collectively, our data not only demonstrated a novel intrinsic mechanism that the HDAC2-TET1 switch critically regulates iPS cell maturation, but also revealed an underlying mechanism of the interplay between histone acetylation and DNA demethylation in gene regulation.

  4. Chondrogenic differentiation of mouse embryonic stem cells promoted by mature chondrocytes

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    In order to direct embryonic stem (ES) cells to differentiate into chondrocytes, a chondrogenic envi-ronment provided by mature chondrocytes was investigated. Flk-1 positive cells sorted from pre-differentiated mouse ES cells were mixed with adult porcine articular chondrocytes, seeded on biodegradable scaffolds, and then implanted subcutaneously into nude mice. The cell-scaffold com-plexes formed cartilage tissues after 4 weeks, which was demonstrated by histology and anti-type II collagen antibody staining. Positive staining of mouse Major Histocompatibility Complex class I molecules confirmed that part of the chondrocytes were derived from mouse ES cells. The current study established a new approach for directing ES cell differentiation.

  5. Chondrogenic differentiation of mouse embryonic stem cells promoted by mature chondrocytes

    Institute of Scientific and Technical Information of China (English)

    XIE Feng; ZHANG WenJie; CHEN FanFan; ZHOU GuangDong; CUI Lei; LIU Wei; CAO YiLin

    2008-01-01

    In order to direct embryonic stem (ES) cells to differentiate into chondrocytes, a chondrogenic envi-ronment provided by mature chondrocytes was investigated. FIk-1 positive cells sorted from pre-differentiated mouse ES cells were mixed with adult porcine articular chondrocytes, seeded on biodegradable scaffolds, and then implanted subcutaneously into nude mice. The cell-scaffold com-plexes formed cartilage tissues after 4 weeks, which was demonstrated by histology and anti-type Ⅱ collagen antibody staining. Positive staining of mouse Major Histocompatibility Complex class Ⅰ molecules confirmed that part of the chondrocytes were derived from mouse ES cells. The current study established a new approach for directing ES cell differentiation.

  6. Birth of lambs after in vitro maturation, fertilization, and coculture with oviductal cells.

    Science.gov (United States)

    Czlonkowska, M; Eysymont, U; Guszkiewicz, A; Kossakowski, M; Dziak, J

    1991-09-01

    Control ovine oocytes matured and fertilized in vitro were transferred to intermediate recipient ewes. After 5 days, 59% of eggs were recovered. Thirty-one (38%) reached morula/blastocyst stage. Twenty-one embryos at the morula or blastocyst stage were transferred to six recipient ewes, resulting in five pregnancies, of which four were maintained. Nine lambs were born (43%). In the experiment, 72 ooctyes matured and fertilized in vitro were cocultured for 5 days with sheep oviductal epithelial cells. Thirty-one eggs (43%) developed to the noncompacted morula stage. Transfer of 26 embryos to 11 recipient ewes resulted in two pregnancies (18%). Two male lambs were born. The result indicates that the coculture of in vitro matured and fertilized ovine eggs with sheep oviductal epithelial cells throughout the preimplantation period is compatible with further development to term.

  7. Preparation of tumor antigen-loaded mature dendritic cells for immunotherapy.

    Science.gov (United States)

    Sabado, Rachel Lubong; Miller, Elizabeth; Spadaccia, Meredith; Vengco, Isabelita; Hasan, Farah; Bhardwaj, Nina

    2013-08-01

    While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors, their clinical efficacy remains to be established. The method described below, prepared in accordance with Good Manufacturing Process (GMP) guidelines, is an optimization of the most common ex vivo preparation method for generating large numbers of DCs for clinical studies. Our method utilizes the synthetic TLR 3 agonist Polyinosinic-Polycytidylic Acid-poly-L-lysine Carboxymethylcellulose (Poly-ICLC) to stimulate the DCs. Our previous study established that Poly-ICLC is the most potent individual maturation stimulus for human DCs as assessed by an upregulation of CD83 and CD86, induction of interleukin-12 (IL-12), tumor necrosis factor (TNF), interferon gamma-induced protein 10 (IP-10), interleukmin 1 (IL-1), and type I interferons (IFN), and minimal interleukin 10 (IL-10) production. DCs are differentiated from frozen peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis. PBMCs are isolated by Ficoll gradient centrifugation and frozen in aliquots. On Day 1, PBMCs are thawed and plated onto tissue culture flasks to select for monocytes which adhere to the plastic surface after 1-2 hr incubation at 37 °C in the tissue culture incubator. After incubation, the lymphocytes are washed off and the adherent monocytes are cultured for 5 days in the presence of interleukin-4 (IL-4) and granulocyte macrophage-colony stimulating factor (GM-CSF) to differentiate to immature DCs. On Day 6, immature DCs are pulsed with the keyhole limpet hemocyanin (KLH) protein which serves as a control for the quality of the vaccine and may boost the immunogenicity of the vaccine. The DCs are stimulated to mature, loaded with peptide antigens, and incubated overnight. On Day 7, the cells are washed, and frozen in 1 ml aliquots containing 4-20 x 10(6) cells using a controlled-rate freezer. Lot release testing for the batches of DCs is performed and must meet minimum

  8. Responsiveness of fetal rat brain cells to glia maturation factor during neoplastic transformation in cell culture

    DEFF Research Database (Denmark)

    Haugen, A; Laerum, O D; Bock, E

    1981-01-01

    of gestation. The brains of the treated fetuses were transferred to cell culture and underwent neoplastic transformation with a characteristic sequence of phenotypic alterations which could be divided into five different stages. During the first 40 days after explantation (stage I & II) BE induced...... morphological differentiation of epitheloid neural cells into astrocytes. This occurred in carcinogen treated cells as well as in untreated control cultures. At the same time cells with astrocyte morphology showed accumulation of glial fibrillary acidic protein (GFA) as tested by indirect immunofluorescence...... with monospecific antibodies against GFA. Thereafter, in the EtNU pre-treated cultures an increased number of cells with astrocyte morphology was seen, and BE further increased the number of cells with long cytoplasmic processes. Control cells were GFA negative, while some few strongly, as well as many weakly...

  9. Development of a standardized protocol for reproducible generation of matured monocyte-derived dendritic cells suitable for clinical application.

    Science.gov (United States)

    Bohnenkamp, H R; Noll, T

    2003-09-01

    There is increasing interest in the generation of dendritic cells (DC) for cancer immunotherapy. In order to utilize DC in clinical trials it is necessary to have standardized, reproducible and easy to use protocols. We describe here the process development for the generation of DC as the result of investigation of culture conditions as well as consumption rates of medium and cytokines. Our studies demonstrate that highly viable DC (93 +/- 2%) can be produced from CD14(+) enriched monocytes via immunomagnetic beads in a high yield (31 +/- 6%) with X-VIVO 15, 400 U ml(-1) GM-CSF and 2000 U ml(-1) IL-4 without serum and feeding. For the maturation of DC different cocktails (TNF-alpha, IL-1beta, IL-6, PGE(2) and TNF-alpha, PGE(2)) were compared. In both cases cells expressed typical surface molecules of mature DC and induced high proliferative responses in mixed lymphocyte reactions which led to IFN-gamma producing T-lymphocytes. The data suggest that the use of this optimized, easy to use protocol results in highly mature DC.

  10. Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin.

    Science.gov (United States)

    Dorn, Isabel; Klich, Katharina; Arauzo-Bravo, Marcos J; Radstaak, Martina; Santourlidis, Simeon; Ghanjati, Foued; Radke, Teja F; Psathaki, Olympia E; Hargus, Gunnar; Kramer, Jan; Einhaus, Martin; Kim, Jeong Beom; Kögler, Gesine; Wernet, Peter; Schöler, Hans R; Schlenke, Peter; Zaehres, Holm

    2015-01-01

    Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34(+) hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex vivo expansion of induced pluripotent stem cells into erythroid cells, we compared induced pluripotent stem cells from human neural stem cells and human cord blood-derived CD34(+) hematopoietic stem cells and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells. Although genome-wide DNA methylation profiling at all promoter regions demonstrates that the epigenetic memory of induced pluripotent stem cells is influenced by the somatic cell type of origin of the stem cells, we found a similar hematopoietic induction potential and erythroid differentiation pattern of induced pluripotent stem cells of different somatic cell origin. All human induced pluripotent stem cell lines showed terminal maturation into normoblasts and enucleated reticulocytes, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the induced pluripotent stem cells derived from CD34(+) hematopoietic stem cells. More detailed methylation analysis of the hematopoietic and erythroid promoters identified similar CpG methylation levels in the induced pluripotent stem cell lines derived from CD34(+) cells and those derived from neural stem cells, which confirms their comparable erythroid differentiation potential.

  11. ENU-induced mutagenesis in grass carp (Ctenopharyngodon idellus by treating mature sperm.

    Directory of Open Access Journals (Sweden)

    Xia-Yun Jiang

    Full Text Available N-ethyl-N-nitrosourea (ENU mutagenesis is a useful approach for genetic improvement of plants, as well as for inducing functional mutants in animal models including mice and zebrafish. In the present study, mature sperm of grass carp (Ctenopharyngodon idellus were treated with a range of ENU concentrations for 45 min, and then wild-type eggs were fertilized. The results indicated that the proportion of embryos with morphological abnormalities at segmentation stage or dead fry at hatching stage increased with increasing ENU dose up to 10 mM. Choosing a dose that was mutagenic, but provided adequate numbers of viable fry, an F1 population was generated from 1 mM ENU-treated sperm for screening purposes. The ENU-treated F1 population showed large variations in growth during the first year. A few bigger mutants with morphologically normal were generated, as compared to the controls. Analysis of DNA from 15 F1 ENU-treated individuals for mutations in partial coding regions of igf-2a, igf-2b, mstn-1, mstn-2, fst-1 and fst-2 loci revealed that most ENU-treated point mutations were GC to AT or AT to GC substitution, which led to nonsense, nonsynonymous and synonymous mutations. The average mutation rate at the examined loci was 0.41%. These results indicate that ENU treatment of mature sperm can efficiently induce point mutations in grass carp, which is a potentially useful approach for genetic improvement of these fish.

  12. Golgi enlargement in Arf-depleted yeast cells is due to altered dynamics of cisternal maturation

    Science.gov (United States)

    Bhave, Madhura; Papanikou, Effrosyni; Iyer, Prasanna; Pandya, Koushal; Jain, Bhawik Kumar; Ganguly, Abira; Sharma, Chandrakala; Pawar, Ketakee; Austin, Jotham; Day, Kasey J.; Rossanese, Olivia W.; Glick, Benjamin S.; Bhattacharyya, Dibyendu

    2014-01-01

    ABSTRACT Regulation of the size and abundance of membrane compartments is a fundamental cellular activity. In Saccharomyces cerevisiae, disruption of the ADP-ribosylation factor 1 (ARF1) gene yields larger and fewer Golgi cisternae by partially depleting the Arf GTPase. We observed a similar phenotype with a thermosensitive mutation in Nmt1, which myristoylates and activates Arf. Therefore, partial depletion of Arf is a convenient tool for dissecting mechanisms that regulate Golgi structure. We found that in arf1Δ cells, late Golgi structure is particularly abnormal, with the number of late Golgi cisternae being severely reduced. This effect can be explained by selective changes in cisternal maturation kinetics. The arf1Δ mutation causes early Golgi cisternae to mature more slowly and less frequently, but does not alter the maturation of late Golgi cisternae. These changes quantitatively explain why late Golgi cisternae are fewer in number and correspondingly larger. With a stacked Golgi, similar changes in maturation kinetics could be used by the cell to modulate the number of cisternae per stack. Thus, the rates of processes that transform a maturing compartment can determine compartmental size and copy number. PMID:24190882

  13. Contact sensitizers decrease 33D1 expression on mature Langerhans cells.

    Science.gov (United States)

    Herouet, C; Cottin, M; Galanaud, P; Leclaire, J; Rousset, F

    1999-01-01

    Langerhans cells play a critical role in allergic contact hypersensitivity. In vivo, these cells capture xenobiotics that penetrate the skin and transport them through the lymphatic vessels into regional lymph nodes for presentation to T cells. During this migration step, Langerhans cells become mature dendritic cells according to their phenotype and their high immunostimulatory capacity. In vitro, when isolated from the skin and cultured for 3 days, Langerhans cells undergo similar phenotypic and functional maturation. In this study, the capacity of sensitizers, irritants and neutral chemicals to modulate the surface marker expression and morphology of pure mature murine Langerhans cells in vitro was examined. Contact with 4 sensitizers (2,4-dinitrobenzenesulfate, 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one, p-phenylenediamine, mercaptobenzo-thiazole) resulted in a rapid, specific, marked fall in 33D1 expression, a murine specific dendritic cell marker. No effect was observed with 2 neutral chemicals (sodium chloride, methyl nicotinate) or 2 irritants (dimethyl sulfoxide, benzalkonium chloride). Nevertheless, sodium lauryl sulfate, a very irritant detergent, altered morphology and down-regulated all membrane markers. These preliminary data suggest that in vitro modulation of 33D1 expression by strong sensitizers may be an approach to the development of an in vitro model for the identification of chemicals that have the potential to cause skin sensitization and to distinguish them as far as possible from irritants.

  14. [Induced-division of neurons derived from neural stem cells].

    Science.gov (United States)

    Lin, Qiu-Xia; Que, Hai-Ping; Lu, Shuang-Hong; Liu, Shao-Jun

    2004-04-25

    In order to explore if mature neurons derived from neural stem cells have the potentiality to divide, we utilized the chemical digestion method to disperse the adult rat brain tissue into single cells, and culture them in serum-free medium. After being cultured for about eight days in vitro, the neural stem cells were induced to differentiate into neurons. The neurons were further induced to divide. Utilizing the method of serial photograph and NF-160 immunocytochemistry, the processes of division of some neurons were recorded. At the same time, PCNA+NF-160 (or Chat, GABA, GAD) double label were used to investigate if the dividing-neurons were mature ones. After the neural stem cells were induced to differentiate in vitro for eight days, they possessed the shape and character of mature neurons. The differentiated neuron had a big nucleus and one or two distinct nucleolus in the nuclear. Within the perikaryon,there were a large amount of dense and Nissl body-like structure. Several long processes emerged from various locations of the cell body. Then, EGF and bFGF were added into the medium to induce division. After two days of induced-division, neuron-like cells were observed to divide; moreover, the number of neuron-like cells in the region increased continually. Immunocytochemistry demonstrated these cells were NF-160-positive. Serial photographs of dividing-process of neuron-like cells were obtained and their daughter cells were also NF-160-positive. After PCNA+NF-160 (or Chat, GABA, GAD) double label, some cells showed brown cell plasma and black nucleus. The above-mentioned results indicate that neurons, which were previously thought to be end-differentiated, can be re-called into cell cycle under appropriate conditions. Mature neurons still have the potential to divide, proliferate and self-renew.

  15. B淋巴细胞诱导成熟蛋白-1对多发性骨髓瘤中浆细胞分化和存活调控作用的研究进展%Research progress of B lymphocyte induced maturation protein-1 on regulation of differentiation and survival of plasma cells in multiple myeloma

    Institute of Scientific and Technical Information of China (English)

    张倩男; 姚瑶; 李振宇

    2016-01-01

    多发性骨髓瘤(MM)是一类浆细胞恶性克隆性疾病.近年其发病率呈逐年上升趋势,且迄今尚无治愈方法.从浆细胞分化和存活的调控机制入手,探索MM治疗的新靶点和新策略是一可行的治疗手段.B淋巴细胞诱导成熟蛋白(Blimp)-1是调控浆细胞分化和存活的关键转录因子,但对其在MM中作用的研究迄今尚少.相关研究结果显示,骨髓瘤细胞高表达Blimp-1,RNA干扰沉默Blimp-1表达,可使浆细胞分化消失并伴各型免疫球蛋白(Ig)合成障碍,从而抑制骨髓瘤细胞增殖并诱导其凋亡.笔者拟就Blimp-1调控浆细胞分化和存活在MM中的作用的研究进展进行综述.%Multiple myeloma (MM) is an incurable plasma malignancy and currently the incidence goes up annually.There is one of feasible methods to explore new targets and strategies for the treatment of MM from the regulation of differentiation and survival of plasma cells.B lymphocyte induced maturation protein(Blimp)-1 is a key transcription factor to regulate differentiation and survival of plasma cells,but up to now,items of research articles on Blimp--1 mechanism in MM are very liffle.Related research has shown that myeloma cells highly expressed Blimp-1 and RNA interference silencing Blimp-1 expression eradicated plasma cell differentiation and made each kind of immunoglobulin (Ig) synthesis disorder,thus inhibiting myeloma cells proliferation and inducing its apoptosis.This review focused on the research progress of Blimp-1 on regulation of differentiation and survival of plasma cells in MM.

  16. Highly efficient transduction of human plasmacytoid dendritic cells without phenotypic and functional maturation

    Directory of Open Access Journals (Sweden)

    Plumas Joel

    2009-01-01

    Full Text Available Abstract Background Gene modified dendritic cells (DC are able to modulate DC functions and induce therapeutic immunity or tolerance in an antigen-specific manner. Among the different DC subsets, plasmacytoid DC (pDC are well known for their ability to recognize and respond to a variety of viruses by secreting high levels of type I interferon. Methods We analyzed here, the transduction efficiency of a pDC cell line, GEN2.2, and of pDC derived from CD34+ progenitors, using lentiviral vectors (LV pseudotyped with different envelope glycoproteins such as the vesicular stomatitis virus envelope (VSVG, the gibbon ape leukaemia virus envelope (GaLV or the feline endogenous virus envelope (RD114. At the same time, we evaluated transgene expression (E-GFP reporter gene under the control of different promoters. Results We found that efficient gene transfer into pDC can be achieved with VSVG-pseudotyped lentiviral vectors (LV under the control of phoshoglycerate kinase (PGK and elongation factor-1 (EF1α promoters (28% to 90% of E-GFP+ cells, respectively in the absence of phenotypic and functional maturation. Surprisingly, promoters (desmin or synthetic C5–12 described as muscle-specific and which drive gene expression in single strand AAV vectors in gene therapy protocols were very highly active in pDC using VSVG-LV. Conclusion Taken together, our results indicate that LV vectors can serve to design pDC-based vaccines in humans, and they are also useful in vitro to evaluate the immunogenicity of the vector preparations, and the specificity and safety of given promoters used in gene therapy protocols.

  17. Postnatal development, maturation and aging in the mouse cochlea and their effects on hair cell regeneration.

    Science.gov (United States)

    Walters, Bradley J; Zuo, Jian

    2013-03-01

    The organ of Corti in the mammalian inner ear is comprised of mechanosensory hair cells (HCs) and nonsensory supporting cells (SCs), both of which are believed to be terminally post-mitotic beyond late embryonic ages. Consequently, regeneration of HCs and SCs does not occur naturally in the adult mammalian cochlea, though recent evidence suggests that these cells may not be completely or irreversibly quiescent at earlier postnatal ages. Furthermore, regenerative processes can be induced by genetic and pharmacological manipulations, but, more and more reports suggest that regenerative potential declines as the organ of Corti continues to age. In numerous mammalian systems, such effects of aging on regenerative potential are well established. However, in the cochlea, the problem of regeneration has not been traditionally viewed as one of aging. This is an important consideration as current models are unable to elicit widespread regeneration or full recovery of function at adult ages yet regenerative therapies will need to be developed specifically for adult populations. Still, the advent of gene targeting and other genetic manipulations has established mice as critically important models for the study of cochlear development and HC regeneration and suggests that auditory HC regeneration in adult mammals may indeed be possible. Thus, this review will focus on the pursuit of regeneration in the postnatal and adult mouse cochlea and highlight processes that occur during postnatal development, maturation, and aging that could contribute to an age-related decline in regenerative potential. Second, we will draw upon the wealth of knowledge pertaining to age related senescence in tissues outside of the ear to synthesize new insights and potentially guide future research aimed at promoting HC regeneration in the adult cochlea.

  18. Global discovery of erythroid long noncoding RNAs reveals novel regulators of red cell maturation

    NARCIS (Netherlands)

    Alvarez-Dominguez, Juan R; Hu, Wenqian; Yuan, Bingbing; Shi, Jiahai; Park, Staphany S; Gromatzky, Austin A; van Oudenaarden, Alexander; Lodish, Harvey F

    2014-01-01

    Erythropoiesis is regulated at multiple levels to ensure the proper generation of mature red cells under multiple physiological conditions. To probe the contribution of long noncoding RNAs (lncRNAs) to this process, we examined >1 billion RNA-seq reads of polyadenylated and nonpolyadenylated RNA fro

  19. Comparison of alpha-Type-1 polarizing and standard dendritic cell cytokine cocktail for maturation of therapeutic monocyte-derived dendritic cell preparations from cancer patients

    DEFF Research Database (Denmark)

    Trepiakas, Redas; Pedersen, Anders Elm; Met, Ozcan

    2008-01-01

    The current "gold standard" for generation of dendritic cell (DC) used in DC-based cancer vaccine studies is maturation of monocyte-derived DCs with tumor necrosis factor-alpha (TNF-alpha)/IL-1beta/IL-6 and prostaglandin E(2) (PGE(2)). Recently, a protocol for producing so-called alpha-Type-1...... of alphaDC1 maturation cocktail to a protocol for clinical grade DC generation from cancer patients performed in X-VIVO 15 medium. We showed that alphaDC1 in this protocol induce lower up-regulation of CD83 and several other maturation markers, co-stimulatory molecules and CCR7 together with higher up......-regulation of inhibitory molecules such as PD-L1, ILT2, ILT3 as compared to sDC. Although alphaDC1 matured DCs secreted more IL-12p70 and IL-23 these DCs had lower or similar stimulatory capacity compared to sDCs when used as stimulating cells in mixed lymphocyte reaction (MLR) or for induction of autologous influenza...

  20. HAIR CELL-LIKE CELL GENERATION INDUCED BY NATURE CULTURE OF ADULT RAT AUDITORY EPITHELIUM

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Hair cells are the mechanosensory cells thatconvert sound and motion signals into electrical i m-pulses in cochlear and vestibular end organs of innerear.Although mature mammals nor mally do notgenerate new hair cells,recentin vivoandin vitrostudies have demonstrated mitotic activity and i m-mature-looking hair cells in mammalian vestibularepithelia after exposure to ototoxic drugs[1-3],sug-gesting that vestibular hair cell regeneration inmammals may be inducible.However,the possibil-ity of auditory hair ce...

  1. Interferon γ Stimulates Cellular Maturation of Dendritic Cell Line DC2.4 Leading to Induction of Efficient Cytotoxic T Cell Responses and Antitumor Immunity

    Institute of Scientific and Technical Information of China (English)

    Tianpei He; Chaoke Tang; Shulin Xu; Terence Moyana; Jim Xiang

    2007-01-01

    Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) for the initiation of antigen (Ag)-specific immune responses. In most studies, mature DCs are generated from bone marrow cells or peripheral monocytes; in either case, the harvested cells are then cultured in medium containing recombinant GM-CSF, IL-4 and TNF-α for 7-10 days and stimulated with lipopolysaccharide (LPS). However, this approach is time-consuming and expensive. There is another less cost approach of using immobilized DC cell lines, which can easily grow in the medium. A disadvantage with the immobilized DC cell lines, however, is that they are immature DCs and lack expression of MHC class Ⅱ and costimulatory CD40 and CD80 molecules. This, therefore, limits their capacity for inducing efficient antitumor immunity. In the current study, we investigated the possible efficacy of various stimuli (IL-1β,IFN-γ, TNF-α, CpG and LPS) in converting the immature dendritic cell line DC2.4 to mature DCs. Our findings were quite interesting since we demonstrated for the first time that IFN-γ was able to stimulate the maturation of DC2.4 cells. The IFN-γ-activated ovalbumin (OVA)-pulsed DC2.4 cells have capacity to upregulate MHC class Ⅱ,CD40, CD80 and CCR7, and to more efficiently stimulate in vitro and in vivo OVA-specific CD8+ T cell responses and antitumor immunity. Therefore, IFN-γ-activated immortal DC2.4 cells may prove to be useful in the study of DC biology and antitumor immunity.

  2. Breast regression protein-39 (BRP-39) promotes dendritic cell maturation in vitro and enhances Th2 inflammation in murine model of asthma

    Institute of Scientific and Technical Information of China (English)

    Qian xu; Shou-jie CHAI; Ying-ying QIAN; Min ZHANG; Kai WANG

    2012-01-01

    Aim: To determine the roles of breast regression protein-39 (BRP-39) in regulating dendritic cell maturation and in pathology of acute asthma.Methods: Mouse bone marrow-derived dendritic cells (BMDCs) were prepared,and infected with adenovirus over-expressing BRP-39.Ovalbumin (OVA)-induced murine model of acute asthma was made in female BALB/c mice by sensitizing and challenging with chicken OVA and Imject Alum.The transfected BMDCs were adoptively transferred into OVA-treated mice via intravenous injection.Airway hyperresponsiveness (AHR),inflammation and pulmonary histopathology were characterized.Results: The expression of BRP-39 mRNA and protein was significantly increased in lung tissues of OVA-treated mice.The BMDCs infected with adenovirus BRP-39 exhibited greater maturation and higher activity in vitro.Adoptive transfer of the cells into OVA-treated mice significantly augmented OVA-induced AHR and eosinophilic inflammation.Meanwhile,BRP-39 further enhanced the production of OVA-induced Th2 cytokines IL-4,IL-5,and IL-13,but significantly attenuated OVA-induced IFN-γ production in bronchoalveolar lavage fluid.Conclusion: In OVA-induced murine model of acute asthma,BRP-39 is over-expressed in lung tissue and augments Th2 inflammatory response and AHR.BRP-39 promotes dendritic cell maturation in vitro.Therefore,BRP-39 may be a potential therapeutic target of asthma.

  3. The perivascular phenotype and behaviors of dedifferentiated cells derived from human mature adipocytes.

    Science.gov (United States)

    Song, Ning; Kou, Liang; Lu, Xiao-Wen; Sugawara, Atsunori; Shimizu, Yutaka; Wu, Min-Ke; Du, Li; Wang, Hang; Sato, Soh; Shen, Jie-Fei

    2015-02-13

    Derived from mature adipocytes, dedifferentiated fat (DFAT) cells represent a special group of multipotent cells. However, their phenotype and cellular nature remain unclear. Our study found that human DFAT cells adopted perivascular characteristics and behaviors. Flow cytometry and immunofluorescent staining revealed that human DFAT cells positively expressed markers highly related to perivascular cell lineages, such as CD140b, NG2 and desmin, but were negative for common endothelial markers, including CD31, CD34, and CD309. Furthermore, DFAT cells displayed vascular network formation ability in Matrigel, and they noticeably promoted and stabilized the vessel structures formed by human umbilical vascular endothelial cells (HUVECs) in vitro. These results provide novel evidence on the pericyte nature of human DFAT cells, further supporting the recent model for the perivascular origin of adult stem cells, in which tissue-specific progenitor cells in mesenchymal tissues associate with blood vessels, exhibiting perivascular characteristics and functions.

  4. Comparison of clinical grade type 1 polarized and standard matured dendritic cells for cancer immunotherapy

    DEFF Research Database (Denmark)

    Hansen, Morten; Hjortø, Gertrud Malene; Donia, Marco

    2013-01-01

    Monocyte-derived dendritic cells (DCs) used for immunotherapy e.g. against cancer are commonly matured by pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and prostaglandin E2 although the absence of Toll-like receptor mediated activation prevents secretion of IL-12 from DCs and subsequent efficient......DCs and strikingly had the highest expression of the inhibitory molecules PD-L1 and CD25. Thus, further studies with type 1 polarized DCs are warranted for use in immunotherapy, but when combined with PGE2 as in mpDCs, they seems to be less optimal for maturation of DCs....

  5. FOXO3-mTOR metabolic cooperation in the regulation of erythroid cell maturation and homeostasis.

    Science.gov (United States)

    Zhang, Xin; Campreciós, Genís; Rimmelé, Pauline; Liang, Raymond; Yalcin, Safak; Mungamuri, Sathish Kumar; Barminko, Jeffrey; D'Escamard, Valentina; Baron, Margaret H; Brugnara, Carlo; Papatsenko, Dmitri; Rivella, Stefano; Ghaffari, Saghi

    2014-10-01

    Ineffective erythropoiesis is observed in many erythroid disorders including β-thalassemia and anemia of chronic disease in which increased production of erythroblasts that fail to mature exacerbate the underlying anemias. As loss of the transcription factor FOXO3 results in erythroblast abnormalities similar to the ones observed in ineffective erythropoiesis, we investigated the underlying mechanisms of the defective Foxo3(-/-) erythroblast cell cycle and maturation. Here we show that loss of Foxo3 results in overactivation of the JAK2/AKT/mTOR signaling pathway in primary bone marrow erythroblasts partly mediated by redox modulation. We further show that hyperactivation of mTOR signaling interferes with cell cycle progression in Foxo3 mutant erythroblasts. Importantly, inhibition of mTOR signaling, in vivo or in vitro enhances significantly Foxo3 mutant erythroid cell maturation. Similarly, in vivo inhibition of mTOR remarkably improves erythroid cell maturation and anemia in a model of β-thalassemia. Finally we show that FOXO3 and mTOR are likely part of a larger metabolic network in erythroblasts as together they control the expression of an array of metabolic genes some of which are implicated in erythroid disorders. These combined findings indicate that a metabolism-mediated regulatory network centered by FOXO3 and mTOR control the balanced production and maturation of erythroid cells. They also highlight physiological interactions between these proteins in regulating erythroblast energy. Our results indicate that alteration in the function of this network might be implicated in the pathogenesis of ineffective erythropoiesis.

  6. Cumulus cells steroidogenesis is influenced by the degree of oocyte maturation

    Directory of Open Access Journals (Sweden)

    Barboni Barbara

    2003-05-01

    Full Text Available Abstract Background The possibility to predict the ability of a germ cell to properly sustain embryo development in vitro or in vivo as early as possible is undoubtedly the main problem of reproductive technologies. To date, only the achievement of nuclear maturation and cumulus expansion is feasible, as all the studies on cytoplasmic maturation are too invasive and have been complicated by the death of the cells analyzed. The authors studied the possibility to test the cytoplasmic quality of pig oocytes by evaluating their ability to produce steroidogenesis enabling factor(s. To this aim, oocytes matured under different culture conditions that allowed to obtain gradable level of cytoplasmic maturation, were used to produce conditioned media (OCM. The secretion of the factor(s in conditioned media was then recorded by evaluating the ability of the spent media to direct granulosa cells (GC steroidogenesis. Methods In order to obtain germ cells characterized by a different degree of developmental competence, selected pig oocytes from prepubertal gilts ovaries were cultured under different IVM protocols; part of the matured oocytes were used to produce OCM, while those remaining were submitted to in vitro fertilization trials to confirm their ability to sustain male pronuclear decondensation. The OCM collected were finally used on cumulus cells grown as monolayers for 5 days. The demonstration that oocytes secreted factor(s can influence GC steroidogenesis in the pig was confirmed in our lab by studying E2 and P4 production by cumulus cells monolayers using a radioimmunoassay technique. Results Monolayers obtained by growing GC surrounding the oocytes for five days represent a tool, which is practical, stable and available in most laboratories; by using this bioassay, we detected the antiluteal effect of immature oocytes, and for the first time, demonstrated that properly matured germ cells are able to direct cumulus cells steroidogenesis by

  7. Chemokine programming dendritic cell antigen response: part I - select chemokine programming of antigen uptake even after maturation.

    Science.gov (United States)

    Park, Jaehyung; Wu, Cindy T; Bryers, James D

    2013-05-01

    Here, we report on the successful programming of dendritic cells (DCs) using selectively applied mixtures of chemokines as a novel protocol for engineering vaccine efficiency. Antigen internalization by DCs is a pivotal step in antigen uptake/presentation for bridging innate and adaptive immunity and in exogenous gene delivery used in vaccine strategies. Contrary to most approaches to improve vaccine efficiency, active enhancement of antigen internalization by DCs as a vaccine strategy has been less studied because DCs naturally down-regulate antigen internalization upon maturation. Whereas chemokines are mainly known as signal proteins that induce leucocyte chemotaxis, very little research has been carried out to identify any additional effects of chemokines on DCs following maturation. Here, immature DCs are pre-treated with select chemokines before intentional maturation using lipopolysaccharide (LPS). When pre-treated with a mixture of CCL3 and CCL19 in a 7 : 3 ratio, then matured with LPS, chemokine pre-treated DCs exhibited 36% higher antigen uptake capacity than immature DCs and 27% higher antigen-processing capacity than immature DCs treated only with LPS. Further, CCL3 : CCL19 (7 : 3) pre-treatment of DCs modulated MHC molecule expression and secretion of various cytokines of DCs. Collectively, DC programming was feasible using a specific chemokine combination and these results provide a novel strategy for enhancing DC-based vaccine efficiency. In Part II, we report on the phenotype changes and antigen presentation capacity of chemokine pre-treated murine bone marrow-derived DCs examined in long-term co-culture with antigen-specific CD4(+) T cells.

  8. Burkholderia pseudomallei enhances maturation of bone marrow-derived dendritic cells.

    Science.gov (United States)

    Williams, Natasha L; Kloeze, Eveline; Govan, Brenda L; Körner, Heinrich; Ketheesan, Natkunam

    2008-12-01

    T-cell activation is essential for protection against Burkholderia pseudomallei infection. Using bone marrow-derived dendritic cells (BMDC) isolated from partially resistant C57BL/6 and susceptible BALB/c mice, the degree of BMDC activation in the presence of B. pseudomallei was investigated. Maturation, cytokine production and internalization of B. pseudomallei by BMDC was assessed in response to infection with a highly virulent and a low-virulent clinical isolate. Maturation was determined by identifying the up-regulation of cell-surface markers CD11c and CD86. IL-1beta and IL-12p40 expression were assessed by reverse-transcriptase PCR. The uptake of B. pseudomallei by BMDC was measured using an internalization assay. This study demonstrated that B. pseudomallei isolates stimulate the maturation of BMDC to the same degree regardless of virulence. However, maturation of BMDC was significantly increased in BALB/c mice compared with C57BL/6 mice. Additionally, the uptake of B. pseudomallei by BMDC was significantly greater with the highly virulent isolate compared with the low-virulent isolate. Expression of IL-12 and IL-1beta following infection with B. pseudomallei was up-regulated. The differences observed may have implications in the development of an effective immune response to B. pseudomallei.

  9. Chronic alcohol consumption enhances iNKT cell maturation and activation.

    Science.gov (United States)

    Zhang, Hui; Zhang, Faya; Zhu, Zhaohui; Luong, Dung; Meadows, Gary G

    2015-01-15

    Alcohol consumption exhibits diverse effects on different types of immune cells. NKT cells are a unique T cell population and play important immunoregulatory roles in different types of immune responses. The effects of chronic alcohol consumption on NKT cells remain to be elucidated. Using a mouse model of chronic alcohol consumption, we found that alcohol increases the percentage of NKT cells, especially iNKT cells in the thymus and liver, but not in the spleen or blood. Alcohol consumption decreases the percentage of NK1.1(-) iNKT cells in the total iNKT cell population in all of the tissues and organs examined. In the thymus, alcohol consumption increases the number of NK1.1(+)CD44(hi) mature iNKT cells but does not alter the number of NK1.1(-) immature iNKT cells. A BrdU incorporation assay shows that alcohol consumption increases the proliferation of thymic NK1.1(-) iNKT cells, especially the NK1.1(-)CD44(lo) Stage I iNKT cells. The percentage of NKG2A(+) iNKT cells increases in all of the tissues and organs examined; whereas CXCR3(+) iNKT cells only increases in the thymus of alcohol-consuming mice. Chronic alcohol consumption increases the percentage of IFN-γ-producing iNKT cells and increases the blood concentration of IFN-γ and IL-12 after in vivo α-galactosylceramide (αGalCer) stimulation. Consistent with the increased cytokine production, the in vivo activation of iNKT cells also enhances the activation of dendritic cells (DC) and NK, B, and T cells in the alcohol-consuming mice. Taken together the data indicate that chronic alcohol consumption enhances iNKT cell maturation and activation, which favors the Th1 immune response.

  10. Changes in homologous and heterologous gap junction contacts during maturation-inducing hormone-dependent meiotic resumption in ovarian follicles of Atlantic croaker

    Science.gov (United States)

    Bolamba, D.; Patino, R.; Yoshizaki, G.; Thomas, P.

    2003-01-01

    Homologous (granulosa cell-granulosa cell) gap junction (GJ) contacts increase in ovarian follicles of Atlantic croaker (Micropogonias undulatus) during the early (first) stage of maturation, but their profile during the second stage [i.e., during maturation-inducing hormone (MIH)-mediated meiotic resumption] is unknown. The profile of homologous GJ contacts during the second stage of maturation in croaker follicles was examined in this study and compared to that of heterologous (granulosa cell-oocyte) GJ, for which changes have been previously documented. Follicles were incubated with human chorionic gonadotropin to induce maturational competence (first stage), and then with MIH to induce meiotic resumption. The follicles were collected for examination immediately before and after different durations of MIH exposure until the oocyte had reached the stage of germinal vesicle breakdown (GVBD; index of meiotic resumption). Ultrathin sections were observed by transmission electron microscopy, and homologous and heterologous GJ contacts were quantified along a 100-??m segment of granulosa cell-zona radiata complex per follicle (three follicles/time/fish, n=3 fish). Relatively high numbers of both types of GJ were observed before and after the first few hours of MIH exposure (up to the stage of oil droplet coalescence). GJ numbers declined during partial yolk globule coalescence (at or near GVBD) and were just under 50% of starting values after the completion of GVBD (Pobservations that GVBD temporally correlates with declining heterologous GJ contacts, and for the first time in teleosts show that there is a parallel decline in homologous GJ. The significance of the changes in homologous and heterologous GJ is uncertain and deserves further study. ?? 2003 Elsevier Science (USA). All rights reserved.

  11. Inhibition of TNF alpha during maturation of dendritic cells results in the development of semi-mature cells: a potential mechanism for the beneficial effects of TNF alpha blockade in rheumatoid arthritis.

    NARCIS (Netherlands)

    Lieshout, A.W.T. van; Barrera Rico, P.; Smeets, R.L.L.; Pesman, G.J.; Riel, P.L.C.M. van; Berg, W.B. van den; Radstake, T.R.D.J.

    2005-01-01

    BACKGROUND: Dendritic cells orchestrate pivotal immunological processes mediated by the production of cytokines and chemokines. OBJECTIVE: To assess whether neutralisation of tumour necrosis factor alpha (TNF alpha) during maturation of dendritic cells affects their phenotype and behaviour, which mi

  12. DNAM-1 Expression Marks an Alternative Program of NK Cell Maturation

    Directory of Open Access Journals (Sweden)

    Ludovic Martinet

    2015-04-01

    Full Text Available Natural killer (NK cells comprise a heterogeneous population of cells important for pathogen defense and cancer surveillance. However, the functional significance of this diversity is not fully understood. Here, we demonstrate through transcriptional profiling and functional studies that the activating receptor DNAM-1 (CD226 identifies two distinct NK cell functional subsets: DNAM-1+ and DNAM-1− NK cells. DNAM-1+ NK cells produce high levels of inflammatory cytokines, have enhanced interleukin 15 signaling, and proliferate vigorously. By contrast, DNAM-1− NK cells that differentiate from DNAM-1+ NK cells have greater expression of NK-cell-receptor-related genes and are higher producers of MIP1 chemokines. Collectively, our data reveal the existence of a functional program of NK cell maturation marked by DNAM-1 expression.

  13. Commonly used prophylactic vaccines as an alternative for synthetically produced TLR ligands to mature monocyte-derived dendritic cells.

    NARCIS (Netherlands)

    Schreibelt, G.; Benitez-Ribas, D.; Schuurhuis, D.; Lambeck, A.J.A.; Hout-Kuijer, M.A. van; Schaft, N.; Punt, C.J.A.; Figdor, C.G.; Adema, G.J.; Vries, I.J.M. de

    2010-01-01

    Currently dendritic cell (DC)-based vaccines are explored in clinical trials, predominantly in cancer patients. Murine studies showed that only maturation with Toll-like receptor (TLR) ligands generates mature DCs that produce interleukin-12 and promote optimal T-cell help. Unfortunately, the limite

  14. B-Cell Depletion Reduces the Maturation of Cerebral Cavernous Malformations in Murine Models.

    Science.gov (United States)

    Shi, Changbin; Shenkar, Robert; Zeineddine, Hussein A; Girard, Romuald; Fam, Maged D; Austin, Cecilia; Moore, Thomas; Lightle, Rhonda; Zhang, Lingjiao; Wu, Meijing; Cao, Ying; Gunel, Murat; Louvi, Angeliki; Rorrer, Autumn; Gallione, Carol; Marchuk, Douglas A; Awad, Issam A

    2016-06-01

    Cerebral cavernous malformations (CCMs) are relatively common vascular malformations, characterized by increased Rho kinase (ROCK) activity, vascular hyper-permeability and the presence of blood degradation products including non-heme iron. Previous studies revealed robust inflammatory cell infiltration, selective synthesis of IgG, in situ antigen driven B-cell clonal expansion, and deposition of immune complexes and complement proteins within CCM lesions. We aimed to evaluate the impact of suppressing the immune response on the formation and maturation of CCM lesions, as well as lesional iron deposition and ROCK activity. Two murine models of heterozygous Ccm3 (Pdcd10), which spontaneously develop CCM lesions with severe and milder phenotypes, were either untreated or received anti-mouse BR3 to deplete B cells. Brains from anti-mouse BR3-treated mice exhibited significantly fewer mature CCM lesions and smaller lesions compared to untreated mice. B cell depletion halted the progression of lesions into mature stage 2 lesions but did not prevent their genesis. Non-heme iron deposition and ROCK activity was decreased in lesions of B cell depleted mice. This represents the first report of the therapeutic benefit of B-cell depletion in the development and progression of CCMs, and provides a proof of principle that B cells play a critical role in CCM lesion genesis and maturation. These findings add biologics to the list of potential therapeutic agents for CCM disease. Future studies would characterize the putative antigenic trigger and further define the mechanism of immune response in the lesions.

  15. Role of Pancreatic Transcription Factors in Maintenance of Mature β-Cell Function

    OpenAIRE

    Hideaki Kaneto; Taka-aki Matsuoka

    2015-01-01

    A variety of pancreatic transcription factors including PDX-1 and MafA play crucial roles in the pancreas and function for the maintenance of mature β-cell function. However, when β-cells are chronically exposed to hyperglycemia, expression and/or activities of such transcription factors are reduced, which leads to deterioration of b-cell function. These phenomena are well known as β-cell glucose toxicity in practical medicine as well as in the islet biology research area. Here we describe th...

  16. Cdc42-dependent actin dynamics controls maturation and secretory activity of dendritic cells

    DEFF Research Database (Denmark)

    Schulz, Anna M; Stutte, Susanne; Hogl, Sebastian

    2015-01-01

    Cell division cycle 42 (Cdc42) is a member of the Rho guanosine triphosphatase family and has pivotal functions in actin organization, cell migration, and proliferation. To further study the molecular mechanisms of dendritic cell (DC) regulation by Cdc42, we used Cdc42-deficient DCs. Cdc42...... disruption, our results propose that Cdc42 control of actin dynamics keeps DCs in an immature state, and cessation of Cdc42 activity during DC maturation facilitates secretion as well as rapid up-regulation of intracellular molecules to the cell surface....

  17. Matured Hop Bittering Components Induce Thermogenesis in Brown Adipose Tissue via Sympathetic Nerve Activity.

    Directory of Open Access Journals (Sweden)

    Yumie Morimoto-Kobayashi

    Full Text Available Obesity is the principal symptom of metabolic syndrome, which refers to a group of risk factors that increase the likelihood of atherosclerosis. In recent decades there has been a sharp rise in the incidence of obesity throughout the developed world. Iso-α-acids, the bitter compounds derived from hops in beer, have been shown to prevent diet-induced obesity by increasing lipid oxidation in the liver and inhibition of lipid absorption from the intestine. Whereas the sharp bitterness induced by effective dose of iso-α-acids precludes their acceptance as a nutrient, matured hop bittering components (MHB appear to be more agreeable. Therefore, we tested MHB for an effect on ameliorating diet-induced body fat accumulation in rodents. MHB ingestion had a beneficial effect but, compared to iso-α-acids and despite containing structurally similar compounds, acted via different mechanisms to reduce body fat accumulation. MHB supplementation significantly reduced body weight gain, epididymal white adipose tissue weight, and plasma non-esterified free fatty acid levels in diet-induced obese mice. We also found that uncoupling protein 1 (UCP1 expression in brown adipose tissue (BAT was significantly increased in MHB-fed mice at both the mRNA and protein levels. In addition, MHB administration in rats induced the β-adrenergic signaling cascade, which is related to cAMP accumulation in BAT, suggesting that MHB could modulate sympathetic nerve activity innervating BAT (BAT-SNA. Indeed, single oral administration of MHB elevated BAT-SNA in rats, and this elevation was dissipated by subdiaphragmatic vagotomy. Single oral administration of MHB maintained BAT temperature at a significantly higher level than in control rats. Taken together, these findings indicate that MHB ameliorates diet-induced body fat accumulation, at least partly, by enhancing thermogenesis in BAT via BAT-SNA activation. Our data suggests that MHB is a useful tool for developing functional

  18. Generation of functionally mature dendritic cells from elutriated monocytes using polyinosinic : polycytidylic acid and soluble CD40 ligand for clinical application.

    Science.gov (United States)

    Kim, S; Kim, H O; Kim, H J; Lee, K; Kim, H-S

    2008-12-01

    Despite the increasing use of dendritic cell (DC) vaccination in clinical trials, optimal conditions for the generation of functionally mature DCs remain to be established. The current standard DC maturation protocol for clinical trials has been used as an inflammatory cytokine cocktail [tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6 and prostaglandin E(2)], but this cocktail induced insufficient maturation of DCs derived from elutriated monocytes when cultured in X-VIVO 15. The aim of this study was to define effective combinations of stimulators for generating functionally mature DCs from elutriated monocytes under current good manufacturing practice conditions. We compared the functional capacity of DCs in response to all possible pairwise combinations of four different classes of stimuli: TNF-alpha, peptidoglycan, polyinosinic : polycytidylic acid [poly(I:C)] and soluble CD40 ligand (CD40L). Maturation status of DCs stimulated with combination of four stimuli was similar to that of the cytokine cocktail as assessed by the cell surface phenotype. However, only the combination of poly(I:C) + CD40L induced complete functional activation of the whole DC population, assessing IL-12p70 production, allostimulatory activity, migratory response to CCL19 and T helper 1-polarizing capacity. Thus, the protocol based on the combination of poly(I:C) and CD40L is more effective for the induction of clinical-grade DCs from elutriated monocytes than the standard cytokine cocktail.

  19. Mature adipocyte-derived dedifferentiated fat cells can trans-differentiate into osteoblasts in vitro and in vivo only by all-trans retinoic acid.

    Science.gov (United States)

    Oki, Yoshinao; Watanabe, Saiko; Endo, Tuyoshi; Kano, Koichiro

    2008-01-01

    We investigated whether de-differentiated fat (DFAT) cells, a mature adipocyte-derived preadipocyte cell line, can be induced to trans-differentiate into osteoblasts in vitro and in vivo. All-trans retinoic acid (RA) induced expression of osteoblast-specific mRNAs encoding Cbfa1/Runx2, osterix, alkaline phosphatase, osteopontin, parathyroid hormone receptor, and osteocalcin in the DFAT cells, but did not induce the expression of adipocyte-specific mRNAs encoding PPARgamma2, C/EBPalpha, and GLUT4. Moreover, alkaline phosphatase activity was expressed in DFAT cells and the cells underwent mineralization of the bone matrix in vitro. Furthermore, when DFAT cells were transplanted subcutaneously into C57BL/6N mice in diffusion chambers, these cells formed ectopic osteoid tissue without any host cell-invasion of the chambers. These results indicate that DFAT cells derived from mature adipocytes can be converted into fully differentiated osteoblasts in vitro and in vivo using RA. DFAT cells provide a unique model for studying the lineage commitment of the adipocytes and osteoblasts derived from mesenchymal stem cells. Identification of the pathways that regulate these processes could lead to the development of new therapeutic strategies for control of unwarranted growth of bone and adipose tissue.

  20. B cell maturation antigen deficiency exacerbates lymphoproliferation and autoimmunity in murine lupus.

    Science.gov (United States)

    Jiang, Chao; Loo, William M; Greenley, Erin J; Tung, Kenneth S; Erickson, Loren D

    2011-06-01

    Systemic lupus erythematosus and its preclinical lupus-prone mouse models are autoimmune disorders involving the production of pathogenic autoantibodies. Genetic predisposition to systemic lupus erythematosus results in B cell hyperactivity, survival of self-reactive B cells, and differentiation to autoantibody-secreting plasma cells (PCs). These corrupt B cell responses are, in part, controlled by excess levels of the cytokine BAFF that normally maintains B cell homeostasis and self-tolerance through limited production. B cell maturation Ag (BCMA) is a receptor for BAFF that, under nonautoimmune conditions, is important for sustaining enduring Ab protection by mediating survival of long-lived PCs but is not required for B cell maturation and homeostasis. Through analysis of two different lupus-prone mouse models deficient in BCMA, we identify BCMA as an important factor in regulating peripheral B cell expansion, differentiation, and survival. We demonstrate that a BCMA deficiency combined with the lpr mutation or the murine lupus susceptibility locus Nba2 causes dramatic B cell and PC lymphoproliferation, accelerated autoantibody production, and early lethality. This study unexpectedly reveals that BCMA works to control B cell homeostasis and self-tolerance in systemic autoimmunity.

  1. Salmonella Infection Drives Promiscuous B Cell Activation Followed by Extrafollicular Affinity Maturation.

    Science.gov (United States)

    Di Niro, Roberto; Lee, Seung-Joo; Vander Heiden, Jason A; Elsner, Rebecca A; Trivedi, Nikita; Bannock, Jason M; Gupta, Namita T; Kleinstein, Steven H; Vigneault, Francois; Gilbert, Tamara J; Meffre, Eric; McSorley, Stephen J; Shlomchik, Mark J

    2015-07-21

    The B cell response to Salmonella typhimurium (STm) occurs massively at extrafollicular sites, without notable germinal centers (GCs). Little is known in terms of its specificity. To expand the knowledge of antigen targets, we screened plasmablast (PB)-derived monoclonal antibodies (mAbs) for Salmonella specificity, using ELISA, flow cytometry, and antigen microarray. Only a small fraction (0.5%-2%) of the response appeared to be Salmonella-specific. Yet, infection of mice with limited B cell receptor (BCR) repertoires impaired the response, suggesting that BCR specificity was important. We showed, using laser microdissection, that somatic hypermutation (SHM) occurred efficiently at extrafollicular sites leading to affinity maturation that in turn led to detectable STm Ag-binding. These results suggest a revised vision of how clonal selection and affinity maturation operate in response to Salmonella. Clonal selection initially is promiscuous, activating cells with virtually undetectable affinity, yet SHM and selection occur during the extrafollicular response yielding higher affinity, detectable antibodies.

  2. Development of a new protocol for 2-day generation of mature dendritic cells from human monocytes.

    Science.gov (United States)

    Obermaier, Bianca; Dauer, Marc; Herten, Jan; Schad, Katharina; Endres, Stefan; Eigler, Andreas

    2003-01-01

    We developed a new 2-day protocol for the generation of dendritic cells (DCs) from human monocytes in vitro. First, we demonstrated that 24 hours of culture with GM-CSF and IL-4 are sufficient to generate immature DCs capable of antigen uptake. We then compared two different strategies for DC maturation: proinflammatory mediators were either added together with GM-CSF and IL-4 from the beginning of cell culture or added after 24 hours of differentiation with GM-CSF and IL-4. After 48 hours of total culture period, expression of activation markers was more pronounced in cells generated by the 2-step differentiation and activation method. Our new protocol for 2-day DC differentiation reduces labor, cost and time and also reliably renders high numbers of mature and viable DCs.

  3. Development of a new protocol for 2-day generation of mature dendritic cells from human monocytes

    Directory of Open Access Journals (Sweden)

    Obermaier Bianca

    2003-01-01

    Full Text Available We developed a new 2-day protocol for the generation of dendritic cells (DCs from human monocytes in vitro. First, we demonstrated that 24 hours of culture with GM-CSF and IL-4 are sufficient to generate immature DCs capable of antigen uptake. We then compared two different strategies for DC maturation: proinflammatory mediators were either added together with GM-CSF and IL-4 from the beginning of cell culture or added after 24 hours of differentiation with GM-CSF and IL-4. After 48 hours of total culture period, expression of activation markers was more pronounced in cells generated by the 2-step differentiation and activation method. Our new protocol for 2-day DC differentiation reduces labor, cost and time and also reliably renders high numbers of mature and viable DCs.

  4. Forced KLF4 expression increases the generation of mature plasma cells and uncovers a network linked with plasma cell stage.

    Science.gov (United States)

    Schoenhals, Matthieu; Jourdan, Michel; Seckinger, Anja; Pantesco, Véronique; Hose, Dirk; Kassambara, Alboukadel; Moreaux, Jérôme; Klein, Bernard

    2016-07-17

    A role of the transcription factor Krüppel-like factor 4 (KLF4) in the generation of mature plasma cells (PC) is unknown. Indeed, KLF4 is critical in controlling the differentiation of various cell linages, particularly monocytes and epithelial cells. KLF4 is expressed at low levels in pro-B cells and its expression increases as they mature into pre-B cells, resting naïve B cells and memory B cells. We show here that KLF4 is expressed in human bone marrow plasma cells and its function was studied using an in vitro model of differentiation of memory B cells into long lived plasma cells. KLF4 is rapidly lost when memory B cells differentiate into highly cell cycling plasmablasts, poorly cycling early plasma cells and then quiescent long-lived plasma cells. A forced expression of KLF4 in plasmablasts enhances the yield of their differentiation into early plasma cell and long lived plasma cells, by inhibiting apoptosis and upregulating previously unknown plasma cell pathways.

  5. Rapamycin Modulates the Maturation of Rat Bone Marrow-derived Dendritic Cells

    Institute of Scientific and Technical Information of China (English)

    Yingjun DING; Xiang CHENG; Tingting TANG; Rui YAO; Yong CHEN; Jiangjiao XIE; Xian YU; Yuhua LIAO

    2008-01-01

    The purpose of the study was to observe the effect of rapamycin (RAPA) on the differentiation and maturation of rat bone marrow-derived dendritic cells (BMDCs) in vitro. BMDCs from Wistar rats were cultured with granulocyte-macrophage colony-stimulating factor plus interleukin-4in the presence or absence of RAPA (20 ng/mL), and stimulated with lipopolysaccharide (LPS) for 24h before cells and supernatants were collected. Surface phenotype of BMDCs was flow-cytometrically detected to determine the expression of maturation markers, MHC class Ⅱ and CD86. Supematants were analyzed for the production of IL-12 and IFN-γ cytokines by using ELISA.BMDCs were co-cultured with T cells from Lewis rats and mixed lymphocyte reaction was assessed by MTT method. The morphology of BMDCs stimulated with LPS remained immature after RAPA pretreatment. RAPA significantly decreased the CD86 expression, impaired the IL-12 and IFN-γproduction of BMDCs stimulated with LPS, and inhibited the proliferation of allogeneic T cells. In conclusion, RAPA can inhibit the maturation of BMDCs stimulated with LPS in terms of the morphology, surface phenotype, cytokine production, and ability of BMDCs to stimulate the proliferation of allogeneic T cells in vitro.

  6. Microrna-221 and microrna-222 modulate differentiation and maturation of skeletal muscle cells.

    Directory of Open Access Journals (Sweden)

    Beatrice Cardinali

    Full Text Available BACKGROUND: MicroRNAs (miRNAs are a class of small non-coding RNAs that have recently emerged as important regulators of gene expression. They negatively regulate gene expression post-transcriptionally by translational repression and target mRNA degradation. miRNAs have been shown to play crucial roles in muscle development and in regulation of muscle cell proliferation and differentiation. METHODOLOGY/PRINCIPAL FINDINGS: By comparing miRNA expression profiling of proliferating myoblasts versus differentiated myotubes, a number of modulated miRNAs, not previously implicated in regulation of myogenic differentiation, were identified. Among these, miR-221 and miR-222 were strongly down-regulated upon differentiation of both primary and established myogenic cells. Conversely, miR-221 and miR-222 expression was restored in post-mitotic, terminally differentiated myotubes subjected to Src tyrosine kinase activation. By the use of specific inhibitors we provide evidence that expression of miR-221 and miR-222 is under the control of the Ras-MAPK pathway. Both in myoblasts and in myotubes, levels of the cell cycle inhibitor p27 inversely correlated with miR-221 and miR-222 expression, and indeed we show that p27 mRNA is a direct target of these miRNAs in myogenic cells. Ectopic expression of miR-221 and miR-222 in myoblasts undergoing differentiation induced a delay in withdrawal from the cell cycle and in myogenin expression, followed by inhibition of sarcomeric protein accumulation. When miR-221 and miR-222 were expressed in myotubes undergoing maturation, a profound alteration of myofibrillar organization was observed. CONCLUSIONS/SIGNIFICANCE: miR-221 and miR-222 have been found to be modulated during myogenesis and to play a role both in the progression from myoblasts to myocytes and in the achievement of the fully differentiated phenotype. Identification of miRNAs modulating muscle gene expression is crucial for the understanding of the circuits

  7. Cortical bone growth and maturational changes in dwarf rats induced by recombinant human growth hormone

    Science.gov (United States)

    Martinez, D. A.; Orth, M. W.; Carr, K. E.; Vanderby, R. Jr; Vailas, A. C.

    1996-01-01

    The growth hormone (GH)-deficient dwarf rat was used to investigate recombinant human (rh) GH-induced bone formation and to determine whether rhGH facilitates simultaneous increases in bone formation and bone maturation during rapid growth. Twenty dwarf rats, 37 days of age, were randomly assigned to dwarf plus rhGH (GH; n = 10) and dwarf plus vehicle (n = 10) groups. The GH group received 1.25 mg rhGH/kg body wt two times daily for 14 days. Biochemical, morphological, and X-ray diffraction measurements were performed on the femur middiaphysis. rhGH stimulated new bone growth in the GH group, as demonstrated by significant increases (P growth.

  8. Effects of gangliosides on cell maturation of murine megakaryocytes in a liquid culture system.

    Science.gov (United States)

    Watanabe, Y; Taniguchi, M; Fukamachi, N; Sakuma, M; Kobayashi, B

    1990-04-01

    Formation of platelet-producing megakaryocytes, the cytoplasm of which showed the terminal stage of cell maturation, heavy granulation and platelet-fields delineated with demarcation membranes, was observed in a short-term culture system, using megakaryocyte-enriched bone marrow cell suspension. Approximately 6-8% of the megakaryocytes changed to the platelet-producing megakaryocytes during 12-hour incubation. In the presence of inhibitors of energy metabolism, formation of the platelet-producing megakaryocytes was inhibited, suggesting that the process is dependent on energy producing systems. Ganglioside GD1a increased both the number of total megakaryocytes and the ratio of the platelet-producing megakaryocytes to total megakaryocytes, while GM1 did not influence the number of total megakaryocytes, but increased the ratio. Gangliosides GM2, GM3 and GD1b showed little effect on either the number of total megakaryocytes or the ratio. The results suggest that ganglioside GD1a stimulates at least two steps of megakaryocyte maturation, the change of megakaryocytic progenitors to megakaryocytes and the subsequent maturation of megakaryocytes to the platelet-producing megakaryocytes, while GM1 stimulates only the latter step of the maturation.

  9. Levamisole enhances immune response by affecting the activation and maturation of human monocyte-derived dendritic cells

    Science.gov (United States)

    Chen, L-Y; Lin, Y-L; Chiang, B-L

    2008-01-01

    Levamisole is a synthetic phenylimidazolthiazole that was first introduced in 1966 as an anti-helmintic agent. Current studies have been focused upon its effect on immune response and on cancer treatment. We examined the molecular mechanisms of levamisole in the activation and maturation of human monocyte-derived dendritic cells (DC) and human T cells. Treatment of DC with levamisole increased the presentation of CD80, CD86, CD83 and human leucocyte antigen D-related (HLA-DR) molecules on the cell membrane, as well as the production of interleukin (IL)-12 p40 and IL-10. Levamisole-treated human DC also enhanced T cell activation towards type 1 T helper immune response by inducing interferon-γ secretion. Neutralization with antibodies against Toll-like receptor (TLR)-2 inhibited levamisole-induced production of IL-12 p40 and IL-10, suggesting a vital role for TLR-2 in signalling DC upon incubation with levamisole. The inhibition of nuclear factor-κB, extracellular signal-regulated kinases 1/2 or c-Jun N-terminal kinases pathways also prevented the effects of levamisole on DC in producing IL-12 p40 or IL-10. Taken together, levamisole could enhance immune response towards T helper 1 development through the activation of dendritic cells or T cell aspects. PMID:18005262

  10. Eco-genetic model to explore fishing-induced ecological and evolutionary effects on growth and maturation schedules.

    Science.gov (United States)

    Wang, Hui-Yu; Höök, Tomas O

    2009-08-01

    Eco-genetic individual-based models involve tracking the ecological dynamics of simulated individual organisms that are in part characterized by heritable parameters. We developed an eco-genetic individual-based model to explore ecological and evolutionary interactions of fish growth and maturation schedules. Our model is flexible and allows for exploration of the effects of heritable growth rates (based on von Bertalanffy and biphasic growth patterns), heritable maturation schedules (based on maturation reaction norm concepts), or both on individual- and population-level traits. In baseline simulations with rather simple ecological trade-offs and over a relatively short time period (<200 simulation years), simulated male and female fish evolve differential genetic growth and maturation. Further, resulting patterns of genetically determined growth and maturation are influenced by mortality rate and density-dependent processes, and maturation and growth parameters interact to mediate the evolution of one another. Subsequent to baseline simulations, we conducted experimental simulations to mimic fisheries harvest with two size-limits (targeting large or small fish), an array of fishing mortality rates, and assuming a deterministic or stochastic environment. Our results suggest that fishing with either size-limit may induce considerable changes in life-history trait expression (maturation schedules and growth rates), recruitment, and population abundance and structure. However, targeting large fish would cause more adverse genetic effects and may lead to a population less resilient to environmental stochasticity.

  11. Induced adult stem (iAS) cells and induced transit amplifying progenitor (iTAP) cells-a possible alternative to induced pluripotent stem (iPS) cells?

    Science.gov (United States)

    Heng, Boon Chin; Richards, Mark; Ge, Zigang; Shu, Yimin

    2010-02-01

    The successful derivation of iPSC lines effectively demonstrates that it is possible to reset the 'developmental clock' of somatic cells all the way back to the initial embryonic state. Hence, it is plausible that this clock may instead be turned back half-way to a less immature developmental stage that is more directly applicable to clinical therapeutic applications or for in vitro pharmacology/toxicology screening assays. Such a suitable developmental state is postulated to be either the putative transit amplifying progenitor stage or adult stem cell stage. It is hypothetically possible to reprogram mature and terminally differentiated somatic cells back to the adult stem cell or transit amplifying progenitor stage, in a manner similar to the derivation of iPSC. It is proposed that the terminology 'Induced Adult Stem Cells' (iASC) or 'Induced Transit Amplifying Progenitor Cells' (iTAPC) be used to described such reprogrammed somatic cells. Of particular interest, is the possibility of resetting the developmental clock of mature differentiated somatic cells of the mesenchymal lineage, explanted from adipose tissue, bone marrow and cartilage. The putative adult stem cell sub-population from which these cells are derived, commonly referred to as 'mesenchymal stem cells', are highly versatile and hold much therapeutic promise in regenerative medicine, as attested to by numerous human clinical trials and animal studies. Perhaps it may be appropriate to term such reprogrammed cells as 'Induced Mesenchymal Stem Cells' (iMSC) or as 'Induced Mesenchumal Progenitor Cells' (iMPC). Given that cells from the same organ/tissue will share some commonalities in gene expression, we hypothesize that the generation of iASC or iTAPC would be more efficient as compared to iPSC generation, since a common epigenetic program must exist between the reprogrammed cells, adult stem cell or progenitor cell types and terminally differentiated cell types from the same organ/tissue.

  12. Acceleration of Functional Maturation and Differentiation of Neonatal Porcine Islet Cell Monolayers Shortly In Vitro Cocultured with Microencapsulated Sertoli Cells

    Directory of Open Access Journals (Sweden)

    Francesca Mancuso

    2010-01-01

    Full Text Available The limited availability of cadaveric human donor pancreata as well as the incomplete success of the Edmonton protocol for human islet allografts fasten search for new sources of insulin the producing cells for substitution cell therapy of insulin-dependent diabetes mellitus (T1DM. Starting from isolated neonatal porcine pancreatic islets (NPIs, we have obtained cell monolayers that were exposed to microencapsulated monolayered Sertoli cells (ESCs for different time periods (7, 14, 21 days. To assess the development of the cocultured cell monolayers, we have studied either endocrine cell phenotype differentiation markers or c-kit, a hematopoietic stem cell marker, has recently been involved with growth and differentiation of β-cell subpopulations in human as well as rodent animal models. ESC which were found to either accelerate maturation and differentiation of the NPIs β-cell phenotype or identify an islet cell subpopulation that was marked positively for c-kit. The insulin/c-kit positive cells might represent a new, still unknown functionally immature β-cell like element in the porcine pancreas. Acceleration of maturation and differentiation of our NPI cell monolayers might generate a potential new opportunity to develop insulin-producing cells that may suite experimental trials for cell therapy of T1DM.

  13. Role for a novel Usher protein complex in hair cell synaptic maturation.

    Directory of Open Access Journals (Sweden)

    Marisa Zallocchi

    Full Text Available The molecular mechanisms underlying hair cell synaptic maturation are not well understood. Cadherin-23 (CDH23, protocadherin-15 (PCDH15 and the very large G-protein coupled receptor 1 (VLGR1 have been implicated in the development of cochlear hair cell stereocilia, while clarin-1 has been suggested to also play a role in synaptogenesis. Mutations in CDH23, PCDH15, VLGR1 and clarin-1 cause Usher syndrome, characterized by congenital deafness, vestibular dysfunction and retinitis pigmentosa. Here we show developmental expression of these Usher proteins in afferent spiral ganglion neurons and hair cell synapses. We identify a novel synaptic Usher complex comprised of clarin-1 and specific isoforms of CDH23, PCDH15 and VLGR1. To establish the in vivo relevance of this complex, we performed morphological and quantitative analysis of the neuronal fibers and their synapses in the Clrn1-/- mouse, which was generated by incomplete deletion of the gene. These mice showed a delay in neuronal/synaptic maturation by both immunostaining and electron microscopy. Analysis of the ribbon synapses in Ames waltzer(av3J mice also suggests a delay in hair cell synaptogenesis. Collectively, these results show that, in addition to the well documented role for Usher proteins in stereocilia development, Usher protein complexes comprised of specific protein isoforms likely function in synaptic maturation as well.

  14. V(D)J recombination in mature B cells: a mechanism for altering antibody responses.

    Science.gov (United States)

    Papavasiliou, F; Casellas, R; Suh, H; Qin, X F; Besmer, E; Pelanda, R; Nemazee, D; Rajewsky, K; Nussenzweig, M C

    1997-10-10

    The clonal selection theory states that B lymphocytes producing high-affinity immunoglobulins are selected from a pool of cells undergoing antibody gene mutation. Somatic hypermutation is a well-documented mechanism for achieving diversification of immune responses in mature B cells. Antibody genes were also found to be modified in such cells in germinal centers by recombination of the variable (V), diversity (D), and joining (J) segments. The ability to alter immunoglobulin expression by V(D)J recombination in the selective environment of the germinal center may be an additional mechanism for inactivation or diversification of immune responses.

  15. MIF Synergizes with Trypanosoma cruzi Antigens to Promote Efficient Dendritic Cell Maturation and IL-12 Production via p38 MAPK

    Science.gov (United States)

    Terrazas, Cesar A.; Huitron, EriK; Vazquez, Alicia; Juarez, Imelda; Camacho, Griselda M.; Calleja, Elsa A.; Rodriguez-Sosa, Miriam

    2011-01-01

    Macrophage migration inhibitory factor (MIF) has been found to be involved in host resistance to several parasitic infections. To determine the mechanisms of the MIF-dependent responses to Trypanosoma cruzi, we investigated host resistance in MIF-/- mice (on the BALB/c background) during an intraperitoneal infection. We focused on the potential involvement of MIF in dendritic cell (DC) maturation and cytokine production. Following a challenge with 5 x 103 T. cruzi parasites, wild type (WT) mice developed a strong IL-12 response and adequate maturation of the draining mesenteric lymph node DCs and were resistant to infection. In contrast, similarly infected MIF-/- mice mounted a weak IL-12 response, displayed immature DCs in the early phases of infection and rapidly succumbed to T. cruzi infection. The lack of maturation and IL-12 production by the DCs in response to total T. cruzi antigen (TcAg) was confirmed by in vitro studies. These effects were reversed following treatment with recombinant MIF. Interestingly, TcAg-stimulated bone marrow-derived DCs from both WT and MIF-/- mice had increased ERK1/2 MAPK phosphorylation. In contrast, p38 phosphorylation was only upregulated in WT DCs. Reconstitution of MIF to MIF-/- DCs upregulated p38 phosphorylation. The MIF-p38 pathway affected MHC-II and CD86 expression as well as IL-12 production. These findings demonstrate that the MIF-induced early DC maturation and IL-12 production mediates resistance to T. cruzi infection, probably by activating the p38 pathway. PMID:22110382

  16. Engineered Microenvironments for the Maturation and Observation of Human Embryonic Stem Cell Derived Cardiomyocytes

    Science.gov (United States)

    Salick, Max R.

    The human heart is a dynamic system that undergoes substantial changes as it develops and adapts to the body's growing needs. To better understand the physiology of the heart, researchers have begun to produce immature heart muscle cells, or cardiomyocytes, from pluripotent stem cell sources with remarkable efficiency. These stem cell-derived cardiomyocytes hold great potential in the understanding and treatment of heart disease; however, even after prolonged culture, these cells continue to exhibit an immature phenotype, as indicated by poor sarcomere organization and calcium handling, among other features. The lack of maturation that is observed in these cardiomyocytes greatly limits their applicability towards drug screening, disease modeling, and cell therapy applications. The mechanical environment surrounding a cell has been repeatedly shown to have a large impact on that cell's behavior. For this reason, we have implemented micropatterning methods to mimic the level of alignment that occurs in the heart in vivo in order to study how this alignment may help the cells to produce a more mature sarcomere phenotype. It was discovered that the level of sarcomere organization of a cardiomyocyte can be strongly influenced by the micropattern lane geometry on which it adheres. Steps were taken to optimize this micropattern platform, and studies of protein organization, gene expression, and myofibrillogenesis were conducted. Additionally, a set of programs was developed to provide quantitative analysis of the level of sarcomere organization, as well as to assist with several other tissue engineering applications.

  17. Delayed functional maturation of natural regulatory T cells in the medulla of postnatal thymus: role of TSLP

    Directory of Open Access Journals (Sweden)

    Knudsen Geoffry

    2006-04-01

    Full Text Available Abstract Background Generation of functional CD4+CD8-CD25+ regulatory T cells (Treg in the murine thymus depends on FoxP3. Removal of the thymus from neonatal mice has been shown to result in a multiple organ autoimmune disease phenotype that can be prevented by introducing the FoxP3+ Treg population to the animal. It has therefore, been proposed that functional FoxP3+ Treg cells are not made in the neonatal thymus; however, it remains unclear when and where functional FoxP3+CD4+CD8-CD25+ thymocytes are generated in postnatal thymus. Results We report that neither FoxP3 mRNA nor protein is expressed in CD4+CD8-CD25+, or CD4+CD8-CD25- thymocytes until 3–4 days post birth, despite the presence of mature CD4+CD8-CD25+/- thymocytes in the thymus by 1–2 days after birth. FoxP3-CD4+CD8-CD25+ thymocytes from day 2 newborn mice show no Treg activity. Interestingly, we are able to detect low numbers of FoxP3+ thymocytes dispersed throughout the medullary region of the thymus as early as 3–4 days post birth. Expression of FoxP3 is induced in embryonic day 17 fetal thymus organ culture (FTOC after 4–6 days of in vitro culture. Treatment of FTOCs with thymic stromal derived lymphopoietin (TSLP enhanced expression of FoxP3, and blocking the TSLP receptor reduces FoxP3 expression in FTOC. Furthermore, TSLP stimulates FoxP3 expression in purified CD4+CD8- thymocytes, but not in CD4+CD8+, CD4-CD8+ and CD4-CD8- thymocytes. Conclusion Expression of FoxP3 or Treg maturation is ontogenically distinct and kinetically delayed from the generation of CD4+CD8-CD25+ or CD4+CD8-CD25- thymocytes in the postnatal thymus. TSLP produced from medullary thymic epithelia cells (mTEC contributes to the expression of FoxP3 and the maturation of natural regulatory T cells. Overall, these results suggest that the development of Treg cells requires paracrine signaling during late stages of thymocyte maturation that is distinct from signaling during positive or negative

  18. Cell signalling pathways underlying induced pluripotent stem cell reprogramming

    Institute of Scientific and Technical Information of China (English)

    Kate; Hawkins; Shona; Joy; Tristan; Mc; Kay

    2014-01-01

    Induced pluripotent stem(i PS) cells, somatic cells reprogrammed to the pluripotent state by forced expression of defined factors, represent a uniquely valuable resource for research and regenerative medicine. However, this methodology remains inefficient due to incomplete mechanistic understanding of the reprogramming process. In recent years, various groups have endeavoured to interrogate the cell signalling that governs the reprogramming process, including LIF/STAT3, BMP, PI3 K, FGF2, Wnt, TGFβ and MAPK pathways, with the aim of increasing our understanding and identifying new mechanisms of improving safety, reproducibility and efficiency. This has led to a unified model of reprogramming that consists of 3 stages: initiation, maturation and stabilisation. Initiation of reprogramming occurs in almost all cells that receive the reprogramming transgenes; most commonly Oct4, Sox2, Klf4 and c Myc, and involves a phenotypic mesenchymal-to-epithelial transition. The initiation stage is also characterised by increased proliferation and a metabolic switch from oxidative phosphorylation to glycolysis. The maturation stage is considered the major bottleneck within the process, resulting in very few "stabilisation competent" cells progressing to the final stabilisation phase. To reach this stage in both mouse and human cells, pre-i PS cells must activate endogenous expression of the core circuitry of pluripotency, comprising Oct4, Sox2, and Nanog, and thus reach a state of transgene independence. By the stabilisation stage, i PS cells generally use the same signalling networks that govern pluripotency in embryonic stem cells. These pathways differ between mouse and human cells although recent work has demonstrated that this is context dependent. As i PS cell generation technologies move forward, tools are being developed to interrogate the process in more detail, thus allowing a greater understanding of this intriguing biological phenomenon.

  19. Developmental maturation of passive electrical properties in retinal ganglion cells of rainbow trout.

    Science.gov (United States)

    Picones, Arturo; Chung, S Clare; Korenbrot, Juan I

    2003-04-01

    We investigated the electrotonic and anatomical features of the dendritic arbor in developing retinal ganglion cells (RGCs). Cell anatomy was studied by filling individual cells with fluorescent, membrane-bound dyes and using computer-assisted image reconstruction. Electrotonic properties were characterized through an analysis of charging membrane currents measured with tight-seal electrodes in the whole-cell mode. We studied developing RGCs in the peripheral growth zone (PGZ) of a fish retina. The PGZ presents a developmental time-line ranging from pluripotent, proliferating cells at the extreme edge, to mature, fully developed retina more centrally. In the PGZ, RGCs mature through three histologically distinct zones (in developmental sequence): bulge, transition and mature zones. In the most peripheral three-quarters of the bulge zone, cells have rounded somas, lack dendritic extensions and some are coupled so that membrane-bound dyes traverse from one cell to its immediate neighbours. In the more central quarter of the bulge, cells' dendrites are few, short and of limited branching. In the transition zone dendritic arbors becomes progressively more expansive and branched and we present a morphometric analysis of these changes. Regardless of the size and branching pattern of the developing RGC dendritic arbor, the ratio of the diameters of parent and progeny dendrites at any branching nodes is well described by Rall's 3/2 power law. Given this anatomical feature, the RGC passive electrical properties are well described by an equivalent electrical circuit consisting of an isopotential cell body in parallel with a single equivalent cylinder of finite length. We measured the values of the electrical parameters that define this equivalent circuit in bulge, transition and mature RGCs. As RGCs develop the electrical properties of their dendritic arbor change in an orderly and tightly regulated manner, not randomly. Electrically, dendritic arbors develop along either of

  20. Gene expression profiling in multipotent DFAT cells derived from mature adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Ono, Hiromasa [Laboratory of Cell and Tissue Biology, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510 (Japan); Database Center for Life Science (DBCLS), Research Organization of Information and Systems (ROIS), Faculty of Engineering Bldg.12 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Oki, Yoshinao [Laboratory of Cell and Tissue Biology, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510 (Japan); Bono, Hidemasa [Database Center for Life Science (DBCLS), Research Organization of Information and Systems (ROIS), Faculty of Engineering Bldg.12 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Kano, Koichiro, E-mail: kkano@brs.nihon-u.ac.jp [Laboratory of Cell and Tissue Biology, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510 (Japan)

    2011-04-15

    Highlights: {yields} Adipocyte dedifferentiation is evident in a significant decrease in typical genes. {yields} Cell proliferation is strongly related to adipocyte dedifferentiation. {yields} Dedifferentiated adipocytes express several lineage-specific genes. {yields} Comparative analyses using publicly available datasets boost the interpretation. -- Abstract: Cellular dedifferentiation signifies the withdrawal of cells from a specific differentiated state to a stem cell-like undifferentiated state. However, the mechanism of dedifferentiation remains obscure. Here we performed comparative transcriptome analyses during dedifferentiation in mature adipocytes (MAs) to identify the transcriptional signatures of multipotent dedifferentiated fat (DFAT) cells derived from MAs. Using microarray systems, we explored similarly expressed as well as significantly differentially expressed genes in MAs during dedifferentiation. This analysis revealed significant changes in gene expression during this process, including a significant reduction in expression of genes for lipid metabolism concomitantly with a significant increase in expression of genes for cell movement, cell migration, tissue developmental processes, cell growth, cell proliferation, cell morphogenesis, altered cell shape, and cell differentiation. Our observations indicate that the transcriptional signatures of DFAT cells derived from MAs are summarized in terms of a significant decrease in functional phenotype-related genes and a parallel increase in cell proliferation, altered cell morphology, and regulation of the differentiation of related genes. A better understanding of the mechanisms involved in dedifferentiation may enable scientists to control and possibly alter the plasticity of the differentiated state, which may lead to benefits not only in stem cell research but also in regenerative medicine.

  1. Giardia duodenalis stimulates partial maturation of bovine dendritic cells associated with altered cytokine secretion and induction of T-cell proliferation.

    Science.gov (United States)

    Grit, G H; Devriendt, B; Van Coppernolle, S; Geurden, T; Hope, J; Vercruysse, J; Cox, E; Geldhof, P; Claerebout, E

    2014-04-01

    Giardia duodenalis is an important intestinal parasite in animals and humans. The role of dendritic cells (DC) in the initiation of the immune response against G. duodenalis is poorly documented. The aim of this study was to test the hypothesis that G. duodenalis interferes with bovine DC function. Therefore, the effect of trophozoites and excretion/secretion products on bovine monocyte-derived dendritic cells (MoDC) was investigated. We assessed MoDC maturation and cytokine production of G. duodenalis-stimulated MoDC and the ability of these MoDC to take up antigen and induce lymphocyte proliferation. Little or no upregulation of maturation markers CD40 and CD80 was measured, but MHCII expression was increased after stimulation with low parasite concentrations. A dose-dependent decrease in ovalbumin uptake was observed in G. duodenalis-stimulated MoDC. In addition, stimulated MoDC induced proliferation of CD3(-) , γδ-T-cells and TCRαβ(+) CD4(+) and CD8(+) T-cells. Increased transcription of TGF-β was shown in CD4(+) T cells, and increased TNF-α, TGF-β, IL-10 and IL-4 were seen in γδ-T-cells. We found no evidence that G. duodenalis has a regulatory or inhibitory effect on bovine MoDC. MoDC stimulated with G. duodenalis are functionally active and able to induce proliferation of T cells that produce both pro- and anti-inflammatory cytokines.

  2. Cell wall chemical characteristics of whole‐crop cereal silages harvested at three maturity stages

    OpenAIRE

    Wallsten, Johanna; Hatfield, Ronald

    2016-01-01

    Abstract Background In cooler climates such as found in Scandinavian countries cereals are important feedstuffs for ruminants often ensiled as whole‐crop cereal silages (WCCS) to preserve nutrients. Animal performance varies with the type of cereal forage and stage of cereal development being ensiled. Cell wall isolation and analysis was undertaken to determine differences among cereal silages harvested at different stages of maturity. Results A set of 27 WCCS samples of barley, wheat and oat...

  3. A preliminary comparison of dendritic cell maturation by total cellular RNA to total cellular lysate derived from breast cancer stem cells

    Directory of Open Access Journals (Sweden)

    Phong Minh Le

    2016-06-01

    Full Text Available Introduction: Dendritic cells (DCs have been widely considered as the most potent antigen-presenting cells. As such, DC-based vaccines are regarded as a promising strategy in cancer vaccination and therapy. This study compared the maturation of DCs induced by total cellular RNA and cell lysate (i.e. nucleic acid and protein. Methods: Both total RNA and cell lysate were isolated from breast cancer stem cells (BCSCs. The lysates were used to incubate with monocyte-derived immature DCs. To track the transfection efficiency, the BCSCs were stably transfected with green fluorescent protein (GFP. The maturation of DCs was evaluated by expression of costimulatory molecules including CD40, CD80, and CD86. Transfections were confirmed by evaluating GFP expression in DCs at 24 hours post transfection. Results: The results of this study showed that GFP is expressed in DCs after both total RNA and lysate incubation. The expression of costimulatory molecules (CD40, CD80, and CD86 was significantly higher in RNA-transfected DCs than in cell lysate-primed DCs. Conclusion: Our findings suggest that total RNA primed BCSCs can be a suitable platform for DC-based vaccine therapy of breast cancer. [Biomed Res Ther 2016; 3(6.000: 679-686

  4. Effects of dextromethorphan on glial cell function: proliferation, maturation, and protection from cytotoxic molecules.

    Science.gov (United States)

    Lisak, Robert P; Nedelkoska, Liljana; Benjamins, Joyce A

    2014-05-01

    Dextromethorphan (DM), a sigma receptor agonist and NMDA receptor antagonist, protects neurons from glutamate excitotoxicity, hypoxia and ischemia, and inhibits microglial activation, but its effects on differentiation and protection of cells in the oligodendroglial lineage are unknown. It is important to protect oligodendroglia (OL) to prevent demyelination and preserve axons, and to protect oligodendroglial progenitors (OPC) to optimize myelination during development and remyelination following damage. Enriched glial cultures from newborn rat brain were used 1-2 days or 6-8 days after shakeoff for OPC or mature OL. DM had large effects on glial proliferation in less mature cultures in contrast to small variable effects in mature cultures; 1 μM DM stimulated proliferation of OPC by 4-fold, microglia (MG) by 2.5-fold and astroglia (AS) by 2-fold. In agreement with increased OPC proliferation, treatment of OPC with DM for 3 days increased the % of OPC relative to OL, with a smaller difference by 5 days, suggesting that maturation of OPC to OL was "catching up" by 5 days. DM at 2 and 20 μM protected both OL and OPC from killing by glutamate as well as NMDA, AMPA, quinolinic acid, staurosporine, and reactive oxygen species (ROS). DM did not protect against kynurenic acid, and only modestly against NO. These agents and DM were not toxic to AS or MG at the concentrations used. Thus, DM stimulates proliferation of OPC, and protects both OL and OPC against excitotoxic and inflammatory insults.

  5. Cdc42-dependent actin dynamics controls maturation and secretory activity of dendritic cells

    Science.gov (United States)

    Schulz, Anna M.; Stutte, Susanne; Hogl, Sebastian; Luckashenak, Nancy; Dudziak, Diana; Leroy, Céline; Forné, Ignasi; Imhof, Axel; Müller, Stephan A.; Brakebusch, Cord H.; Lichtenthaler, Stefan F.

    2015-01-01

    Cell division cycle 42 (Cdc42) is a member of the Rho guanosine triphosphatase family and has pivotal functions in actin organization, cell migration, and proliferation. To further study the molecular mechanisms of dendritic cell (DC) regulation by Cdc42, we used Cdc42-deficient DCs. Cdc42 deficiency renders DCs phenotypically mature as they up-regulate the co-stimulatory molecule CD86 from intracellular storages to the cell surface. Cdc42 knockout DCs also accumulate high amounts of invariant chain–major histocompatibility complex (MHC) class II complexes at the cell surface, which cannot efficiently present peptide antigens (Ag’s) for priming of Ag-specific CD4 T cells. Proteome analyses showed a significant reduction in lysosomal MHC class II–processing proteins, such as cathepsins, which are lost from DCs by enhanced secretion. As these effects on DCs can be mimicked by chemical actin disruption, our results propose that Cdc42 control of actin dynamics keeps DCs in an immature state, and cessation of Cdc42 activity during DC maturation facilitates secretion as well as rapid up-regulation of intracellular molecules to the cell surface. PMID:26553928

  6. Shh promotes sweat gland cell maturation in three-dimensional culture.

    Science.gov (United States)

    Huang, Zhijian; Zhen, Yunfang; Yin, Wei; Ma, Zhourui; Zhang, Liya

    2016-06-01

    Sweat gland (SG) cells forming SG tubule-like structures in 3D culture, this is one of the most important methods to identify the biological function of SG cells and stem cells-derived SG-like cells, but also the important way on research of SG regeneration in vitro. In this study, we seeded human fibroblasts and SG cells in gels and used immunohistochemistry to confirm whether SG tubule-like structures formed. Fibroblasts are necessary factor in the process of SG cells maturation and forming SG's secretory region in 3D culture. Further experimentation revealed that Sonic hedgehog (Shh) was secreted by fibroblasts within the 3D culture. By adding Shh protein to 3D culture, there had more SG tubule-like structures formed. These results suggest that Shh is an important factor during the process of forming SG tubule-like structures in 3D cultures, and adding Shh recommbinant protein could promote SG cell maturation and enhance the efficiency of structure formation.

  7. Expression of tryptophan 2,3-dioxygenase in mature granule cells of the adult mouse dentate gyrus

    Directory of Open Access Journals (Sweden)

    Ohira, Koji

    2010-09-01

    Full Text Available Abstract New granule cells are continuously generated in the dentate gyrus of the adult hippocampus. During granule cell maturation, the mechanisms that differentiate new cells not only describe the degree of cell differentiation, but also crucially regulate the progression of cell differentiation. Here, we describe a gene, tryptophan 2,3-dioxygenase (TDO, whose expression distinguishes stem cells from more differentiated cells among the granule cells of the adult mouse dentate gyrus. The use of markers for proliferation, neural progenitors, and immature and mature granule cells indicated that TDO was expressed in mature cells and in some immature cells. In mice heterozygous for the alpha-isoform of calcium/calmodulin-dependent protein kinase II, in which dentate gyrus granule cells fail to mature normally, TDO immunoreactivity was substantially downregulated in the dentate gyrus granule cells. Moreover, a 5-bromo-2'-deoxyuridine labeling experiment revealed that new neurons began to express TDO between 2 and 4 wk after the neurons were generated, when the axons and dendrites of the granule cells developed and synaptogenesis occurred. These findings indicate that TDO might be required at a late-stage of granule cell development, such as during axonal and dendritic growth, synaptogenesis and its maturation.

  8. Regulation of cathepsins S and L by cystatin F during maturation of dendritic cells.

    Science.gov (United States)

    Magister, Spela; Obermajer, Nataša; Mirković, Bojana; Svajger, Urban; Renko, Miha; Softić, Adaleta; Romih, Rok; Colbert, Jeff D; Watts, Colin; Kos, Janko

    2012-05-01

    In dendritic cells (DCs) cysteine cathepsins play a key role in antigen processing, invariant chain (Ii) cleavage and regulation of cell adhesion after maturation stimuli. Cystatin F, a cysteine protease inhibitor, is present in DCs in endosomal/lysosomal vesicles and thus has a potential to modulate cathepsin activity. In immature DCs cystatin F colocalizes with cathepsin S. After induction of DC maturation however, it is translocated into lysosomes and colocalizes with cathepsin L. The inhibitory potential of cystatin F depends on the properties of the monomer. We showed that the full-length monomeric cystatin F was a 12-fold stronger inhibitor of cathepsin S than the N-terminally processed cystatin F, whereas no significant difference in inhibition was observed for cathepsins L, H and X. Therefore, the role of cystatin F in regulating the main cathepsin S function in DCs, i.e. the processing of Ii, may depend on the form of the monomer present in endosomal/lysosomal vesicles. On the other hand, intact and truncated monomeric cystatin F are both potent inhibitors of cathepsin L and it is likely that cystatin F could regulate its activity in maturing, adherent DCs, controlling the processing of procathepsin X, which promotes cell adhesion via activation of Mac-1 (CD11b/CD18) integrin receptor.

  9. Jak3 is involved in dendritic cell maturation and CCR7-dependent migration.

    Directory of Open Access Journals (Sweden)

    Ana Rivas-Caicedo

    Full Text Available BACKGROUND: CCR7-mediated signalling is important for dendritic cell maturation and homing to the lymph nodes. We have previously demonstrated that Jak3 participates in the signalling pathway of CCR7 in T lymphocytes. METHODOLOGY AND PRINCIPAL FINDINGS: Here, we used Jak3(-/- mice to analyze the role of Jak3 in CCR7-mediated dendritic cells migration and function. First, we found no differences in the generation of DCs from Jak3(-/- bone marrow progenitors, when compared to wild type cells. However, phenotypic analysis of the bone marrow derived DCs obtained from Jak3(-/- mice showed reduced expression of co-stimulatory molecules compared to wild type (Jak3(+/+. In addition, when we analyzed the migration of Jak3(-/- and Jak3(+/+ mature DCs in response to CCL19 and CCL21 chemokines, we found that the absence of Jak3 results in impaired chemotactic responses both in vitro and in vivo. Moreover, lymphocyte proliferation and contact hypersensitivity experiments showed that DC-mediated T lymphocyte activation is reduced in the absence of Jak3. CONCLUSION/SIGNIFICANCE: Altogether, our data provide strong evidence that Jak3 is important for DC maturation, migration and function, through a CCR7-mediated signalling pathway.

  10. A novel MEK-ERK-AMPK signaling axis controls chemokine receptor CCR7-dependent survival in human mature dendritic cells.

    Science.gov (United States)

    López-Cotarelo, Pilar; Escribano-Díaz, Cristina; González-Bethencourt, Ivan Luis; Gómez-Moreira, Carolina; Deguiz, María Laura; Torres-Bacete, Jesús; Gómez-Cabañas, Laura; Fernández-Barrera, Jaime; Delgado-Martín, Cristina; Mellado, Mario; Regueiro, José Ramón; Miranda-Carús, María Eugenia; Rodríguez-Fernández, José Luis

    2015-01-09

    Chemokine receptor CCR7 directs mature dendritic cells (mDCs) to secondary lymph nodes where these cells regulate the activation of T cells. CCR7 also promotes survival in mDCs, which is believed to take place largely through Akt-dependent signaling mechanisms. We have analyzed the involvement of the AMP-dependent kinase (AMPK) in the control of CCR7-dependent survival. A pro-apoptotic role for AMPK is suggested by the finding that pharmacological activators induce apoptosis, whereas knocking down of AMPK with siRNA extends mDC survival. Pharmacological activation of AMPK also induces apoptosis of mDCs in the lymph nodes. Stimulation of CCR7 leads to inhibition of AMPK, through phosphorylation of Ser-485, which was mediated by G(i)/Gβγ, but not by Akt or S6K, two kinases that control the phosphorylation of AMPK on Ser-485 in other settings. Using selective pharmacological inhibitors, we show that CCR7-induced phosphorylation of AMPK on Ser-485 is mediated by MEK and ERK. Coimmunoprecipitation analysis and proximity ligation assays indicate that AMPK associates with ERK, but not with MEK. These results suggest that in addition to Akt-dependent signaling mechanisms, CCR7 can also promote survival of mDCs through a novel MEK1/2-ERK1/2-AMPK signaling axis. The data also suggest that AMPK may be a potential target to modulate mDC lifespan and the immune response.

  11. Gene expression profiling in multipotent DFAT cells derived from mature adipocytes.

    Science.gov (United States)

    Ono, Hiromasa; Oki, Yoshinao; Bono, Hidemasa; Kano, Koichiro

    2011-04-15

    Cellular dedifferentiation signifies the withdrawal of cells from a specific differentiated state to a stem cell-like undifferentiated state. However, the mechanism of dedifferentiation remains obscure. Here we performed comparative transcriptome analyses during dedifferentiation in mature adipocytes (MAs) to identify the transcriptional signatures of multipotent dedifferentiated fat (DFAT) cells derived from MAs. Using microarray systems, we explored similarly expressed as well as significantly differentially expressed genes in MAs during dedifferentiation. This analysis revealed significant changes in gene expression during this process, including a significant reduction in expression of genes for lipid metabolism concomitantly with a significant increase in expression of genes for cell movement, cell migration, tissue developmental processes, cell growth, cell proliferation, cell morphogenesis, altered cell shape, and cell differentiation. Our observations indicate that the transcriptional signatures of DFAT cells derived from MAs are summarized in terms of a significant decrease in functional phenotype-related genes and a parallel increase in cell proliferation, altered cell morphology, and regulation of the differentiation of related genes. A better understanding of the mechanisms involved in dedifferentiation may enable scientists to control and possibly alter the plasticity of the differentiated state, which may lead to benefits not only in stem cell research but also in regenerative medicine.

  12. Accumulation of cytolytic CD8{sup +} T cells in B16-melanoma and proliferation of mature T cells in TIS21-knockout mice after T cell receptor stimulation

    Energy Technology Data Exchange (ETDEWEB)

    Ryu, Min Sook [Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, 164, World cul-ro, Yeongtong-gu, Suwon, Gyeonggi-do 443-380 (Korea, Republic of); Woo, Min-Yeong [Department of Microbiology, Ajou University School of Medicine, 164, World cul-ro, Yeongtong-gu, Suwon, Gyeonggi-do 443-380 (Korea, Republic of); Department of Biomedical Sciences, The Graduate School, Ajou University (Korea, Republic of); Kwon, Daeho [Department of Microbiology, Kwandong University College of Medicine, Gangneung, Gangwon-do 210-701 (Korea, Republic of); Hong, Allen E. [Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, 164, World cul-ro, Yeongtong-gu, Suwon, Gyeonggi-do 443-380 (Korea, Republic of); Song, Kye Yong [Department of Pathology, Chung-Ang University College of Medicine, Dongjak-gu, Seoul 156-756 (Korea, Republic of); Park, Sun [Department of Microbiology, Ajou University School of Medicine, 164, World cul-ro, Yeongtong-gu, Suwon, Gyeonggi-do 443-380 (Korea, Republic of); Lim, In Kyoung [Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, 164, World cul-ro, Yeongtong-gu, Suwon, Gyeonggi-do 443-380 (Korea, Republic of)

    2014-10-01

    In vivo and in vitro effects of TIS21 gene on the mature T cell activation and antitumor activities were explored by employing MO5 melanoma orthograft and splenocytes isolated from the TIS21-knockout (KO) mice. Proliferation and survival of mature T cells were significantly increased in the KO than the wild type (WT) cells, indicating that TIS21 inhibits the rate of mature T cell proliferation and its survival. In MO5 melanoma orthograft model, the KO mice recruited much more CD8{sup +} T cells into the tumors at around day 14 after tumor cell injection along with reduced tumor volumes compared with the WT. The increased frequency of granzyme B{sup +} CD8{sup +} T cells in splenocytes of the KO mice compared with the WT may account for antitumor-immunity of TIS21 gene in the melanoma orthograft. In contrast, reduced frequencies of CD107a{sup +} CD8{sup +} T cells in the splenocytes of KO mice may affect the loss of CD8{sup +} T cell infiltration in the orthograft at around day 19. These results indicate that TIS21 exhibits antiproliferative and proapoptotic effects in mature T cells, and differentially affects the frequencies of granzyme B{sup +} CD8{sup +} T-cells and CD107a{sup +} CD8{sup +} T-cells, thus transiently regulating in vivo anti-tumor immunity. - Highlights: • Constitutive expression of TIS21 in splenocytes and upregulation by TCR stimulation. • Proliferation of mature T-cells in spleen of TIS21KO mice after TCR stimulation. • Inhibition of cell death in mature T-cells of TIS21KO mice compared with the wild type. • Inhibition of melanoma growth in TIS21KO mice and CD8{sup +} T cell infiltration in tumor. • Reduction of CD 107{sup +}CD8{sup +} T cells, but increased granzyme B{sup +} CD8{sup +} T cells in TIS21KO mice.

  13. Modulation of phenotypic and functional maturation of murine dendritic cells (DCs) by purified Achyranthes bidentata polysaccharide (ABP).

    Science.gov (United States)

    Zou, Yaxuan; Meng, Jingjuan; Chen, Wenna; Liu, Jingling; Li, Xuan; Li, Weiwei; Lu, Changlong; Shan, Fengping

    2011-08-01

    There are a large number of interactions at molecular and cellular levels between the plant polysaccharides and immune system. Plant polysaccharides present an interesting effects as immunomodulators, particularly in the induction of the cells both in innate and adaptive immune systems. Activation of DCs could improve antitumoral responses usually diminished in cancer patients, and natural adjuvants provide a possibility of inducing this activation. ABP is a purified polysaccharide isolated from Achyranthes bidentata, a traditional Chinese medicine (TCM). The aim of this study is to investigate modulation of phenotypic and functional maturation of murine DCs by ABP. Both phenotypic and functional activities were assessed with use of conventional scanning electronic microscopy (SEM) for the morphology of the DC, transmitted electron microscopy (TEM) for intracellular lysosomes inside the DC, cellular immunohistochemistry for phagocytosis by the DCs, flow cytometry (FCM) for the changes in key surface molecules, bio-assay for the activity of acidic phosphatases (ACP), and ELISA for the production of pro-inflammatory cytokine IL-12. In fact, we found that purified ABP induced phenotypic maturation revealed by increased expression of CD86, CD40, and MHC II. Functional experiments showed the down-regulation of ACP inside DCs (which occurs when phagocytosis of DCs is decreased, and antigen presentation increased with maturation). Finally, ABP increased the production of IL-12. These data reveal that ABP promotes effective activation of murine DCs. This adjuvant-like activity may have therapeutic applications in clinical settings where immune responses need boosting. It is therefore concluded that ABP can exert positive modulation to murine DCs.

  14. WIP and WICH/WIRE co-ordinately control invadopodium formation and maturation in human breast cancer cell invasion

    Science.gov (United States)

    García, Esther; Ragazzini, Chiara; Yu, Xinzi; Cuesta-García, Elena; Bernardino de la Serna, Jorge; Zech, Tobias; Sarrió, David; Machesky, Laura M.; Antón, Inés M.

    2016-01-01

    Cancer cells form actin-rich degradative protrusions (invasive pseudopods and invadopodia), which allows their efficient dispersal during metastasis. Using biochemical and advanced imaging approaches, we demonstrate that the N-WASP-interactors WIP and WICH/WIRE play non-redundant roles in cancer cell invasion. WIP interacts with N-WASP and cortactin and is essential for invadopodium assembly, whereas WICH/WIRE regulates N-WASP activation to control invadopodium maturation and degradative activity. Our data also show that Nck interaction with WIP and WICH/WIRE modulates invadopodium maturation; changes in WIP and WICH/WIRE levels induce differential distribution of Nck. We show that WIP can replace WICH/WIRE functions and that elevated WIP levels correlate with high invasiveness. These findings identify a role for WICH/WIRE in invasiveness and highlight WIP as a hub for signaling molecule recruitment during invadopodium generation and cancer progression, as well as a potential diagnostic biomarker and an optimal target for therapeutic approaches. PMID:27009365

  15. Maturation and upregulation of functions of murine dendritic cells (DCs) under the influence of purified aromatic-turmerone (AR).

    Science.gov (United States)

    Yonggang, Tan; Yiming, Meng; Heying, Zhang; Cheng, Sun; Qiushi, Wang; Xianghong, Yang; Wei, Zheng; Huawei, Zhou; Shan, Fengping

    2012-10-01

    The aim of this work is to evaluate the effects of purified aromatic-turmerone (ar-turmerione, AR) on murine dendritic cells (DCs). These impacts of AR on DCs from bone marrow derived DCs(BMDCs) were assessed with use of conventional scanning electron microscopy (SEM), fluorescence activated cell sorting (FACS), transmission electron microscopy (TEM), cytochemistry assay, FITC-dextran, bio-assay and enzyme linked immunosorbent assay (ELISA). We found that AR induced phenotypic maturation as evidenced by increased expression of CD86, CD40, CD83, CD80 and major histocompatibility complex II (MHC II). The functional tests showed the activity of acidic phosphatase (ACP) inside the DCs were downregulated after treatment with AR (which occurs when phagocytosis of DCs were decreased). Finally, we proved that AR increased the production of IL-12 and tumor necrosis factor α (TNF-α). These data suggested that AR could promote phenotypic and functional maturation of DCs and this adjuvant-like activity may have potential therapeutic value. It is therefore concluded that AR could exert positive modulation on murine DCs.

  16. Lycium barbarum polysaccharides regulate phenotypic and functional maturation of murine dendritic cells.

    Science.gov (United States)

    Zhu, Jie; Zhao, Lu-Hang; Zhao, Xiao-Ping; Chen, Zhi

    2007-06-01

    Lycium barbarum polysaccharides (LBPs) have been known to have a variety of immunomodulatory functions including activation of T cells, B cells and NK cells. Dendritic cells (DC) are potent antigen-presenting cells that play pivotal roles in the initiation of the primary immune response. However, little is known about the immunomodulatory effects of LBPs on murine bone marrow derived dendritic cells (BMDC). In the present study, the effects of LBPs on the phenotypic and functional maturation of murine BMDC were investigated in vitro. Compared to the BMDC that were only subjected to treatment with RPMI1640, the co-expression of I-A/I-E, CD11c and secretion of IL-12 p40 by BMDC stimulated with LBPs (100 microg/ml) were increased. In addition, the endocytosis of FITC-dextran by LBPs-treated BMDC (100 microg/ml) was impaired, whereas the activation of proliferation of allogenic lymphocytes by BMDC was enhanced. Our results strongly suggest that LBPs are capable of promoting both the phenotypic and functional maturation of murine BMDC in vitro.

  17. Suppression of dendritic cells' maturation and functions by daidzein, a phytoestrogen

    Energy Technology Data Exchange (ETDEWEB)

    Yum, Min Kyu; Jung, Mi Young [School of Life Sciences and Biotechnology, Korea University, Seoul 136-701 (Korea, Republic of); Cho, Daeho [Department of Life Science, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Kim, Tae Sung, E-mail: tskim@korea.ac.kr [School of Life Sciences and Biotechnology, Korea University, Seoul 136-701 (Korea, Republic of)

    2011-12-15

    Isoflavones are ubiquitous compounds in foods and in the environment in general. Daidzein and genistein, the best known of isoflavones, are structurally similar to 17{beta}-estradiol and known to exert estrogenic effects. They also evidence a broad variety of biological properties, including antioxidant, anti-carcinogenic, anti-atherogenic and anti-osteoporotic activities. Previously, daidzein was reported to increase the phagocytic activity of peritoneal macrophages and splenocyte proliferation, and to inhibit nitric oxide (NO) production in macrophages. However, its potential impacts on immune response in dendritic cells (DCs), antigen-presenting cells that link innate and adaptive immunity, have yet to be clearly elucidated. In this study, we evaluated the effects of isoflavones on the maturation and activation of DCs. Isoflavones (formononetin, daidzein, equol, biochanin A, genistein) were found to differentially affect the expression of CD86, a costimulatory molecule, on lipopolysaccharide (LPS)-stimulated DCs. In particular, daidzein significantly and dose-dependently inhibited the expression levels of maturation-associated cell surface markers including CD40, costimulatory molecules (CD80, CD86), and major histocompatibility complex class II (I-A{sup b}) molecule on LPS-stimulated DCs. Daidzein also suppressed pro-inflammatory cytokine production such as IL-12p40, IL-6 and TNF-{alpha}, whereas it didn't affect IL-10 and IL-1{beta} expression. Furthermore, daidzein enhanced endocytosis and inhibited the allo-stimulatory ability of LPS-stimulated DCs on T cells, indicating that daidzein treatment can inhibit the functional maturation of DCs. These results demonstrate that daidzein may exhibit immunosuppressive activity by inhibiting the maturation and activation of DCs. -- Highlights: Black-Right-Pointing-Pointer Daidzein inhibited expression of maturation-associated cell surface markers in DCs. Black-Right-Pointing-Pointer Daidzein suppressed expression

  18. The effects of 6-gingerol on proliferation, differentiation, and maturation of osteoblast-like MG-63 cells

    Energy Technology Data Exchange (ETDEWEB)

    Fan, J.Z.; Yang, X.; Bi, Z.G. [Department of Orthopedic Surgery, First Affiliated Hospital, Harbin Medicine University, Harbin (China)

    2015-04-28

    We investigated whether 6-gingerol affects the maturation and proliferation of osteoblast-like MG63 cells in vitro. Osteoblast-like MG63 cells were treated with 6-gingerol under control conditions, and experimental inflammation was induced by tumor necrosis factor-α (TNF-α). Expression of different osteogenic markers and cytokines was analyzed by real-time PCR, Western blotting, and enzyme-linked immunosorbent assay. In addition, alkaline phosphatase (ALP) enzyme activity and biomineralization as markers for differentiation were measured. Treatment with 6-gingerol resulted in insignificant effects on the proliferation rate. 6-Gingerol induced the differentiation of osteoblast-like cells with increased transcription levels of osteogenic markers, upregulated ALP enzyme activity, and enhanced mineralized nodule formation. Stimulation with TNF-α led to enhanced interleukin-6 and nuclear factor-κB expression and downregulated markers of osteoblastic differentiation. 6-Gingerol reduced the degree of inflammation in TNF-α-treated MG-63 cells. In conclusion, 6-gingerol stimulated osteoblast differentiation in normal physiological and inflammatory settings, and therefore, 6-gingerol represents a promising agent for treating osteoporosis or bone inflammation.

  19. Development of a new protocol for 2-day generation of mature dendritic cells from human monocytes

    OpenAIRE

    Obermaier Bianca; Dauer Marc; Herten Jan; Schad Katharina; Endres Stefan; Eigler Andreas

    2003-01-01

    We developed a new 2-day protocol for the generation of dendritic cells (DCs) from human monocytes in vitro. First, we demonstrated that 24 hours of culture with GM-CSF and IL-4 are sufficient to generate immature DCs capable of antigen uptake. We then compared two different strategies for DC maturation: proinflammatory mediators were either added together with GM-CSF and IL-4 from the beginning of cell culture or added after 24 hours of differentiation with GM-CSF and IL-4. After 48 hours of...

  20. Coupled electrophysiological recording and single cell transcriptome analyses revealed molecular mechanisms underlying neuronal maturation

    Directory of Open Access Journals (Sweden)

    Xiaoying Chen

    2016-02-01

    Full Text Available ABSTRACT The mammalian brain is heterogeneous, containing billions of neurons and trillions of synapses forming various neural circuitries, through which sense, movement, thought, and emotion arise. The cellular heterogeneity of the brain has made it difficult to study the molecular logic of neural circuitry wiring, pruning, activation, and plasticity, until recently, transcriptome analyses with single cell resolution makes decoding of gene regulatory networks underlying aforementioned circuitry properties possible. Here we report success in performing both electrophysiological and whole-genome transcriptome analyses on single human neurons in culture. Using Weighted Gene Coexpression Network Analyses (WGCNA, we identified gene clusters highly correlated with neuronal maturation judged by electrophysiological characteristics. A tight link between neuronal maturation and genes involved in ubiquitination and mitochondrial function was revealed. Moreover, we identified a list of candidate genes, which could potentially serve as biomarkers for neuronal maturation. Coupled electrophysiological recording and single cell transcriptome analysis will serve as powerful tools in the future to unveil molecular logics for neural circuitry functions.

  1. Pluripotent stem cells derived from mouse and human white mature adipocytes.

    Science.gov (United States)

    Jumabay, Medet; Abdmaulen, Raushan; Ly, Albert; Cubberly, Mark R; Shahmirian, Laurine J; Heydarkhan-Hagvall, Sepideh; Dumesic, Daniel A; Yao, Yucheng; Boström, Kristina I

    2014-02-01

    White mature adipocytes give rise to so-called dedifferentiated fat (DFAT) cells that spontaneously undergo multilineage differentiation. In this study, we defined stem cell characteristics of DFAT cells as they are generated from adipocytes and the relationship between these characteristics and lineage differentiation. Both mouse and human DFAT cells, prepared from adipose tissue and lipoaspirate, respectively, showed evidence of pluripotency, with a maximum 5-7 days after adipocyte isolation. The DFAT cells spontaneously formed clusters in culture, which transiently expressed multiple stem cell markers, including stage-specific embryonic antigens, and Sca-1 (mouse) and CD105 (human), as determined by real-time polymerase chain reaction, fluorescence-activated cell sorting, and immunostaining. As the stem cell markers decreased, markers characteristic of the three germ layers and specific lineage differentiation, such as α-fetoprotein (endoderm, hepatic), Neurofilament-66 (ectoderm, neurogenic), and Troponin I (mesoderm, cardiomyogenic), increased. However, no teratoma formation was detected after injection in immunodeficient mice. A novel modification of the adipocyte isolation aimed at ensuring the initial purity of the adipocytes and avoiding ceiling culture allowed isolation of DFAT cells with pluripotent characteristics. Thus, the adipocyte-derived DFAT cells represent a plastic stem cell population that is highly responsive to changes in culture conditions and may benefit cell-based therapies.

  2. The A2B adenosine receptor impairs the maturation and immunogenicity of dendritic cells.

    Science.gov (United States)

    Wilson, Jeffrey M; Ross, William G; Agbai, Oma N; Frazier, Renea; Figler, Robert A; Rieger, Jayson; Linden, Joel; Ernst, Peter B

    2009-04-15

    The endogenous purine nucleoside adenosine is an important antiinflammatory mediator that contributes to the control of CD4(+) T cell responses. While adenosine clearly has direct effects on CD4(+) T cells, it remains to be determined whether actions on APC such as dendritic cells (DC) are also important. In this report we characterize DC maturation and function in BMDC stimulated with LPS in the presence or absence of the nonselective adenosine receptor agonist NECA (5'-N-ethylcarboxamidoadenosine). We found that NECA inhibited TNF-alpha and IL-12 in a concentration-dependent manner, whereas IL-10 production was increased. NECA-treated BMDC also expressed reduced levels of MHC class II and CD86 and were less effective at stimulating CD4(+) T cell proliferation and IL-2 production compared with BMDC exposed to vehicle control. Based on real-time RT-PCR, the A(2A) adenosine receptor (A(2A)AR) and A(2B)AR were the predominant adenosine receptors expressed in BMDC. Using adenosine receptor subtype selective antagonists and BMDC derived from A(2A)AR(-/-) and A(2B)AR(-/-)mice, it was shown that NECA modulates TNF-alpha, IL-12, IL-10, and CD86 responses predominantly via A(2B)AR. These data indicate that engagement of A(2B)AR modifies murine BMDC maturation and suggest that adenosine regulates CD4(+) T cell responses by selecting for DC with impaired immunogencity.

  3. Endometrial aspiration biopsy: a non-invasive method of obtaining functional lymphoid progenitor cells and mature natural killer cells.

    LENUS (Irish Health Repository)

    McMenamin, Moya

    2012-09-01

    The aim of this study was to compare the efficacy of endometrial aspiration biopsy (EAB) with the more traditional dilatation and curettage (D&C) for the procurement of lymphoid progenitor cells and uterine natural killer (NK) populations in endometrial tissue. This prospective observational study conducted in a tertiary referral university hospital examined endometrium obtained from 32 women admitted for laparoscopic gynaecological procedures. Each participant had endometrium sampled using both EAB and D&C. Both methods were assessed as a source of uterine NK and lymphoid progenitor cells. Similar proportions of mature CD45+CD56+ NK cells (range 25.4-36.2%) and CD45+CD34+ lymphoid progenitors (range 1.2-2.0%) were found in tissue obtained using both EAB and D&C. These cells were adequate for flow cytometric analysis, magnetic bead separation and culture. Colony formation by the CD34+ population demonstrated maturational potential. Tissues obtained via endometrial biopsy and D&C are equivalent, by analysis of uterine NK and lymphoid progenitor cells. The aim of this study was to compare two methods of endometrial sampling - endometrial aspiration biopsy and traditional dilatation and curettage - for the procurement of haematopoietic stem cells and uterine natural killer (NK) populations in endometrial tissue. Thirty-two women who had gynaecological procedures in a tertiary referral hospital participated in this study and had endometrial tissue collected via both methods. Similar populations of mature NK cells and haematopoietic stem cells were found in tissue obtained using both endometrial aspiration biopsy and dilatation and curettage. Tissue obtained via endometrial aspiration biopsy was adequate for the culture and growth of haematopoietic stem cells. We conclude that tissue obtained via endometrial biopsy and dilatation and curettage is equivalent, by analysis of uterine NK and haematopoietic stem cells using flow cytometry. This has implications for further

  4. Scrapie affects the maturation cycle and immune complex trapping by follicular dendritic cells in mice.

    Directory of Open Access Journals (Sweden)

    Gillian McGovern

    Full Text Available Transmissible spongiform encephalopathies (TSEs or prion diseases are infectious neurological disorders of man and animals, characterised by abnormal disease-associated prion protein (PrP(d accumulations in the brain and lymphoreticular system (LRS. Prior to neuroinvasion, TSE agents often accumulate to high levels within the LRS, apparently without affecting immune function. However, our analysis of scrapie-affected sheep shows that PrP(d accumulations within the LRS are associated with morphological changes to follicular dendritic cells (FDCs and tingible body macrophages (TBMs. Here we examined FDCs and TBMs in the mesenteric lymph nodes (MLNs of scrapie-affected mice by light and electron microscopy. In MLNs from uninfected mice, FDCs could be morphologically categorised into immature, mature and regressing forms. However, in scrapie-affected MLNs this maturation cycle was adversely affected. FDCs characteristically trap and retain immune complexes on their surfaces, which they display to B-lymphocytes. In scrapie-affected MLNs, some FDCs were found where areas of normal and abnormal immune complex retention occurred side by side. The latter co-localised with PrP(d plasmalemmal accumulations. Our data suggest this previously unrecognised morphology represents the initial stage of an abnormal FDC maturation cycle. Alterations to the FDCs included PrP(d accumulation, abnormal cell membrane ubiquitin and excess immunoglobulin accumulation. Regressing FDCs, in contrast, appeared to lose their membrane-attached PrP(d. Together, these data suggest that TSE infection adversely affects the maturation and regression cycle of FDCs, and that PrP(d accumulation is causally linked to the abnormal pathology observed. We therefore support the hypothesis that TSEs cause an abnormality in immune function.

  5. SOX2+ cell population from normal human brain white matter is able to generate mature oligodendrocytes.

    Directory of Open Access Journals (Sweden)

    Jorge Oliver-De La Cruz

    Full Text Available OBJECTIVES: A number of neurodegenerative diseases progress with a loss of myelin, which makes them candidate diseases for the development of cell-replacement therapies based on mobilisation or isolation of the endogenous neural/glial progenitor cells, in vitro expansion, and further implantation. Cells expressing A2B5 or PDGFRA/CNP have been isolated within the pool of glial progenitor cells in the subcortical white matter of the normal adult human brain, all of which demonstrate glial progenitor features. However, the heterogeneity and differentiation potential of this pool of cells is not yet well established. METHODS: We used diffusion tensor images, histopathology, and immunostaining analysis to demonstrate normal cytoarchitecture and the absence of abnormalities in human temporal lobe samples from patients with mesial temporal sclerosis. These samples were used to isolate and enrich glial progenitor cells in vitro, and later to detect such cells in vivo. RESULTS: We have identified a subpopulation of SOX2+ cells, most of them co-localising with OLIG2, in the white matter of the normal adult human brain in vivo. These cells can be isolated and enriched in vitro, where they proliferate and generate immature (O4+ and mature (MBP+ oligodendrocytes and, to a lesser extent, astrocytes (GFAP+. CONCLUSION: Our results demonstrate the existence of a new glial progenitor cell subpopulation that expresses SOX2 in the white matter of the normal adult human brain. These cells might be of use for tissue regeneration procedures.

  6. The active translation of MHCII mRNA during dendritic cells maturation supplies new molecules to the cell surface pool.

    Science.gov (United States)

    Malanga, Donatella; Barba, Pasquale; Harris, Paul E; Maffei, Antonella; Del Pozzo, Giovanna

    2007-04-01

    The transition of human dendritic cells (DCs) from the immature to the mature phenotype is characterized by an increased density of MHC class II (MHCII) molecules on the plasma membrane, a key requirement of their competence as professional antigen presenting cells (APCs). MHCII molecules on the cell surface derive from newly synthesized as well as from preexisting proteins. So far, all the studies done on DCs during maturation, to establish the relative contribution of newly synthesized MHCII molecules to the cell surface pool did not produced a clear, unified scenario. We report that, in human DCs stimulated ex vivo with LPS, the changes in the RNA accumulation specific for at least two MHCII genes (HLA-DRA and HLA-DQA1) due to transcriptional upregulation, is associated with the active translation at high rate of these transcripts. Our finding reveals that, across the 24h of the maturation process in human DCs, newly synthesized MHCII proteins are supplied to the APCs cell surface pool.

  7. A gene expression signature shared by human mature oocytes and embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Pantesco Véronique

    2009-01-01

    Full Text Available Abstract Background The first week of human pre-embryo development is characterized by the induction of totipotency and then pluripotency. The understanding of this delicate process will have far reaching implication for in vitro fertilization and regenerative medicine. Human mature MII oocytes and embryonic stem (ES cells are both able to achieve the feat of cell reprogramming towards pluripotency, either by somatic cell nuclear transfer or by cell fusion, respectively. Comparison of the transcriptome of these two cell types may highlight genes that are involved in pluripotency initiation. Results Based on a microarray compendium of 205 samples, we compared the gene expression profile of mature MII oocytes and human ES cells (hESC to that of somatic tissues. We identified a common oocyte/hESC gene expression profile, which included a strong cell cycle signature, genes associated with pluripotency such as LIN28 and TDGF1, a large chromatin remodelling network (TOP2A, DNMT3B, JARID2, SMARCA5, CBX1, CBX5, 18 different zinc finger transcription factors, including ZNF84, and several still poorly annotated genes such as KLHL7, MRS2, or the Selenophosphate synthetase 1 (SEPHS1. Interestingly, a large set of genes was also found to code for proteins involved in the ubiquitination and proteasome pathway. Upon hESC differentiation into embryoid bodies, the transcription of this pathway declined. In vitro, we observed a selective sensitivity of hESC to the inhibition of the activity of the proteasome. Conclusion These results shed light on the gene networks that are concurrently overexpressed by the two human cell types with somatic cell reprogramming properties.

  8. Age and size at maturity: a quantitative review of diet-induced reaction norms in insects.

    Science.gov (United States)

    Teder, Tiit; Vellau, Helen; Tammaru, Toomas

    2014-11-01

    Optimality models predict that diet-induced bivariate reaction norms for age and size at maturity can have diverse shapes, with the slope varying from negative to positive. To evaluate these predictions, we perform a quantitative review of relevant data, using a literature-derived database of body sizes and development times for over 200 insect species. We show that bivariate reaction norms with a negative slope prevail in nearly all taxonomic and ecological categories of insects as well as in some other ectotherm taxa with comparable life histories (arachnids and amphibians). In insects, positive slopes are largely limited to species, which feed on discrete resource items, parasitoids in particular. By contrast, with virtually no meaningful exceptions, herbivorous and predatory insects display reaction norms with a negative slope. This is consistent with the idea that predictable resource depletion, a scenario selecting for positively sloped reaction norms, is not frequent for these insects. Another source of such selection-a positive correlation between resource levels and juvenile mortality rates-should similarly be rare among insects. Positive slopes can also be predicted by models which integrate life-history evolution and population dynamics. As bottom-up regulation is not common in most insect groups, such models may not be most appropriate for insects.

  9. Dual kappa and lambda expressing in mature B-cell neoplasm: An unusual case

    Directory of Open Access Journals (Sweden)

    Ashish Gupta

    2015-01-01

    Full Text Available A 57 years old male presented with transfusion dependent anemia for last 2 months with mild weight loss and on and off fever. On examination he was found to have small axillary lymph nodes with moderate splenomegaly. Flow cytometry revealed 63.2% cells in the lymphocyte window in cluster of differentiation (CD 45 versus side scatter plot. Further analysis on CD-19 gated cells showed that - 75.9% of these cells expressed CD-19 plus the expression of CD23, CD25, CD43, CD20 and CD22. The entire cluster showed dual expression of kappa and lambda light chains. Final diagnosis of low-grade B non-Hodgkin lymphoma was made. Given that dual kappa and lambda expressing in mature B cell neoplasm has been described in literature, it is important to differentiate such cases from reactive lymphoid proliferations.

  10. Intratesticular signals regulate germ cell progression and production of qualitatively mature spermatozoa in vertebrates

    Directory of Open Access Journals (Sweden)

    Rosaria eMeccariello

    2014-05-01

    Full Text Available Spermatogenesis, a highly conserved process in vertebrates, is mainly under the hypothalamic-pituitary control, being regulated by the secretion of pituitary gonadotropins, FSH and LH, in response to stimulation exerted by Gonadotropin Releasing Hormone (GnRH from hypothalamic neurons. At testicular level, gonadotropins bind specific receptors located on the somatic cells regulating the production of steroids and factors necessary to ensure a correct spermatogenesis. Indeed, besides the endocrine route, a complex network of cell-to-cell communications regulates germ cell progression, and a combination of endocrine and intragonadal signals sustains the production of high quality mature spermatozoa. In this review we focus on the recent advances in the area of the intragonadal signals supporting sperm development.

  11. In vivo targeting of antigens to maturing dendritic cells via the DEC-205 receptor improves T cell vaccination.

    Science.gov (United States)

    Bonifaz, Laura C; Bonnyay, David P; Charalambous, Anna; Darguste, Dara I; Fujii, Shin-Ichiro; Soares, Helena; Brimnes, Marie K; Moltedo, Bruno; Moran, Thomas M; Steinman, Ralph M

    2004-03-15

    The prevention and treatment of prevalent infectious diseases and tumors should benefit from improvements in the induction of antigen-specific T cell immunity. To assess the potential of antigen targeting to dendritic cells to improve immunity, we incorporated ovalbumin protein into a monoclonal antibody to the DEC-205 receptor, an endocytic receptor that is abundant on these cells in lymphoid tissues. Simultaneously, we injected agonistic alpha-CD40 antibody to mature the dendritic cells. We found that a single low dose of antibody-conjugated ovalbumin initiated immunity from the naive CD4+ and CD8+ T cell repertoire. Unexpectedly, the alphaDEC-205 antigen conjugates, given s.c., targeted to dendritic cells systemically and for long periods, and ovalbumin peptide was presented on MHC class I for 2 weeks. This was associated with stronger CD8+ T cell-mediated immunity relative to other forms of antigen delivery, even when the latter was given at a thousand times higher doses. In parallel, the mice showed enhanced resistance to an established rapidly growing tumor and to viral infection at a mucosal site. By better harnessing the immunizing functions of maturing dendritic cells, antibody-mediated antigen targeting via the DEC-205 receptor increases the efficiency of vaccination for T cell immunity, including systemic and mucosal resistance in disease models.

  12. Genetic alterations in systemic nodal and extranodal non-cutaneous lymphomas derived from mature T cells and natural killer cells.

    Science.gov (United States)

    Boi, Michela; Stathis, Anastasios; Zucca, Emanuele; Inghirami, Giorgio; Bertoni, Francesco

    2012-08-01

    Mature (peripheral) T-cell and natural killer (NK)-cell lymphomas comprise a series of rather different neoplasms. Based on morphologic, immunophenotypic, genetic, and clinical data, the World Health Organization classification recognizes more than 20 entities or provisional entities. The variable clinical presentations, the objective recognition and pathological stratification, the difficulties regarding treatment, and the hardly predictable response to therapy indicate that the management of these entities requires novel tools. In contrast to B-cell lymphomas or precursor T-cell neoplasms, few recurrent translocations have been identified so far in T-cell non-Hodgkin's and NK-cell lymphomas. Additionally, some of the entities recognized by the World Health Organization classification are very rare and very scarce molecular data are available for T-cell lymphomas. Here, we have reviewed published reports focusing on the genetic lesions and gene expression profiling underlying systemic nodal and extranodal non-cutaneous mature T-cell and NK-cell lymphomas. We also provide a summary of new agents in clinical development and outline some future directions.

  13. Endothelial differentiation in multipotent cells derived from mouse and human white mature adipocytes.

    Science.gov (United States)

    Jumabay, Medet; Abdmaulen, Raushan; Urs, Sumithra; Heydarkhan-Hagvall, Sepideh; Chazenbalk, Gregorio D; Jordan, Maria C; Roos, Kenneth P; Yao, Yucheng; Boström, Kristina I

    2012-12-01

    White mature adipocytes give rise to multipotent cells, so-called de-differentiated fat (DFAT) cells, when losing their fat in culture. The objective of this study was to examine the ability of DFAT cells to give rise to endothelial cells (ECs) in vitro and vivo. We demonstrate that mouse and human DFAT cells, derived from adipose tissue and lipospirate, respectively, initially lack expression of CD34, CD31, CD146, CD45 and pericyte markers, distinguishing them from progenitor cells previously identified in adipose stroma. The DFAT cells spontaneously differentiate into vascular ECs in vitro, as determined by real-time PCR, fluorescence activated cell sorting, immunostaining, and formation of tube structures. Treatment with bone morphogenetic protein (BMP)4 and BMP9, important in regulating angiogenesis, significantly enhances the EC differentiation. Furthermore, adipocyte-derived cells from Green Fluorescent Protein-transgenic mice were detected in the vasculature of infarcted myocardium up to 6 weeks after ligation of the left anterior descending artery in mice. We conclude that adipocyte-derived multipotent cells are able to spontaneously give rise to ECs, a process that is promoted by BMPs and may be important in cardiovascular regeneration and in physiological and pathological changes in fat and other tissues.

  14. Peripheral tolerance through clonal deletion of mature CD4-CD8+ T cells.

    Science.gov (United States)

    Carlow, D A; Teh, S J; van Oers, N S; Miller, R G; Teh, H S

    1992-05-01

    Transgenic mice bearing the alpha beta transgenes encoding a defined T cell receptor specific for the male (H-Y) antigen presented by the H-2Db class I MHC molecule were used to study mechanisms of peripheral tolerance. Female transgenic mice produce large numbers of functionally homogeneous CD8+ male antigen-reactive T cells in the thymus that subsequently accumulate in the peripheral lymphoid organs. We have used three experimental approaches to show that male reactive CD8+ T cells can be eliminated from peripheral lymphoid organs after exposure to male antigen. (i) In female transgenic mice that were neonatally tolerized with male spleen cells, male reactive CD8+ T cells continued to be produced in large numbers in the thymus but were virtually absent in the lymph nodes. (ii) Injection of thymocytes from female transgenic mice into female mice neonatally tolerized with the male antigen, or into normal male mice, led to the specific elimination of male-reactive CD8+ T cells in the lymph nodes. (iii) Four days after male lymphoid cells were injected intravenously into female transgenic mice, male antigen-reactive CD8+ T cells recovered from the lymph nodes of recipient mice were highly apoptotic when compared to CD4+ (non-male reactive) T cells. These data indicate that tolerance to extrathymic antigen can be achieved through elimination of mature T cells in the peripheral lymphoid organs.

  15. Otoferlin couples to clathrin-mediated endocytosis in mature cochlear inner hair cells.

    Science.gov (United States)

    Duncker, Susanne V; Franz, Christoph; Kuhn, Stephanie; Schulte, Uwe; Campanelli, Dario; Brandt, Niels; Hirt, Bernhard; Fakler, Bernd; Blin, Nikolaus; Ruth, Peter; Engel, Jutta; Marcotti, Walter; Zimmermann, Ulrike; Knipper, Marlies

    2013-05-29

    The encoding of auditory information with indefatigable precision requires efficient resupply of vesicles at inner hair cell (IHC) ribbon synapses. Otoferlin, a transmembrane protein responsible for deafness in DFNB9 families, has been postulated to act as a calcium sensor for exocytosis as well as to be involved in rapid vesicle replenishment of IHCs. However, the molecular basis of vesicle recycling in IHCs is largely unknown. In the present study, we used high-resolution liquid chromatography coupled with mass spectrometry to copurify otoferlin interaction partners in the mammalian cochlea. We identified multiple subunits of the adaptor protein complex AP-2 (CLAP), an essential component of clathrin-mediated endocytosis, as binding partners of otoferlin in rats and mice. The interaction between otoferlin and AP-2 was confirmed by coimmunoprecipitation. We also found that AP-2 interacts with myosin VI, another otoferlin binding partner important for clathrin-mediated endocytosis (CME). The expression of AP-2 in IHCs was verified by reverse transcription PCR. Confocal microscopy experiments revealed that the expression of AP-2 and its colocalization with otoferlin is confined to mature IHCs. When CME was inhibited by blocking dynamin action, real-time changes in membrane capacitance showed impaired synaptic vesicle replenishment in mature but not immature IHCs. We suggest that an otoferlin-AP-2 interaction drives Ca(2+)- and stimulus-dependent compensating CME in mature IHCs.

  16. In Vivo Targeting of Antigens to Maturing Dendritic Cells via the DEC-205 Receptor Improves T Cell Vaccination

    Science.gov (United States)

    Bonifaz, Laura C.; Bonnyay, David P.; Charalambous, Anna; Darguste, Dara I.; Fujii, Shin-Ichiro; Soares, Helena; Brimnes, Marie K.; Moltedo, Bruno; Moran, Thomas M.; Steinman, Ralph M.

    2004-01-01

    The prevention and treatment of prevalent infectious diseases and tumors should benefit from improvements in the induction of antigen-specific T cell immunity. To assess the potential of antigen targeting to dendritic cells to improve immunity, we incorporated ovalbumin protein into a monoclonal antibody to the DEC-205 receptor, an endocytic receptor that is abundant on these cells in lymphoid tissues. Simultaneously, we injected agonistic α-CD40 antibody to mature the dendritic cells. We found that a single low dose of antibody-conjugated ovalbumin initiated immunity from the naive CD4+ and CD8+ T cell repertoire. Unexpectedly, the αDEC-205 antigen conjugates, given s.c., targeted to dendritic cells systemically and for long periods, and ovalbumin peptide was presented on MHC class I for 2 weeks. This was associated with stronger CD8+ T cell–mediated immunity relative to other forms of antigen delivery, even when the latter was given at a thousand times higher doses. In parallel, the mice showed enhanced resistance to an established rapidly growing tumor and to viral infection at a mucosal site. By better harnessing the immunizing functions of maturing dendritic cells, antibody-mediated antigen targeting via the DEC-205 receptor increases the efficiency of vaccination for T cell immunity, including systemic and mucosal resistance in disease models. PMID:15024047

  17. Influence of streptozotocin-induced diabetes and insulin treatment on the pituitary-testicular axis during sexual maturation in rats.

    Science.gov (United States)

    Sudha, S; Valli, G; Julie, P M; Arunakaran, J; Govindarajulu, P; Balasubramanian, K

    2000-01-01

    Effects of streptozotocin (STZ)-diabetes and insulin treatment on the functioning of pituitary-testicular axis during sexual maturation was studied. Prepubertal (30 days old) and pubertal (50 days old) male Wistar rats were made diabetic by a single injection of STZ. A group of diabetic rats was given insulin (3U/100 g b.wt./day in 2 equally divided doses), 3 days after STZ treatment. Prepubertal and pubertal rats of all groups were killed on postnatal days 51 and 71, respectively. STZ-diabetes caused marked reduction in serum LH, FSH, prolactin, testosterone and testicular interstitial fluid testosterone as well as the activities of Leydig cellular steroidogenic enzymes (3beta-and 17beta-hydroxysteroid dehydrogenases). Insulin treatment to diabetic rats maintained these changes at control range except FSH and prolactin in prepubertal rats. The results indicate that (i) diabetes-induced steroidogenic lesions in Leydig cells represent a direct consequence of dysfunctioning of pituitary-testicular axis, (ii) the adverse effects of diabetes on pituitary-testicular functions are influenced by age of its induction and (iii) optimum insulin level is essential for the acquisition of Leydig cellular steroidogenic efficacy during sexual development.

  18. Ctr2 Regulates Mast Cell Maturation by Affecting the Storage and Expression of Tryptase and Proteoglycans.

    Science.gov (United States)

    Öhrvik, Helena; Logeman, Brandon; Noguchi, Glyn; Eriksson, Inger; Kjellén, Lena; Thiele, Dennis J; Pejler, Gunnar

    2015-10-15

    Copper (Cu) is essential for multiple cellular functions. Cellular uptake of Cu(+) is carried out by the Ctr1 high-affinity Cu transporter. The mobilization of endosomal Cu pools is regulated by a protein structurally similar to Ctr1, called Ctr2. It was recently shown that ablation of Ctr2 caused an increase in the concentration of Cu localized to endolysosomes. However, the biological significance of excess endolysosomal Cu accumulation has not been assessed. In this study, we addressed this issue by investigating the impact of Ctr2 deficiency on mast cells, a cell type unusually rich in endolysosomal organelles (secretory granules). We show that Ctr2(-/-) mast cells have increased intracellular Cu concentrations and that the absence of Ctr2 results in increased metachromatic staining, the latter indicating an impact of Ctr2 on the storage of proteoglycans in the secretory granules. In agreement with this, the absence of Ctr2 caused a skewed ratio between proteoglycans of heparin and chondroitin sulfate type, with increased amounts of heparin accompanied by a reduction of chondroitin sulfate. Moreover, transmission electron microscopy analysis revealed a higher number of electron-dense granules in Ctr2(-/-) mast cells than in wild-type cells. The increase in granular staining and heparin content is compatible with an impact of Ctr2 on mast cell maturation and, in support of this, the absence of Ctr2 resulted in markedly increased mRNA expression, storage, and enzymatic activity of tryptase. Taken together, the present study introduces Ctr2 and Cu as novel actors in the regulation of mast cell maturation and granule homeostasis.

  19. CD25(+) Bcl6(low) T follicular helper cells provide help to maturingcells in germinal centers of human tonsil.

    Science.gov (United States)

    Li, Haishan; Pauza, C David

    2015-01-01

    The majority of CXCR5(+) PD1(+) CD4(+) T follicular helper (Tfh) cells (>90%) are CD25(-) Bcl6(hi) , while a small subpopulation (<10%) are CD25(+) Bcl6(low) but do not express FoxP3 and are not T regulatory cells. We purified T:B-cell conjugates from tonsils and found they were enriched for the CD25(+) Bcl6(low) Tfh-cell subpopulation. In response to IL-2, these CD25(+) Tfh cells increased expression of costimulatory molecules ICOS or OX40, upregulated transcription factor cMaf, produced cytokines IL-21, IL-17, and IL-10, and raised the levels of antiapoptotic protein Bcl2. Conjugates formed with CD25(+) BCl6(low) Tfh cells included B cells expressing higher levels of activation-induced cytidine deaminase (AID), memory marker CD45RO, surface IgG or IgA, and MHC class II compared to B-cell conjugates including CD25(-) Bcl6(hi) Tfh cells. While IL-2 suppresses early Tfh-cell differentiation, Tfh-cell recognition of antigen-presenting B cells and signaling through the T-cell receptor likely triggers expression of the high-affinity IL-2 receptor and responses to IL-2 including downregulation of Bcl6. CD25 expression on Tfh cells and local production of IL-2 in tonsil or lymph node may support B helper T-cell function during later stages of B-cell maturation and the development of immune memory.

  20. Global discovery of erythroid long noncoding RNAs reveals novel regulators of red cell maturation.

    Science.gov (United States)

    Alvarez-Dominguez, Juan R; Hu, Wenqian; Yuan, Bingbing; Shi, Jiahai; Park, Staphany S; Gromatzky, Austin A; van Oudenaarden, Alexander; Lodish, Harvey F

    2014-01-23

    Erythropoiesis is regulated at multiple levels to ensure the proper generation of mature red cells under multiple physiological conditions. To probe the contribution of long noncoding RNAs (lncRNAs) to this process, we examined >1 billion RNA-seq reads of polyadenylated and nonpolyadenylated RNA from differentiating mouse fetal liver red blood cells and identified 655 lncRNA genes including not only intergenic, antisense, and intronic but also pseudogene and enhancer loci. More than 100 of these genes are previously unrecognized and highly erythroid specific. By integrating genome-wide surveys of chromatin states, transcription factor occupancy, and tissue expression patterns, we identify multiple lncRNAs that are dynamically expressed during erythropoiesis, show epigenetic regulation, and are targeted by key erythroid transcription factors GATA1, TAL1, or KLF1. We focus on 12 such candidates and find that they are nuclear-localized and exhibit complex developmental expression patterns. Depleting them severely impaired erythrocyte maturation, inhibiting cell size reduction and subsequent enucleation. One of them, alncRNA-EC7, is transcribed from an enhancer and is specifically needed for activation of the neighboring gene encoding BAND 3. Our study provides an annotated catalog of erythroid lncRNAs, readily available through an online resource, and shows that diverse types of lncRNAs participate in the regulatory circuitry underlying erythropoiesis.

  1. Mature cystic teratoma with malignant transformation of teratomatous urothelial cells: Rare case presentation

    Directory of Open Access Journals (Sweden)

    Senjuti Dasgupta

    2015-01-01

    Full Text Available The occurrence of malignancies in somatic elements of mature cystic teratoma of ovary is rare. The malignancies that may be encountered in dermoid cyst include squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, melanoma, sarcoma, carcinoid, and germ cell neoplasms. The development of transitional cell carcinoma (TCC in dermoid cyst is extremely rare with only four such cases having been reported in literature so far. Here we report the fifth case of such an occurrence in a 50-year-old postmenopausal multiparous female patient. She presented with pain and gradual swelling of abdomen for 1 month. Abdominal computed tomography revealed a solid space occupying lesion with few cystic components at right pelvis, raising the possibility of an ovarian neoplasm. The level of CA-125 was slightly raised (56∙45 U/ml. Total abdominal hysterectomy and bilateral salpingo-oopherectomy was performed. Microscopic examination showed cyst wall lined by stratified squamous epithelium. Beneath the cyst wall, a tumor mass was present, histological features of which resembled that of high-grade TCC (stage pT1aNXMX. On immunohistochemical analysis, the tumor was found to be positive for CK7 and CK20 and negative for WT-1. These results were consistent with a diagnosis of TCC arising in urothelium of mature cystic teratoma. Reporting of such extremely rare cases is important for the assessment of prognostic factors and treatment protocols.

  2. Establishment of a preadipocyte cell line derived from mature adipocytes of GFP transgenic mice and formation of adipose tissue.

    Science.gov (United States)

    Nobusue, Hiroyuki; Endo, Tsuyoshi; Kano, Koichiro

    2008-06-01

    We established a preadipocyte cell line from mature adipocytes obtained from subcutaneous fat tissue of green fluorescent protein (GFP) transgenic mice. The floating top layer, containing mature adipocytes, was isolated from subcutaneous fat tissue by collagenase digestion and filtration. Fluorescence-activated cell sorting and microscopic analysis revealed that the floating cell fraction comprised a highly homogeneous adipocyte population with no adipose stromal-vascular cells. Isolated mature adipocytes dedifferentiated into fibroblast-like cells and actively proliferated in ceiling culture. In vitro studies showed that the cells could redifferentiate into mature adipocytes in an identical way to 3T3-L1 preadipocytes. No changes in the differentiation pattern were observed during the propagation of our cells. They were successfully maintained and differentiated for at least 22 passages. We named these cells dedifferentiated fat (DFAT-GFP) cells. When DFAT-GFP cells were implanted subcutaneously into C57BL/6N mice, they developed highly vascularized fat pads that morphologically resembled normal subcutaneous adipose tissue and consisted of GFP-positive cells; however, implanted 3T3-L1 cells did not have such an effect on the mice. We conclude that DFAT-GFP cells provide a model that should enable us to study the mechanisms of adipocyte differentiation and adipose tissue formation in vivo and in vitro.

  3. Thymic and peripheral microenvironments differentially mediate development and maturation of iNKT cells by IL-15 transpresentation.

    Science.gov (United States)

    Castillo, Eliseo F; Acero, Luis F; Stonier, Spencer W; Zhou, Dapeng; Schluns, Kimberly S

    2010-10-01

    Invariant NKT (iNKT) cells are an innate type of T cells, which respond rapidly on activation. iNKT cells acquire these innate-like abilities during development; however, the signals driving development and functional maturation remain only partially understood. Because interleukin-15 (IL-15) is crucial for iNKT development and is delivered by transpresentation, we set out to identify the cell types providing IL-15 to developing iNKT cells and determine their role at the various states of development and maturation. We report here that transpresentation of IL-15 by parenchymal cells was crucial for generating normal number of iNKTs in the thymus, whereas both hematopoietic and parenchymal cells regulated iNKT cell numbers in the periphery, particularly in the liver. Specifically, dendritic cells contributed to peripheral iNKT cell numbers by up-regulating Bcl-2 expression and promoting extrathymic iNKT cell ex-pansion and their homeostatic proliferation. Whether IL-15 affects functional maturation of iNKT cells was also examined. In IL-15Rα(-/-) mice, CD44(High)NK1.1(+) iNKT cells displayed decreased T-bet expression and in response to α-galactosylceramide, had deficient interferon-γ expression. Such defects could be reversed by exogenous IL-15 signals. Overall, these studies identify stage-specific functions of IL-15, which are determined by the tissue microenvironment and elucidate the importance of IL-15 in functional maturation.

  4. 3'uridylation controls mature microRNA turnover during CD4 T cell activation.

    Science.gov (United States)

    Gutierrez-Vazquez, Cristina; Enright, Anton J; Rodríguez-Galán, Ana; Perez-García, Arantxa; Collier, Paul; Jones, Matthew R; Benes, Vladimir; Mizgerd, Joseph P; Mittelbrunn, María; Ramiro, Almudena R; Sanchez-Madrid, Francisco

    2017-03-28

    Activation of T lymphocytes requires a tight regulation of microRNAs (miRNAs) expression. Terminal uridyltransferases (TUTases) catalyze 3' non-templated nucleotide addition (3'NTA) to miRNAs which may influence miRNA stability and function. Here, we investigated 3'NTA to mature miRNA in CD4 T lymphocytes by deep sequencing. Upon T cell activation, miRNA sequences bearing terminal uridines are specifically decreased, concomitantly with downregulation of TUT4 and TUT7 enzymes. Analyzing TUT4 deficient T lymphocytes, we proved that this terminal uridyltransferase is essential for the maintenance of miRNA uridylation in steady state of T lymphocytes. Analysis of synthetic uridylated miRNAs shows that 3' addition of uridine promotes degradation of these uridylated miRNAs after T cell activation. Our data underline post-transcriptional uridylation as a mechanism to fine tune miRNA levels during T cell activation.

  5. Effects of vitamin A on in vitro maturation of pre-pubertal mouse spermatogonial stem cells.

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    Albanne Travers

    Full Text Available Testicular tissue cryopreservation is the only potential option for fertility preservation in pre-pubertal boys exposed to gonadotoxic treatment. Completion of spermatogenesis after in vitro maturation is one of the future uses of harvested testicular tissue. The purpose of the current study was to evaluate the effects of vitamin A on in vitro maturation of fresh and frozen-thawed mouse pre-pubertal spermatogonial stem cells in an organ culture system. Pre-pubertal CD1 mouse fresh testes were cultured for 7 (D7, 9 (D9 and 11 (D11 days using an organ culture system. Basal medium was supplemented with different concentrations of retinol (Re or retinoic acid (RA alone or in combination. Seminiferous tubule morphology (tubule diameter, intra-tubular cell type, intra-tubular cell death and proliferation (PCNA antibody and testosterone level were assessed at D7, D9 and D11. Pre-pubertal mouse testicular tissue were frozen after a soaking temperature performed at -7 °C, -8 °C or -9 °C and after thawing, were cultured for 9 days, using the culture medium preserving the best fresh tissue functionality. Retinoic acid at 10(-6M and retinol at 3.3.10(-7M, as well as retinol 10(-6M are favourable for seminiferous tubule growth, maintenance of intra-tubular cell proliferation and germ cell differentiation of fresh pre-pubertal mouse spermatogonia. Structural and functional integrity of frozen-thawed testicular tissue appeared to be well-preserved after soaking temperature at -8 °C, after 9 days of organotypic culture using 10(-6M retinol. RA and Re can control in vitro germ cell proliferation and differentiation. Re at a concentration of 10(-6M maintains intra-tubular cell proliferation and the ability of spermatogonia to initiate spermatogenesis in fresh and frozen pre-pubertal mouse testicular tissue using a soaking temperature at -8 °C. Our data suggested a possible human application for in vitro maturation of cryopreserved pre-pubertal testicular

  6. Effects of mature Sertoli cells on allogeneic islets cocultured in vitro

    Institute of Scientific and Technical Information of China (English)

    Heli Xiang; Wujun Xue; Yan Teng; Xinshun Feng; Puxun Tian; Xiaoming Ding

    2006-01-01

    Objective: To set up a method for isolation and culture of mature Sertoli cells and to estimate their effects on allogeneic islets cocultured in vitro. Methods: Adult SD rat testicular Sertoli cells were prepared successfully by three-step enzyme digestion. Then they were cocultured respectively with allogeneic islets and activated Wistar rat splenocytes. 24-hour cumulative insulin release and glucose-stimulated insulin secretion test were performed to detect islet function between pure islets culture group and coculture group. Splenocyte proliferation activity was determined by MTT colorimetry assay to observe the inhibition effect of Sertoli cells in different densities. Result: Firstly, in pure islet culture group, the 24-hour cumulative insulin release was gradually decreased in 21-day culture time. Compared to day 3, this change was significant on day 7 (P < 0.05) and on day 10,14,21 (P < 0.01). In contrast, in coculture group, compared to day 3, the 24-hour cumulative insulin release was increased significantly on day 7 (P < 0.01 ), and then gradually decreased on day 10 and 14, but still higher than that of day 3. It was on day 21 that it began to decrease compared to day 3 (P < 0.05). During the culture time in vitro, the 24-hour cumulative insulin release of islet coculture group was significantly higher than that of pure islets culture group (P < 0.01). In the case of stimulation index(SI), there was a similar tendency as insulin release in the two groups. Secondly, mature Sertoli cells(1×106/mL)pretreated by 15 grays irradiation could decrease proliferation activity of activated splenocytes compared to that of control group (P < 0.01 ). This inhibition effect was dose-dependent. Conclusion: Mature Sertoli cells can improve the function and prolong the survival of islet cells cultured in vitro. They can also provide an immune protection to islet cells. The approach described above might be applicable to human islet transplantation as soon as

  7. Relation between clinical mature and immature lymphocyte cells in human peripheral blood and their spatial label free scattering patterns

    Science.gov (United States)

    Zhang, Lu; Zhao, Xin; Zhang, Zhenxi; Zhao, Hong; Chen, Wei; Yuan, Li

    2016-07-01

    A single living cell's light scattering pattern (LSP) in the horizontal plane, which has been denoted as the cell's "2D fingerprint," may provide a powerful label-free detection tool in clinical applications. We have recently studied the LSP in spatial scattering planes, denoted as the cell's "3D fingerprint," for mature and immature lymphocyte cells in human peripheral blood. The effects of membrane size, morphology, and the existence of the nucleus on the spatial LSP are discussed. In order to distinguish clinical label-free mature and immature lymphocytes, the special features of the spatial LSP are studied by statistical method in both the spatial and frequency domains. Spatial LSP provides rich information on the cell's morphology and contents, which can distinguish mature from immature lymphocyte cells and hence ultimately it may be a useful label-free technique for clinical leukemia diagnosis.

  8. Generation of immortal cell lines from the adult pituitary: role of cAMP on differentiation of SOX2-expressing progenitor cells to mature gonadotropes.

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    Ginah L Kim

    Full Text Available The pituitary is a complex endocrine tissue composed of a number of unique cell types distinguished by the expression and secretion of specific hormones, which in turn control critical components of overall physiology. The basic function of these cells is understood; however, the molecular events involved in their hormonal regulation are not yet fully defined. While previously established cell lines have provided much insight into these regulatory mechanisms, the availability of representative cell lines from each cell lineage is limited, and currently none are derived from adult pituitary. We have therefore used retroviral transfer of SV40 T-antigen to mass immortalize primary pituitary cell culture from an adult mouse. We have generated 19 mixed cell cultures that contain cells from pituitary cell lineages, as determined by RT-PCR analysis and immunocytochemistry for specific hormones. Some lines expressed markers associated with multipotent adult progenitor cells or transit-amplifying cells, including SOX2, nestin, S100, and SOX9. The progenitor lines were exposed to an adenylate cyclase activator, forskolin, over 7 days and were induced to differentiate to a more mature gonadotrope cell, expressing significant levels of α-subunit, LHβ, and FSHβ mRNAs. Additionally, clonal populations of differentiated gonadotropes were exposed to 30 nM gonadotropin-releasing hormone and responded appropriately with a significant increase in α-subunit and LHβ transcription. Further, exposure of the lines to a pulse paradigm of GnRH, in combination with 17β-estradiol and dexamethasone, significantly increased GnRH receptor mRNA levels. This array of adult-derived pituitary cell models will be valuable for both studies of progenitor cell characteristics and modulation, and the molecular analysis of individual pituitary cell lineages.

  9. An affinity matured minibody for PET imaging of prostate stem cell antigen (PSCA)-expressing tumors

    Energy Technology Data Exchange (ETDEWEB)

    Lepin, Eric J.; Leyton, Jeffrey V.; Olafsen, Tove; Salazar, Felix B.; McCabe, Katelyn E.; Wu, Anna M. [University of California, Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, Los Angeles, CA (United States); Zhou, Yu; Marks, James D. [University of California, Department of Anesthesia, San Francisco, CA (United States); Hahm, Scott; Reiter, Robert E. [University of California, Department of Urology, David Geffen School of Medicine, Los Angeles, CA (United States)

    2010-08-15

    Prostate stem cell antigen (PSCA), a cell surface glycoprotein expressed in normal human prostate and bladder, is over-expressed in the majority of localized prostate cancer and most bone metastases. We have previously shown that the hu1G8 minibody, a humanized anti-PSCA antibody fragment (single-chain Fv-C{sub H}3 dimer, 80 kDa), can localize specifically and image PSCA-expressing xenografts at 21 h post-injection. However, the humanization and antibody fragment reformatting decreased its apparent affinity. Here, we sought to evaluate PET imaging contrast with affinity matured minibodies. Yeast scFv display, involving four rounds of selection, was used to generate the three affinity matured antibody fragments (A2, A11, and C5) that were reformatted into minibodies. These three affinity matured anti-PSCA minibodies were characterized in vitro, and following radiolabeling with {sup 124}I were evaluated in vivo for microPET imaging of PSCA-expressing tumors. The A2, A11, and C5 minibody variants all demonstrated improved affinity compared to the parental (P) minibody and were ranked as follows: A2 > A11 > C5 > P. The {sup 124}I-labeled A11 minibody demonstrated higher immunoreactivity than the parental minibody and also achieved the best microPET imaging contrast in two xenograft models, LAPC-9 (prostate cancer) and Capan-1 (pancreatic cancer), when evaluated in vivo. Of the affinity variant minibodies tested, the A11 minibody that ranked second in affinity was selected as the best immunoPET tracer to image PSCA-expressing xenografts. This candidate is currently under development for evaluation in a pilot clinical imaging study. (orig.)

  10. Role of Pancreatic Transcription Factors in Maintenance of Mature β-Cell Function

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    Hideaki Kaneto

    2015-03-01

    Full Text Available A variety of pancreatic transcription factors including PDX-1 and MafA play crucial roles in the pancreas and function for the maintenance of mature β-cell function. However, when β-cells are chronically exposed to hyperglycemia, expression and/or activities of such transcription factors are reduced, which leads to deterioration of b-cell function. These phenomena are well known as β-cell glucose toxicity in practical medicine as well as in the islet biology research area. Here we describe the possible mechanism for β-cell glucose toxicity found in type 2 diabetes. It is likely that reduced expression levels of PDX-1 and MafA lead to suppression of insulin biosynthesis and secretion. In addition, expression levels of incretin receptors (GLP-1 and GIP receptors in β-cells are decreased, which likely contributes to the impaired incretin effects found in diabetes. Taken together, down-regulation of insulin gene transcription factors and incretin receptors explains, at least in part, the molecular mechanism for β-cell glucose toxicity.

  11. B cell development in the bone marrow is regulated by homeostatic feedback exerted by mature B cells

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    Gitit eShahaf

    2016-03-01

    Full Text Available Cellular homeostasis in the B cell compartment is strictly imposed to balance cell production and cell loss. However, it is not clear whether B cell development in the bone marrow (BM is an autonomous process or subjected to regulation by the peripheral B cell compartment. To specifically address this question, we used mice transgenic for human CD20, where effective depletion of B lineage cells is obtained upon administration of mouse-anti-human CD20 antibodies, in the absence of any effect on other cell lineages and/or tissues. We followed the kinetics of B cell return to equilibrium by BrdU labeling and flow cytometry and analyzed the resulting data by mathematical modeling. Labeling was much faster in depleted mice. Compared to control mice, B cell-depleted mice exhibited a higher proliferation rate in the pro-/pre-B compartment, and higher cell death and lower differentiation in the immature B cell compartment. We validated the first result by analysis of the expression of Ki67, the nuclear protein expressed in proliferating cells, and the second using Annexin-V staining. Collectively, our results suggest that B lymphopoiesis is subjected to homeostatic feedback mechanisms imposed by mature B cells in the peripheral compartment.

  12. Involvement of Phosphatases in Proliferation, Maturation, and Hemoglobinization of Developing Erythroid Cells

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    Eitan Fibach

    2011-01-01

    Full Text Available Production of RBCs is triggered by the action of erythropoietin (Epo through its binding to surface receptors (Epo-R on erythroid precursors in the bone marrow. The intensity and the duration of the Epo signal are regulated by several factors, including the balance between the activities of kinesase and phosphatases. The Epo signal determines the proliferation and maturation of the precursors into hemoglobin (Hb-containing RBCs. The activity of various protein tyrosine phosphatases, including those involved in the Epo pathway, can be inhibited by sodium orthovanadate (Na3VO4, vanadate. Adding vanadate to cultured erythroid precursors of normal donors and patients with β-thalassemia enhanced cell proliferation and arrested maturation. This was associated with an increased production of fetal hemoglobin (HbF. Increased HbF in patients with β-hemoglobinopathies (β-thalassemia and sickle cell disease ameliorates the clinical symptoms of the disease. These results raise the possibility that specific and nontoxic inhibitors of phosphatases may be considered as a therapeutic modality for elevating HbF in patients with β-hemoglobinopathies as well as for intensifying the Epo response in other forms of anemia.

  13. Myosin VI is required for the proper maturation and function of inner hair cell ribbon synapses.

    Science.gov (United States)

    Roux, Isabelle; Hosie, Suzanne; Johnson, Stuart L; Bahloul, Amel; Cayet, Nadège; Nouaille, Sylvie; Kros, Corné J; Petit, Christine; Safieddine, Saaid

    2009-12-01

    The ribbon synapses of auditory inner hair cells (IHCs) undergo morphological and electrophysiological transitions during cochlear development. Here we report that myosin VI (Myo6), an actin-based motor protein involved in genetic forms of deafness, is necessary for some of these changes to occur. By using post-embedding immunogold electron microscopy, we showed that Myo6 is present at the IHC synaptic active zone. In Snell's waltzer mutant mice, which lack Myo6, IHC ionic currents and ribbon synapse maturation proceeded normally until at least post-natal day 6. In adult mutant mice, however, the IHCs displayed immature potassium currents and still fired action potentials, as normally only observed in immature IHCs. In addition, the number of ribbons per IHC was reduced by 30%, and 30% of the remaining ribbons were morphologically immature. Ca2+-dependent exocytosis probed by capacitance measurement was markedly reduced despite normal Ca2+ currents and the large proportion of morphologically mature synapses, which suggests additional defects, such as loose Ca2+-exocytosis coupling or inefficient vesicular supply. Finally, we provide evidence that Myo6 and otoferlin, a putative Ca2+ sensor of synaptic exocytosis also involved in a genetic form of deafness, interact at the IHC ribbon synapse, and we suggest that this interaction is involved in the recycling of synaptic vesicles. Our findings thus uncover essential roles for Myo6 at the IHC ribbon synapse, in addition to that proposed in membrane turnover and anchoring at the apical surface of the hair cells.

  14. Intestinal colonization of IL-2 deficient mice with non-colitogenic B. vulgatus prevents DC maturation and T-cell polarization.

    Directory of Open Access Journals (Sweden)

    Martina Müller

    Full Text Available BACKGROUND: IL-2 deficient (IL-2(-/- mice mono-colonized with E. coli mpk develop colitis whereas IL-2(-/--mice mono-colonized with B. vulgatus mpk do not and are even protected from E. coli mpk induced colitis. METHODOLOGY/PRINCIPAL FINDINGS: We investigated if mono-colonization with E. coli mpk or B. vulgatus mpk differentially modulates distribution, activation and maturation of intestinal lamina propria (LP dendritic cells (DC. LP DC in mice mono-colonized with protective B. vulgatus mpk or co-colonized with E. coli mpk/B. vulgatus mpk featured a semi-mature LP DC phenotype (CD40(loCD80(loMHC-II(hi whereas mono-colonization with colitogenic E. coli mpk induced LP DC activation and maturation prior to onset of colitis. Accordingly, chemokine receptor (CCR 7 surface expression was more strikingly enhanced in mesenteric lymph node DC from E. coli mpk than B. vulgatus mpk mono- or co-colonized mice. Mature but not semi-mature LP DC promoted Th1 polarization. As B. vulgatus mpk promotes differentiation of semi-mature DC presumably by IL-6, mRNA and protein expression of IL-6 was investigated in LP DC. The data demonstrated that IL-6 mRNA and protein was increased in LP DC of B. vulgatus mpk as compared to E. coli mpk mono-colonized IL-2(-/--mice. The B. vulgatus mpk mediated suppression of CCR7 expression and DC migration was abolished in IL-6(-/--DC in vitro. CONCLUSIONS/SIGNIFICANCE: From this data we conclude that the B. vulgatus triggered IL-6 secretion by LP DC in absence of proinflammatory cytokines such as IL-12 or TNF-alpha induces a semi-mature LP DC phenotype, which might prevent T-cell activation and thereby the induction of colitis in IL-2(-/--mice. The data provide new evidence that IL-6 might act as an immune regulatory cytokine in the mucosa by targeting intestinal DC.

  15. PGE2对LPS诱导小鼠骨髓源性树突细胞成熟及EP4受体表达影响%The effect of prostaglandin E2 on maturation and EP4 expression on dendritic cell induced by LPS

    Institute of Scientific and Technical Information of China (English)

    盛康亮; 李影; 付静静; 陈镜宇; 张玲玲; 魏伟

    2015-01-01

    Objective To investigate the effects of PGE2 in the different concentrations on the maturation of bone marrow-derived dendritic cells ( DCs) of mouse stimulated by LPS and EP4 expression. Methods The bone mar-row-derived DCs were induced in the presence of recombinant granulocyte macrophage colony stimulating factor (rmGM-CSF) and rmIL-4. Maturated DCs were induced by LPS (100 ng/ml), and then were treated with PGE2 in different concentrations (0. 625, 1. 25, 2. 5, 5, 10, 20 nmol/L) for 24 h. The ability of antigen uptake and the expressions of CD40 , CD83 and MHC-Ⅱon DCs surface were analyzed by flow cytometry;EP4 R expression was al-so measured by flow cytometry. T cell proliferation in a mixed lymphocyte reaction was analyzed by MTT assay. Re-sults PGE2 (2. 5, 5, 10 nmol/L) significantly enhanced the expression of CD40, CD83, MHC class II molec- ules. However,PGE2 had no effect on CD80 expression in all of concentrations. PGE2(1. 25,2. 5,5,10,20 nmol/L) significantly inhibited the ability of antigen uptake of DCs. DCs stimulated by PGE2 (2. 5,5,10 nmol/L) could promote T cell proliferation. PGE2 (2. 5,5,10 nmol/L) increased EP4 expression on DCs surface. Conclusion PGE2 could regulate DCs function,which might be related to EP4 receptor expression affected by PGE2.%目的:观察不同浓度前列腺素E2(PGE2)刺激对脂多糖( LPS)诱导小鼠骨髓源性树突细胞( DCs)成熟及EP4受体表达的影响。方法采用重组小鼠粒细胞-巨噬细胞集落刺激因子( rmGM-CSF)、重组小鼠白细胞介素4( rmIL-4)刺激小鼠骨髓源细胞,诱导生成DCs;经LPS(100 ng/ml)诱导成熟后,用不同浓度 PGE2(0.625、1.25、2.5、5、10、20 nmol/L)刺激 DCs 24 h,流式细胞术检测 DCs 表型 CD40、CD83、MHC-Ⅱ的表达和抗原摄取功能,荧光间标法检测小鼠骨髓源DCs细胞膜上EP4的表达,以平均荧光强度表示表达的高低;MTT法检测经PGE2及LPS诱导成熟的DCs对T细胞增殖的作

  16. In vitro maturation (IVM as a new technique to treat polycystic ovary syndrome (PCOS and induce pregnancy in Indonesia

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    Soegiharto Soebijanto

    2009-12-01

    Full Text Available Aim: To assesse the success of inducing pregnancies in the treatment of PCOS (Poly Cystic Ovary Syndrome cases with in vitro maturation as a newly application technique in Indonesia.Methods This paper is a report of 7 cases in Indonesia that used the newly developed technique. There were 7 cases confi rmed PCOS, in which 1 patient with a history of OHSS (Ovary Hyper Stimulation Syndrome in a previous IVF procedure and 1 patient with PCOS characteristics, suspected hyper responder, in the Family Hospital from January to May of 2009. Follicular induction was performed with a minimum dose, primming with HCG 10.000 IU, on the 10th day and 40 hours later ovum pick up was  performed, followed by in vitro maturation. Subsequently, insemination was performed and the inseminated follicle was assessed. Well qualifi ed embryos then transferred them into the uterus. We then performed assessment of pregnancy biochemically, by the presence of embryonic sac and embryonic heart beat.Results: We have performed the IVM (In Vitro Maturation technique in the Family Hospital, West Jakarta, along with the TRB team of the Family Hospital in seven PCOS cases. From these patients, we have found 156 antral follicles (average of 22 follicles per patient, 82 oocytes, and after maturation, 61  nature oocytes (75 %. In three cases, in vitro fertilization was performed, while in 4 cases ICSI (In Cystoplasmic Sperm Infection was performed. In these serial cases we obtained 41 embryos, and 22 fertilized embryos were transferred. Of 7 cases, we achieved two successful pregnancies (29%.Conclusion: In vitro maturation is an alternative procedures in solving infertility problems for PCOS patients with lower risk of OHSS and more cost effective than conventional IVF. (Med J Indones 2009; 18: 269-75Keywords: pregnancy, PCOS, in vitro maturation

  17. Multicenter phase II study of matured dendritic cells pulsed with melanoma cell line lysates in patients with advanced melanoma

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    Hernandez Jackie

    2010-09-01

    Full Text Available Abstract Background Several single center studies have provided evidence of immune activation and antitumor activity of therapeutic vaccination with dendritic cells (DC in patients with metastatic melanoma. The efficacy of this approach in patients with favorable prognosis metastatic melanoma limited to the skin, subcutaneous tissues and lung (stages IIIc, M1a, M1b was tested in a multicenter two stage phase 2 study with centralized DC manufacturing. Methods The vaccine (IDD-3 consisted 8 doses of autologous monocyte-derived matured DC generated in serum-free medium with granulocyte macrophage colony stimulating factor (GM-CSF and interleukin-13 (IL-13, pulsed with lysates of three allogeneic melanoma cell lines, and matured with interferon gamma. The primary endpoint was antitumor activity. Results Among 33 patients who received IDD-3 there was one complete response (CR, two partial responses (PR, and six patients had stable disease (SD lasting more than eight weeks. The overall prospectively defined tumor growth control rate was 27% (90% confidence interval of 13-46%. IDD-3 administration had minimal toxicity and it resulted in a high frequency of immune activation to immunizing melanoma antigens as assessed by in vitro immune monitoring assays. Conclusions The administration of matured DC loaded with tumor lysates has significant immunogenicity and antitumor activity in patients with limited metastatic melanoma. Clinical trial registration NCT00107159.

  18. Msx genes define a population of mural cell precursors required for head blood vessel maturation.

    Science.gov (United States)

    Lopes, Miguel; Goupille, Olivier; Saint Cloment, Cécile; Lallemand, Yvan; Cumano, Ana; Robert, Benoît

    2011-07-01

    Vessels are primarily formed from an inner endothelial layer that is secondarily covered by mural cells, namely vascular smooth muscle cells (VSMCs) in arteries and veins and pericytes in capillaries and veinules. We previously showed that, in the mouse embryo, Msx1(lacZ) and Msx2(lacZ) are expressed in mural cells and in a few endothelial cells. To unravel the role of Msx genes in vascular development, we have inactivated the two Msx genes specifically in mural cells by combining the Msx1(lacZ), Msx2(lox) and Sm22α-Cre alleles. Optical projection tomography demonstrated abnormal branching of the cephalic vessels in E11.5 mutant embryos. The carotid and vertebral arteries showed an increase in caliber that was related to reduced vascular smooth muscle coverage. Taking advantage of a newly constructed Msx1(CreERT2) allele, we demonstrated by lineage tracing that the primary defect lies in a population of VSMC precursors. The abnormal phenotype that ensues is a consequence of impaired BMP signaling in the VSMC precursors that leads to downregulation of the metalloprotease 2 (Mmp2) and Mmp9 genes, which are essential for cell migration and integration into the mural layer. Improper coverage by VSMCs secondarily leads to incomplete maturation of the endothelial layer. Our results demonstrate that both Msx1 and Msx2 are required for the recruitment of a population of neural crest-derived VSMCs.

  19. Flotillins are involved in the polarization of primitive and mature hematopoietic cells.

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    Lawrence Rajendran

    Full Text Available BACKGROUND: Migration of mature and immature leukocytes in response to chemokines is not only essential during inflammation and host defense, but also during development of the hematopoietic system. Many molecules implicated in migratory polarity show uniform cellular distribution under non-activated conditions, but acquire a polarized localization upon exposure to migratory cues. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present evidence that raft-associated endocytic proteins (flotillins are pre-assembled in lymphoid, myeloid and primitive hematopoietic cells and accumulate in the uropod during migration. Furthermore, flotillins display a polarized distribution during immunological synapse formation. Employing the membrane lipid-order sensitive probe Laurdan, we show that flotillin accumulation in the immunological synapse is concomittant with membrane ordering in these regions. CONCLUSIONS: Together with the observation that flotillin polarization does not occur in other polarized cell types such as polarized epithelial cells, our results suggest a specific role for flotillins in hematopoietic cell polarization. Based on our results, we propose that in hematopoietic cells, flotillins provide intrinsic cues that govern segregation of certain microdomain-associated molecules during immune cell polarization.

  20. Variability of doublecortin-associated dendrite maturation in adult hippocampal neurogenesis is independent of the regulation of precursor cell proliferation

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    Jessberger Sebastian

    2006-11-01

    Full Text Available Abstract Background In the course of adult hippocampal neurogenesis most regulation takes place during the phase of doublecortin (DCX expression, either as pro-proliferative effect on precursor cells or as survival-promoting effect on postmitotic cells. We here obtained quantitative data about the proliferative population and the dynamics of postmitotic dendrite development during the period of DCX expression. The question was, whether any indication could be obtained that the initiation of dendrite development is timely bound to the exit from the cell cycle. Alternatively, the temporal course of morphological maturation might be subject to additional regulatory events. Results We found that (1 20% of the DCX population were precursor cells in cell cycle, whereas more than 70% were postmitotic, (2 the time span until newborn cells had reached the most mature stage associated with DCX expression varied between 3 days and several weeks, (3 positive or negative regulation of precursor cell proliferation did not alter the pattern and dynamics of dendrite development. Dendrite maturation was largely independent of close contacts to astrocytes. Conclusion These data imply that dendrite maturation of immature neurons is initiated at varying times after cell cycle exit, is variable in duration, and is controlled independently of the regulation of precursor cell proliferation. We conclude that in addition to the major regulatory events in cell proliferation and selective survival, additional micro-regulatory events influence the course of adult hippocampal neurogenesis.

  1. An induced junction photovoltaic cell

    Science.gov (United States)

    Call, R. L.

    1974-01-01

    Silicon solar cells operating with induced junctions rather than diffused junctions have been fabricated and tested. Induced junctions were created by forming an inversion layer near the surface of the silicon by supplying a sheet of positive charge above the surface. Measurements of the response of the inversion layer cell to light of different wavelengths indicated it to be more sensitive to the shorter wavelengths of the sun's spectrum than conventional cells. The greater sensitivity occurs because of the shallow junction and the strong electric field at the surface.

  2. New insights into the trophic and cytoprotective effects of creatine in in vitro and in vivo models of cell maturation.

    Science.gov (United States)

    Sestili, Piero; Ambrogini, Patrizia; Barbieri, Elena; Sartini, Stefano; Fimognari, Carmela; Calcabrini, Cinzia; Diaz, Anna Rita; Guescini, Michele; Polidori, Emanuela; Luchetti, Francesca; Canonico, Barbara; Lattanzi, Davide; Cuppini, Riccardo; Papa, Stefano; Stocchi, Vilberto

    2016-08-01

    A growing body of scientific reports indicates that the role of creatine (Cr) in cellular biochemistry and physiology goes beyond its contribution to cell energy. Indeed Cr has been shown to exert multiple effects promoting a wide range of physiological responses in vitro as well as in vivo. Included in these, Cr promotes in vitro neuron and muscle cell differentiation, viability and survival under normal or adverse conditions; anabolic, protective and pro-differentiative effects have also been observed in vivo. For example Cr has been shown to accelerate in vitro differentiation of cultured C2C12 myoblasts into myotubes, where it also induces a slight but significant hypertrophic effect as compared to unsupplemented cultures; Cr also prevents the anti-differentiation effects caused by oxidative stress in the same cells. In trained adults, Cr increases the mRNA expression of relevant myogemic factors, protein synthesis, muscle strength and size, in cooperation with physical exercise. As to neurons and central nervous system, Cr favors the electrophysiological maturation of chick neuroblasts in vitro and protects them from oxidative stress-caused killing; similarly, Cr promotes the survival and differentiation of GABA-ergic neurons in fetal spinal cord cultures in vitro; in vivo, maternal Cr supplementation promotes the morpho-functional development of hippocampal neurons in rat offsprings. This article, which presents also some new experimental data, focuses on the trophic, pro-survival and pro-differentiation effects of Cr and examines the ensuing preventive and therapeutic potential in pathological muscle and brain conditions.

  3. Monocyte derived dendritic cells generated by IFN-α acquire mature dendritic and natural killer cell properties as shown by gene expression analysis

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    Czibere Akos

    2007-09-01

    Full Text Available Abstract Background Dendritic cell (DC vaccines can induce antitumor immune responses in patients with malignant diseases, while the most suitable DC culture conditions have not been established yet. In this study we compared monocyte derived human DC from conventional cultures containing GM-CSF and IL-4/TNF-α (IL-4/TNF-DC with DC generated by the novel protocol using GM-CSF and IFN-α (IFN-DC. Methods To characterise the molecular differences of both DC preparations, gene expression profiling was performed using Affymetrix microarrays. The data were conformed on a protein level by immunophenotyping, and functional tests for T cell stimulation, migration and cytolytic activity were performed. Results Both methods resulted in CD11c+ CD86+ HLA-DR+ cells with a typical DC morphology that could efficiently stimulate T cells. But gene expression profiling revealed two distinct DC populations. Whereas IL-4/TNF-DC showed a higher expression of genes envolved in phagocytosis IFN-DC had higher RNA levels for markers of DC maturity and migration to the lymph nodes like DCLAMP, CCR7 and CD49d. This different orientation of both DC populations was confined by a 2.3 fold greater migration in transwell experiments (p = 0.01. Most interestingly, IFN-DC also showed higher RNA levels for markers of NK cells such as TRAIL, granzymes, KLRs and other NK cell receptors. On a protein level, intracytoplasmatic TRAIL and granzyme B were observed in 90% of IFN-DC. This translated into a cytolytic activity against K562 cells with a median specific lysis of 26% at high effector cell numbers as determined by propidium iodide uptake, whereas IL-4/TNF-DC did not induce any tumor cell lysis (p = 0.006. Thus, IFN-DC combined characteristics of mature DC and natural killer cells. Conclusion Our results suggest that IFN-DC not only stimulate adaptive but also mediate innate antitumor immune responses. Therefore, IFN-DC should be evaluated in clinical vaccination trials. In

  4. Germinal Center B-Cell-Associated Nuclear Protein (GANP) Involved in RNA Metabolism for B Cell Maturation.

    Science.gov (United States)

    Sakaguchi, N; Maeda, K

    2016-01-01

    Germinal center B-cell-associated nuclear protein (GANP) is upregulated in germinal center B cells against T-cell-dependent antigens in mice and humans. In mice, GANP depletion in B cells impairs antibody affinity maturation. Conversely, its transgenic overexpression augments the generation of high-affinity antigen-specific B cells. GANP associates with AID in the cytoplasm, shepherds AID into the nucleus, and augments its access to the rearranged immunoglobulin (Ig) variable (V) region of the genome in B cells, thereby precipitating the somatic hypermutation of V region genes. GANP is also upregulated in human CD4(+) T cells and is associated with APOBEC3G (A3G). GANP interacts with A3G and escorts it to the virion cores to potentiate its antiretroviral activity by inactivating HIV-1 genomic cDNA. Thus, GANP is characterized as a cofactor associated with AID/APOBEC cytidine deaminase family molecules in generating diversity of the IgV region of the genome and genetic alterations of exogenously introduced viral targets. GANP, encoded by human chromosome 21, as well as its mouse equivalent on chromosome 10, contains a region homologous to Saccharomyces Sac3 that was characterized as a component of the transcription/export 2 (TREX-2) complex and was predicted to be involved in RNA export and metabolism in mammalian cells. The metabolism of RNA during its maturation, from the transcription site at the chromosome within the nucleus to the cytoplasmic translation apparatus, needs to be elaborated with regard to acquired and innate immunity. In this review, we summarize the current knowledge on GANP as a component of TREX-2 in mammalian cells.

  5. Chronic mast cell leukemia (MCL) with KIT S476I: a rare entity defined by leukemic expansion of mature mast cells and absence of organ damage.

    Science.gov (United States)

    Valent, Peter; Berger, Jörg; Cerny-Reiterer, Sabine; Peter, Barbara; Eisenwort, Gregor; Hoermann, Gregor; Müllauer, Leonhard; Mannhalter, Christine; Steurer, Michael; Bettelheim, Peter; Horny, Hans-Peter; Arock, Michel

    2015-02-01

    Mast cell leukemia (MCL) is a rare, life-threatening malignancy defined by a substantial increase in neoplastic mast cells (MCs) in bone marrow (BM) smears, drug-resistance, and a poor prognosis. In most patients, the survival time is less than 1 year. However, exceptional cases may present with a less malignant course. We report on a 49-year-old female patient with MCL diagnosed in 2013. In February 2013, first symptoms, including flushing, headache, and diarrhea, were recorded. In addition, mild anemia was detected. The disease was characterized by a massive increase in well-granulated, mature, and often spindle-shaped MCs (80 %) in BM smears. The serum tryptase level amounted to 332 ng/mL. Like in most other MCL patients, no skin lesions were detected. However, unlike in other patients, tryptase levels remained stable, and no other signs or symptoms of MCL-induced organ damage were found. Sequencing studies revealed an isolated S476I point mutation in KIT but no mutation in codon 816. The patient received histamine receptor blockers but refused cytoreductive therapy. After 9 months, still no progression or organ damage was detected. However, progression with transformation to acute MCL occurred after 12 months. We propose that the chronic type of MCL with stable conditions, absence of organ damage, and a mature MC morphology is recognized as a distinct entity that should be distinguished from the acute variant of MCL.

  6. Galactosylated collagen matrix enhanced in vitro maturation of human embryonic stem cell-derived hepatocyte-like cells.

    Science.gov (United States)

    Ghodsizadeh, Arefeh; Hosseinkhani, Hossein; Piryaei, Abbas; Pournasr, Behshad; Najarasl, Mostafa; Hiraoka, Yosuke; Baharvand, Hossein

    2014-05-01

    Due to their important biomedical applications, functional human embryonic stem cell-derived hepatocyte-like cells (hESC-HLCs) are an attractive topic in the field of stem cell differentiation. Here, we have initially differentiated hESCs into functional hepatic endoderm (HE) and continued the differentiation by replating them onto galactosylated collagen (GC) and collagen matrices. The differentiation of hESC-HE cells into HLCs on GC substrate showed significant up-regulation of hepatic-specific genes such as ALB, HNF4α, CYP3A4, G6P, and ASGR1. There was more albumin secretion and urea synthesis, as well as more cytochrome p450 activity, in differentiated HLCs on GC compared to the collagen-coated substrate. These results suggested that GC substrate has the potential to be used for in vitro maturation of hESC-HLCs.

  7. Maturation of shark single-domain (IgNAR) antibodies: evidence for induced-fit binding.

    Science.gov (United States)

    Stanfield, Robyn L; Dooley, Helen; Verdino, Petra; Flajnik, Martin F; Wilson, Ian A

    2007-03-23

    Sharks express an unusual heavy-chain isotype called IgNAR, whose variable regions bind antigen as independent soluble domains. To further probe affinity maturation of the IgNAR response, we structurally characterized the germline and somatically matured versions of a type II variable (V) region, both in the presence and absence of its antigen, hen egg-white lysozyme. Despite a disulfide bond linking complementarity determining regions (CDRs) 1 and 3, both germline and somatically matured V regions displayed significant structural changes in these CDRs upon complex formation with antigen. Somatic mutations in the IgNAR V region serve to increase the number of contacts with antigen, as reflected by a tenfold increase in affinity, and one of these mutations appears to stabilize the CDR3 region. In addition, a residue in the HV4 loop plays an important role in antibody-antigen interaction, consistent with the high rate of somatic mutations in this non-CDR loop.

  8. Maturation of Shark Single-Domain (IgNAR) Antibodies: Evidence for Induced-Fit Binding

    Energy Technology Data Exchange (ETDEWEB)

    Stanfield, R.L.; Dooley, H.; Verdino, P.; Flajnik, M.F.; Wilson, I.A.; /Scripps Res. Inst. /Maryland U.

    2007-07-13

    Sharks express an unusual heavy-chain isotype called IgNAR, whose variable regions bind antigen as independent soluble domains. To further probe affinity maturation of the IgNAR response, we structurally characterized the germline and somatically matured versions of a type II variable (V) region, both in the presence and absence of its antigen, hen egg-white lysozyme. Despite a disulfide bond linking complementarity determining regions (CDRs) 1 and 3, both germline and somatically matured V regions displayed significant structural changes in these CDRs upon complex formation with antigen. Somatic mutations in the IgNAR V region serve to increase the number of contacts with antigen, as reflected by a tenfold increase in affinity, and one of these mutations appears to stabilize the CDR3 region. In addition, a residue in the HV4 loop plays an important role in antibody-antigen interaction, consistent with the high rate of somatic mutations in this non-CDR loop.

  9. Giardia lamblia binding immunoglobulin protein triggers maturation of dendritic cells via activation of TLR4-MyD88-p38 and ERK1/2 MAPKs.

    Science.gov (United States)

    Lee, H-Y; Kim, J; Noh, H J; Kim, H-P; Park, S-J

    2014-12-01

    Much remains unknown about the mammalian immune response to Giardia lamblia, a protozoan pathogen that causes diarrhoeal outbreaks. We fractionated protein extracts of G. lamblia trophozoites by Viva-spin centrifugation, DEAE ion exchange and gel filtration chromatography. Resultant fractions were screened for antigenic molecules by western blots analysis using anti-G. lamblia antibodies (Abs), resulting in identification of G. lamblia binding immunoglobulin protein (GlBiP). Maturation of mouse dendritic cells (DCs) in response to recombinant GlBiP (rGlBiP) was detected by increased expression of surface molecules such as CD80, CD86 and MHC class II; these mature DCs, produced pro-inflammatory cytokines (TNF-α, IL-12 and IL-6). Especially, the truncated rGlBiP containing the heat-shock protein 70 domain-induced cytokine production from mouse DCs. rGlBiP-induced DC activation was initiated by TLR4 in a MyD88-dependent way and occurred through activation of p38 and ERK1/2 MAPKs as well as increased activity of NF-κB and AP-1. Moreover, CD4(+) T cells stimulated with rGlBiP-treated DCs produced high levels of IL-2 and IFN-γ. Together, our results suggest that GlBiP contributes to maturation of DCs via activation of TLR4-MyD88-p38, ERK1/2 MAPK, NF-κB and AP-1.

  10. Lymph nodes from HIV-infected individuals harbor mature dendritic cells and increased numbers of PD-L1+ conventional dendritic cells.

    Science.gov (United States)

    Carranza, Paloma; Del Río Estrada, Perla M; Díaz Rivera, Dafne; Ablanedo-Terrazas, Yuria; Reyes-Terán, Gustavo

    2016-07-01

    The immune response induced by dendritic cells (DC) during the HIV infection has been of remarkable interest because of the therapeutic potential of DC for vaccine development. However, their beneficial or detrimental contribution in HIV infection remains unclear. The activation state of DC in lymph nodes (LN) is essential to induce T cell responses against HIV. In the present study, we characterized the immunophenotype and function of conventional (cDC) and plasmacytoid (pDC) dendritic cells from peripheral blood (PB) and LN of HIV(+) individuals. We observed that the frequency of PB pDC was decreased and exhibited an immature phenotype; whereas in the LN, activated pDC accumulated (CD40(+) and CD83(+)). In addition, the frequency of PB cDC from HIV(+) individuals was decreased and exhibited an immature phenotype, whereas LN harbored activated and mature cDC (CD40(+), CD83(+), CD80(+) and CD86(+)). However, an increased number of PD-L1(+) cDC was also observed in the LN. Moreover, pDC and cDC were able to produce inflammatory cytokines (IFN-α, TNF-α and IL-12) after TLR stimulation. These findings suggests that LN cDC expressing PD-L1 from HIV(+) individuals may negatively impact the generation of HIV-specific T cells and that DC might be contributing to tissue chronic immune activation.

  11. Two-step differentiation of mast cells from induced pluripotent stem cells.

    Science.gov (United States)

    Yamaguchi, Tomoko; Tashiro, Katsuhisa; Tanaka, Satoshi; Katayama, Sumie; Ishida, Waka; Fukuda, Ken; Fukushima, Atsuki; Araki, Ryoko; Abe, Masumi; Mizuguchi, Hiroyuki; Kawabata, Kenji

    2013-03-01

    Mast cells play important roles in the pathogenesis of allergic diseases. They are generally classified into 2 phenotypically distinct populations: connective tissue-type mast cells (CTMCs) and mucosal-type mast cells (MMCs). The number of mast cells that can be obtained from tissues is limited, making it difficult to study the function of mast cells. Here, we report the generation and characterization of CTMC-like mast cells derived from mouse induced pluripotent stem (iPS) cells. iPS cell-derived mast cells (iPSMCs) were generated by the OP9 coculture method or embryoid body formation method. The number of Safranin O-positive cells, expression levels of CD81 protein and histidine decarboxylase mRNA, and protease activities were elevated in the iPSMCs differentiated by both methods as compared with those in bone marrow-derived mast cells (BMMCs). Electron microscopic analysis revealed that iPSMCs contained more granules than BMMCs. Degranulation was induced in iPSMCs after stimulation with cationic secretagogues or vancomycin. In addition, iPSMCs had the ability to respond to stimulation with the IgE/antigen complex in vitro and in vivo. Moreover, when iPSMCs generated on OP9 cells were cocultured with Swiss 3T3 fibroblasts, protease activities as maturation index were more elevated, demonstrating that mature mast cells were differentiated from iPS cells. iPSMCs can be used as an in vitro model of CTMCs to investigate their functions.

  12. Use of rat mature adipocyte-derived dedifferentiated fat cells as a cell source for periodontal tissue regeneration

    Directory of Open Access Journals (Sweden)

    Daisuke eAkita

    2016-02-01

    Full Text Available Lipid-free fibroblast-like cells, known as dedifferentiated fat (DFAT cells, can be generated from mature adipocytes with a large single lipid droplet. DFAT cells can re-establish their active proliferation ability and can transdifferentiate into various cell types under appropriate culture conditions. The first objective of this study was to compare the multilineage differentiation potential of DFAT cells with that of adipose-derived stem cells (ASCs on mesenchymal stem cellsWe obtained DFAT cells and ASCs from inbred rats and found that rat DFAT cells possess higher osteogenic differentiation potential than rat ASCs. On the other hand, DFAT cells show similar adipogenic differentiation, and chondrogenic differentiation potential in comparison with ASCs. The second objective of this study was to assess the regenerative potential of DFAT cells combined with novel solid scaffolds composed of PLGA (Poly d, l-lactic-co-glycolic acid on periodontal tissue, and to compare this with the regenerative potential of ASCs combined with PLGA scaffolds. Cultured DFAT cells and ASCs were seeded onto PLGA scaffolds (DFAT/PLGA and ASCs/PLGA and transplanted into periodontal fenestration defects in rat mandible. Micro computed tomography analysis revealed a significantly higher amount of bone regeneration in the DFAT/PLGA group compared with that of ASCs/PLGA and PLGA-alone groups at 2, 3 and 5 weeks after transplantation. Similarly, histomorphometric analysis showed that DFAT/PLGA groups had significantly greater width of cementum, periodontal ligament and alveolar bone than ASCs/PLGA and PLGA-alone groups. In addition, transplanted fluorescent-labeled DFAT cells were observed in the periodontal ligament beside the newly formed bone and cementum. These findings suggest that DFAT cells have a greater potential for enhancing periodontal tissue regeneration than ASCs. Therefore, DFAT cells are a promising cell source for periodontium regeneration.

  13. Use of Rat Mature Adipocyte-Derived Dedifferentiated Fat Cells as a Cell Source for Periodontal Tissue Regeneration.

    Science.gov (United States)

    Akita, Daisuke; Kano, Koichiro; Saito-Tamura, Yoko; Mashimo, Takayuki; Sato-Shionome, Momoko; Tsurumachi, Niina; Yamanaka, Katsuyuki; Kaneko, Tadashi; Toriumi, Taku; Arai, Yoshinori; Tsukimura, Naoki; Matsumoto, Taro; Ishigami, Tomohiko; Isokawa, Keitaro; Honda, Masaki

    2016-01-01

    Lipid-free fibroblast-like cells, known as dedifferentiated fat (DFAT) cells, can be generated from mature adipocytes with a large single lipid droplet. DFAT cells can re-establish their active proliferation ability and can transdifferentiate into various cell types under appropriate culture conditions. The first objective of this study was to compare the multilineage differentiation potential of DFAT cells with that of adipose-derived stem cells (ASCs) on mesenchymal stem cells. We obtained DFAT cells and ASCs from inbred rats and found that rat DFAT cells possess higher osteogenic differentiation potential than rat ASCs. On the other hand, DFAT cells show similar adipogenic differentiation, and chondrogenic differentiation potential in comparison with ASCs. The second objective of this study was to assess the regenerative potential of DFAT cells combined with novel solid scaffolds composed of PLGA (Poly d, l-lactic-co-glycolic acid) on periodontal tissue, and to compare this with the regenerative potential of ASCs combined with PLGA scaffolds. Cultured DFAT cells and ASCs were seeded onto PLGA scaffolds (DFAT/PLGA and ASCs/PLGA) and transplanted into periodontal fenestration defects in rat mandible. Micro computed tomography analysis revealed a significantly higher amount of bone regeneration in the DFAT/PLGA group compared with that of ASCs/PLGA and PLGA-alone groups at 2, 3, and 5 weeks after transplantation. Similarly, histomorphometric analysis showed that DFAT/PLGA groups had significantly greater width of cementum, periodontal ligament and alveolar bone than ASCs/PLGA and PLGA-alone groups. In addition, transplanted fluorescent-labeled DFAT cells were observed in the periodontal ligament beside the newly formed bone and cementum. These findings suggest that DFAT cells have a greater potential for enhancing periodontal tissue regeneration than ASCs. Therefore, DFAT cells are a promising cell source for periodontium regeneration.

  14. Regulation of Oligodendrocyte Progenitor Cell Maturation by PPARδ: Effects on Bone Morphogenetic Proteins

    Directory of Open Access Journals (Sweden)

    Maria Vittoria Simonini

    2009-12-01

    Full Text Available In EAE (experimental autoimmune encephalomyelitis, agonists of PPARs (peroxisome proliferator-activated receptors provide clinical benefit and reduce damage. In contrast with PPARγ, agonists of PPARδ are more effective when given at later stages of EAE and increase myelin gene expression, suggesting effects on OL (oligodendrocyte maturation. In the present study we examined effects of the PPARδ agonist GW0742 on OPCs (OL progenitor cells, and tested whether the effects involve modulation of BMPs (bone morphogenetic proteins. We show that effects of GW0742 are mediated through PPARδ since no amelioration of EAE clinical scores was observed in PPARδ-null mice. In OPCs derived from E13 mice (where E is embryonic day, GW0742, but not the PPARγ agonist pioglitazone, increased the number of myelin-producing OLs. This was due to activation of PPARδ since process formation was reduced in PPARδ-null compared with wild-type OPCs. In both OPCs and enriched astrocyte cultures, GW0742 increased noggin protein expression; however, noggin mRNA was only increased in astrocytes. In contrast, GW0742 reduced BMP2 and BMP4 mRNA levels in OPCs, with lesser effects in astrocytes. These findings demonstrate that PPARδ plays a role in OPC maturation, mediated, in part, by regulation of BMP and BMP antagonists.

  15. Isolation of Mature (Peritoneum-Derived Mast Cells and Immature (Bone Marrow-Derived Mast Cell Precursors from Mice.

    Directory of Open Access Journals (Sweden)

    Steffen K Meurer

    Full Text Available Mast cells (MCs are a versatile cell type playing key roles in tissue morphogenesis and host defence against bacteria and parasites. Furthermore, they can enhance immunological danger signals and are implicated in inflammatory disorders like fibrosis. This granulated cell type originates from the myeloid lineage and has similarities to basophilic granulocytes, both containing large quantities of histamine and heparin. Immature murine mast cells mature in their destination tissue and adopt either the connective tissue (CTMC or mucosal (MMC type. Some effector functions are executed by activation/degranulation of MCs which lead to secretion of a typical set of MC proteases (MCPT and of the preformed or newly synthesized mediators from its granules into the local microenvironment. Due to the potential accumulation of mutations in key signalling pathway components of corresponding MC cell-lines, primary cultured MCs are an attractive mean to study general features of MC biology and aspects of MC functions relevant to human disease. Here, we describe a simple protocol for the simultaneous isolation of mature CTMC-like murine MCs from the peritoneum (PMCs and immature MC precursors from the bone marrow (BM. The latter are differentiated in vitro to yield BM-derived MCs (BMMC. These cells display the typical morphological and phenotypic features of MCs, express the typical MC surface markers, and can be propagated and kept in culture for several weeks. The provided protocol allows simple amplification of large quantities of homogenous, non-transformed MCs from the peritoneum and bone marrow-derived mast cells for cell- and tissue-based biomedical research.

  16. Mature dendritic cells generated from patient-derived peripheral blood monocytes in one-step culture using streptococcal preparation OK-432 exert an enhanced antigen-presenting capacity.

    Science.gov (United States)

    Naito, Kei; Ueda, Yuji; Itoh, Tsuyoshi; Fuji, Nobuaki; Shimizu, Keiji; Yano, Yutaro; Yamamoto, Yoshiki; Imura, Kenichiro; Kohara, Junji; Iwamoto, Arihiro; Shiozaki, Atsushi; Tamai, Hidemasa; Shimizu, Takeshi; Mazda, Osam; Yamagishi, Hisakazu

    2006-06-01

    Dendritic cells (DCs) have been shown to be potent in inducing cytotoxic T cell (CTL) response leading to the efficient anti-tumor effect in active immunotherapy. Myeloid DCs are conventionally generated from human peripheral blood monocytes in the presence of interleukin (IL)-4 and granulocyte/macrophage colony-stimulating factor (GM-CSF). Streptococcal preparation OK-432, which is known to be a multiple cytokine inducer, has been extensively studied as to its maturation effects on immature DCs using an in vitro culture system. The purpose of this study was to examine whether it could be possible to generate mature DCs directly from peripheral monocytes using OK-432. We specifically focused on the possibility that recombinant cytokines, which are considered to be essential for in vitro DC generation, could be substituted by OK-432. Human peripheral monocytes, which were obtained from patients with advanced cancer, were cultured with IL-4 and OK-432 for 7 days. Cultured cells were compared with DCs generated in the presence of IL-4 and GM-CSF with or without OK-432 with regard to the surface phenotype as well as the antigen-presenting capacity. As a result, the culture of monocytes in the presence of IL-4 followed by the addition of OK-432 on day 4 (IL-4/OK-DC) induced cells with a fully mature DC phenotype. Functional assays also demonstrated that IL-4/OK-DCs had a strong antigen-presenting capacity determined by their enhanced antigen-specific CTL response and exerted a Th1-type T cell response which is critical for the induction of anti-tumor response. In conclusion, human peripheral blood monocytes cultured in the presence of IL-4 and OK-432 without exogenous GM-CSF demonstrated a fully mature DC phenotype and strong antigen-presenting capacity. This one-step culture protocol allows us to generate fully mature DCs directly from monocytes in 7 days and thus, this protocol can be applicable for DC-based anti-tumor immunotherapy.

  17. Polyphosphate induces matrix metalloproteinase-3-mediated proliferation of odontoblast-like cells derived from induced pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Ozeki, Nobuaki; Hase, Naoko; Yamaguchi, Hideyuki; Hiyama, Taiki; Kawai, Rie [Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya, Aichi 464-8651 (Japan); Kondo, Ayami [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto, Chikusa-ku, Nagoya 464-8650 (Japan); Nakata, Kazuhiko [Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya, Aichi 464-8651 (Japan); Mogi, Makio, E-mail: makio@dpc.agu.ac.jp [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto, Chikusa-ku, Nagoya 464-8650 (Japan)

    2015-05-01

    Inorganic polyphosphate [Poly(P)] may represent a physiological source of phosphate and has the ability to induce bone differentiation in osteoblasts. We previously reported that cytokine-induced matrix metalloproteinase (MMP)-3 accelerates the proliferation of purified odontoblast-like cells. In this study, MMP-3 small interfering RNA (siRNA) was transfected into odontoblast-like cells derived from induced pluripotent stem cells to investigate whether MMP-3 activity is induced by Poly(P) and/or is associated with cell proliferation and differentiation into odontoblast-like cells. Treatment with Poly(P) led to an increase in both cell proliferation and additional odontoblastic differentiation. Poly(P)-treated cells showed a small but significant increase in dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1) mRNA expression, which are markers of mature odontoblasts. The cells also acquired additional odontoblast-specific properties including adoption of an odontoblastic phenotype typified by high alkaline phosphatase (ALP) activity and a calcification capacity. In addition, Poly(P) induced expression of MMP-3 mRNA and protein, and increased MMP-3 activity. MMP-3 siRNA-mediated disruption of the expression of these effectors potently suppressed the expression of odontoblastic biomarkers ALP, DSPP, and DMP-1, and blocked calcification. Interestingly, upon siRNA-mediated silencing of MMP-3, we noted a potent and significant decrease in cell proliferation. Using specific siRNAs, we revealed that a unique signaling cascade, Poly(P)→MMP-3→DSPP and/or DMP-1, was intimately involved in the proliferation of odontoblast-like cells. - Highlights: • Polyphosphate increases proliferation of iPS cell-derived odontoblast-like cells. • Polyphosphate-induced MMP-3 results in an increase of cell proliferation. • Induced cell proliferation involves MMP-3, DSPP, and/or DMP-1 sequentially. • Induced MMP-3 also results in an increase of odontoblastic

  18. The microvesicle component of HIV-1 inocula modulates dendritic cell infection and maturation and enhances adhesion to and activation of T lymphocytes.

    Directory of Open Access Journals (Sweden)

    Sarah K Mercier

    2013-10-01

    Full Text Available HIV-1 is taken up by immature monocyte derived dendritic cells (iMDDCs into tetraspanin rich caves from which the virus can either be transferred to T lymphocytes or enter into endosomes resulting in degradation. HIV-1 binding and fusion with the DC membrane results in low level de novo infection that can also be transferred to T lymphocytes at a later stage. We have previously reported that HIV-1 can induce partial maturation of iMDDCs at both stages of trafficking. Here we show that CD45⁺ microvesicles (MV which contaminate purified HIV-1 inocula due to similar size and density, affect DC maturation, de novo HIV-1 infection and transfer to T lymphocytes. Comparing iMDDCs infected with CD45-depleted HIV-1BaL or matched non-depleted preparations, the presence of CD45⁺ MVs was shown to enhance DC maturation and ICAM-1 (CD54 expression, which is involved in DC∶T lymphocyte interactions, while restricting HIV-1 infection of MDDCs. Furthermore, in the DC culture HIV-1 infected (p24⁺ MDDCs were more mature than bystander cells. Depletion of MVs from the HIV-1 inoculum markedly inhibited DC∶T lymphocyte clustering and the induction of alloproliferation as well as limiting HIV-1 transfer from DCs to T lymphocytes. The effects of MV depletion on these functions were reversed by the re-addition of purified MVs from activated but not non-activated SUPT1.CCR5-CL.30 or primary T cells. Analysis of the protein complement of these MVs and of these HIV-1 inocula before and after MV depletion showed that Heat Shock Proteins (HSPs and nef were the likely DC maturation candidates. Recombinant HSP90α and β and nef all induced DC maturation and ICAM-1 expression, greater when combined. These results suggest that MVs contaminating HIV-1 released from infected T lymphocytes may be biologically important, especially in enhancing T cell activation, during uptake by DCs in vitro and in vivo, particularly as MVs have been detected in the circulation of HIV-1

  19. Heterogeneity in predisposition of hepatic cells to be induced into pancreatic endocrine cells by PDX-1

    Institute of Scientific and Technical Information of China (English)

    Shun Lu; Wei-Ping Wang; Xiao-Fei Wang; Zong-Mei Zheng; Ping Chen; Kang-Tao Ma; Chun-Yan Zhou

    2005-01-01

    AIM: The role of Pancreatic and Duodenal Homeobox-1(PDX-1) as a major regulator of pancreatic development determines the function and phenotype of β cell. In this study, potential plasticity of liver cells into pancreatic endocrine cells induced by PDX-1 was evaluated.METHODS: Human hepatoma cell line HepG2 was stably transfected with mammalian expression plasmid pcDNA3-PDX encoding human PDX-1 gene. Ectopic expression of PDX-1 and insulin were detected by RT-PCR,Western blot and/or immunostaining. PDX-1+ HepG2 cells were transplanted under renal capsule of STZ-induced diabetic nude mice (n = 16) to examine the inducing effect in vivo.RESULTS: Exogenous PDX-1 transgene was proved to express effectively in HepG2 cell at both mRNA and protein levels. The expression of endogenous insulin and some βcell-specific differentiation markers and transcription factors were not induced in PDX-1+ HepG2 cells. When transplanted under renal capsule of STZ-induced diabetic nude mice, PDX-1+ HepG2 cells did not generate insulinproducing cells. These data indicated that stable transfected PDX-1 could not convert hepatoma cell line HepG2 to pancreatic cells in vitro or in vivo. Mature hepatocytes might need much more complicated or rigorous conditions to be shifted to insulin-producing cells.CONCLUSION: The expression of exogenous PDX-1 is not sufficient to induce relatively mature hepatocytes differentiating into insulin-producing cells.

  20. Studies on mRNA electroporation of immature and mature dendritic cells: Effects on their immunogenic potential

    DEFF Research Database (Denmark)

    Met, O.; Eriksen, J.; Svane, Inge Marie

    2008-01-01

    Previous studies have shown that mRNA-electroporated dendritic cells (DCs) are able to process and present tumor-associated antigens, leading to the activation of tumor-specific T cells in vitro and in vivo. However, the optimal maturation state of antigen loading and half-life of the m...

  1. Dexamethasone impairs the differentiation and maturation of murine dendritic cells by Toll-like receptor 4-nuclear factor-κB pathway

    Institute of Scientific and Technical Information of China (English)

    HE Xiao-kui; WU Xiang-ling; HE Xiu-juan; LI Bo; HU Yong-xiu

    2010-01-01

    Background Recent studies have demonstrated that dexamethasone (DEX) interferes with immune responses by targeting key functions of dendritic cells (DCs) at the earliest stage. However, the cellular and molecular mechanisms are still incompletely understood. This study aimed to explore the possible mechanisms by investigating the roles of DEX on differentiation, maturation & function of murine DCs and the effects of DEX on DCs via Toll-like receptor 4 (TLR4)-nuclear factor (NF)-κB mediated signal pathway.Methods Immature DCs (imDCs) were cultured from murine bone marrow (BM) cells. We added DEX into culture medium at different time. The expression of CD11c, CD86 and I-A~b (mouse MHC class Ⅱ molecule) was determined by flow cytometry. We determined the expression of NF-κB and its inhibitory protein I-κBα by electrophoretic mobility shift assay (EMSA) and Western blotting, respectively. The productions of interleukin (IL)-12p70 and IL-10 in cell culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA).Results DEX impaired differentiation of DCs from murine bone marrow progenitors, and inhibited lipopolysaccharide (LPS) induced maturation of DCs. DEX significantly inhibited NF-κB expression of normal DCs, the higher the DEX concentration or the longer the DEX treatment time, the more obvious the effect. However, DEX had little effect on LPS-induced NF-κB activation, and partially impaired LPS-induced I-κBα degradation. DEX significantly decreased LPS induced IL-12p70 production by DCs. Interestingly, our results showed a synergistic effect between DEX and LPS on the production of IL-10 by DCs.Conclusions DEX inhibits the differentiation and maturation of murine DCs involved in TLR4-I-κB-NF-κB pathway, and also indirectly impairs Th1 development and interferes with the Th1-Th2 balance through IL-12 and/or IL-10 secretion by DCs.

  2. SAM pointed domain ETS factor (SPDEF) regulates terminal differentiation and maturation of intestinal goblet cells

    Energy Technology Data Exchange (ETDEWEB)

    Noah, Taeko K.; Kazanjian, Avedis [Gastroenterology, Hepatology and Nutrition, Cincinnati Children' s Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH (United States); Whitsett, Jeffrey [Developmental Biology, Cincinnati Children' s Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH (United States); Neonatology and Pulmonary Biology, Cincinnati Children' s Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH (United States); Shroyer, Noah F., E-mail: noah.shroyer@cchmc.org [Gastroenterology, Hepatology and Nutrition, Cincinnati Children' s Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH (United States); Developmental Biology, Cincinnati Children' s Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH (United States)

    2010-02-01

    Background and Aims: SPDEF (also termed PDEF or PSE) is an ETS family transcription factor that regulates gene expression in the prostate and goblet cell hyperplasia in the lung. Spdef has been reported to be expressed in the intestine. In this paper, we identify an important role for Spdef in regulating intestinal epithelial cell homeostasis and differentiation. Methods: SPDEF expression was inhibited in colon cancer cells to determine its ability to control goblet cell gene activation. The effects of transgenic expression of Spdef on intestinal differentiation and homeostasis were determined. Results: In LS174T colon cancer cells treated with Notch/{gamma}-secretase inhibitor to activate goblet cell gene expression, shRNAs that inhibited SPDEF also repressed expression of goblet cell genes AGR2, MUC2, RETLNB, and SPINK4. Transgenic expression of Spdef caused the expansion of intestinal goblet cells and corresponding reduction in Paneth, enteroendocrine, and absorptive enterocytes. Spdef inhibited proliferation of intestinal crypt cells without induction of apoptosis. Prolonged expression of the Spdef transgene caused a progressive reduction in the number of crypts that expressed Spdef, consistent with its inhibitory effects on cell proliferation. Conclusions: Spdef was sufficient to inhibit proliferation of intestinal progenitors and induce differentiation into goblet cells; SPDEF was required for activation of goblet cell associated genes in vitro. These data support a model in which Spdef promotes terminal differentiation into goblet cells of a common goblet/Paneth progenitor.

  3. Inducement of chromosome translocation with small alien segments by irradiating mature female gametes of the whole arm translocation line

    Institute of Scientific and Technical Information of China (English)

    CHEN ShengWei; CHEN PeiDu; WANG XiuE

    2008-01-01

    Haynaldia villosa Schur. (syn. Dasypyrum villosum Candargy, 2n=14, VV) has been proved to be an Important genetic resource for wheat improvement. The development of translocation with small alien chromosome segments, especially interstitial translocation, will be helpful for better utilization of its useful genes. Up to now, most of the reported Triticum aestivum - H. villosa translocation lines are involved in a whole arm or large alien fragments. In this paper, we report a highly efficient approach for the creation of small chromosome segment translocation lines. Before flowering, the female gametes of wheat-H, villosa 6VS/6AL trsnslocation line were irradiated by 60Co-γ ray at 160 Rad/M dosage rate and three dosages (1600, 1920, 2240 Rad). Anthers were removed from the irradiated florets on the same day and the florets were pollinated with normal fresh pollens of T. aestivum cv. Chinese Spring after 2-3 days. Genomic in situ hybridization (GISH) at mitosis metaphase of root-tip cell of M1 plants was used to detect the chromosome structural changes involving 6VS of H. villosa. Among the 534 M1 plants screened, 97 plants contained small segment chromosome structural changes of 6VS, including 80 interstitial translocation chromosomes, 57 terminal translocation chromosomes and 55 deletion chromosomes. For the 2240 Rad dosage treatment, the inducement frequencies of interstitial translocation, terminal translocation and deletion were 21.02%, 14.01%, and 14.65%, respectively, which were much higher than those previously reported. The M2 seeds were obtained by bsckcrossing of 74 M1 plants involving 146 chromosomes structural changes of 6VS, and it was found that the structural aberrations in the M1 plants could be transmitted to their progenies. Irradiating mature female gametes of whole arm translocation is a new and highly efficient approach for creation of small segment chromosome structural changes, especially for interstitial translocations.

  4. Early vessel destabilization mediated by Angiopoietin-2 and subsequent vessel maturation via Angiopoietin-1 induce functional neovasculature after ischemia.

    Directory of Open Access Journals (Sweden)

    Di Qin

    Full Text Available BACKGROUND: We assessed whether Angiopoietin-2 (Ang2, a Tie2 ligand and partial antagonist of Angiopoietin-1 (Ang1, is required for early vessel destabilization during postischemic angiogenesis, when combined with vascular growth factors. METHODS: In vitro, matrigel co-cultures assessed endothelial-cell tube formation and pericyte recruitment after stimulation of VEGF-A, Apelin (APLN, Ang1 with or without Ang2. In a murine hindlimb ischemia model, adeno-associated virus (rAAV, 3×10(12 virusparticles transduction of VEGF-A, APLN and Ang1 with or without Ang2 (continuous or early expression d0-3 was performed intramuscularly (d-14. Femoral artery ligation was performed at d0, followed by laser doppler perfusion meassurements (LDI 7 and 14. At d7 (early timepoint and d14 (late timepoint, histological analysis of capillary/muscle fiber ratio (CMF-R, PECAM-1 and pericyte/capillary ratio (PC-R, NG2 was performed. RESULTS: In vitro, VEGF-A, APLN and Ang1 induced ring formation, but only APLN and Ang1 recruited pericytes. Ang2 did not affect tube formation by APLN, but reduced pericyte recruitment after APLN or Ang1 overexpression. In vivo, rAAV.VEGF-A did not alter LDI-perfusion at d14, consistent with an impaired PC-R despite a rise in CMF-R. rAAV.APLN improved perfusion at d14, with or without continuous Ang2, increasing CMF-R and PC-R. rAAV.Ang1 improved perfusion at d14, when combined with rAAV.Ang2 (d0-3, accompanied by an increased CMF-R and PC-R. CONCLUSION: The combination of early vessel destabilization (Ang2 d0-3 and continuous Ang1 overexpression improves hindlimb perfusion, pointing to the importance of early vessel destabilization and subsequent vessel maturation for enhanced therapeutic neovascularization.

  5. Inducement of chromosome translocation with small alien segments by irradiating mature female gametes of the whole arm translocation line

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Haynaldia villosa Schur. (syn. Dasypyrum villosum Candargy, 2n=14, VV) has been proved to be an important genetic resource for wheat improvement. The development of translocation with small alien chromosome segments, especially interstitial translocation, will be helpful for better utilization of its useful genes. Up to now, most of the reported Triticum aestivum – H. villosa translocation lines are involved in a whole arm or large alien fragments. In this paper, we report a highly efficient approach for the creation of small chromosome segment translocation lines. Before flowering, the female gametes of wheat-H. villosa 6VS/6AL translocation line were irradiated by 60CO-γ ray at 160 Rad/M dosage rate and three dosages (1600, 1920, 2240 Rad). Anthers were removed from the irradiated florets on the same day and the florets were pollinated with normal fresh pollens of T. aestivum cv. Chinese Spring after 2-3 days. Genomic in situ hybridization (GISH) at mitosis metaphase of root-tip cell of M1 plants was used to detect the chromosome structural changes involving 6VS of H. villosa. Among the 534 M1 plants screened, 97 plants contained small segment chromosome structural changes of 6VS, including 80 interstitial translocation chromosomes, 57 terminal translocation chromosomes and 55 deletion chromosomes. For the 2240 Rad dosage treatment, the inducement frequencies of interstitial translo-cation, terminal translocation and deletion were 21.02%, 14.01%, and 14.65%, respectively, which were much higher than those previously reported. The M2 seeds were obtained by backcrossing of 74 M1 plants involving 146 chromosomes structural changes of 6VS, and it was found that the structural aberrations in the M1 plants could be transmitted to their progenies. Irradiating mature female gametes of whole arm translocation is a new and highly efficient approach for creation of small segment chromosome struc-tural changes, especially for interstitial translocations.

  6. Observation of hydrocarbon generation and migration of highly-matured carbonates by means of laser-induced fluorescence microscopy

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Some important information on hydrocarbon generation, inclusion and migration in highly-matured carbonates of lower Palaeozoic age from the Ordos Basin and Tarim Basin hasbeen analyzed by a newly-combined laser-induced fluorescence microscope (LFM) designed by our laboratory. The following information has been obtained from the lower Ordovician lamellar carbonates with equivalent vitrinite reflectance (Ro) as high as 1.6%-1.7% and residual TOC of 0.14%-0.35% from the Ordos Basin: wide occurrences of oil and source macerals with strong fluorescence, including G. Prisca alginite, lamalginite, telalginite and algae-detrinite; fluorescing asphalt among mineral crystals; some groundmass and spheroid-like reservoir bitumen with high maturation levels in the pores of dolomites. Various kinds of fluorescing organic inclusions and asphalt have been found in the carbonates, calcareous shales and silt-shales with high maturation levels from the Cambrian-Ordovician strata in the Tarim Basin. All this helps us find and evaluate significant and excellent source rocks for large-and middle-scale gas fields. The net and micro-net systems for hydrocarbon generation, expulsion and migration in carbonates have been revealed by the highly-powered laser-induced fluorescence microscopy.

  7. Statistical study of biomechanics of living brain cells during growth and maturation on artificial substrates.

    Science.gov (United States)

    Chen, La; Li, Wenfang; Maybeck, Vanessa; Offenhäusser, Andreas; Krause, Hans-Joachim

    2016-11-01

    There is increasing evidence that mechanical issues play a vital role in neuron growth and brain development. The importance of this grows as novel devices, whose material properties differ from cells, are increasingly implanted in the body. In this work, we studied the mechanical properties of rat brain cells over time and on different materials by using a high throughput magnetic tweezers system. It was found that the elastic moduli of both neurite and soma in networked neurons increased with growth. However, neurites at DIV4 exhibited a relatively high stiffness, which could be ascribed to the high outgrowth tension. The power-law exponents (viscoelasticity) of both neurites and somas of neurons decreased with culture time. On the other hand, the stiffness of glial cells also increased with maturity. Furthermore, both neurites and glia become softer when cultured on compliant substrates. Especially, the glial cells cultured on a soft substrate obviously showed a less dense and more porous actin and GFAP mesh. In addition, the viscoelasticity of both neurites and glia did not show a significant dependence on the substrates' stiffness.

  8. Impaired maturation of large dense-core vesicles in muted-deficient adrenal chromaffin cells.

    Science.gov (United States)

    Hao, Zhenhua; Wei, Lisi; Feng, Yaqin; Chen, Xiaowei; Du, Wen; Ma, Jing; Zhou, Zhuan; Chen, Liangyi; Li, Wei

    2015-04-01

    The large dense-core vesicle (LDCV), a type of lysosome-related organelle, is involved in the secretion of hormones and neuropeptides in specialized secretory cells. The granin family is a driving force in LDCV biogenesis, but the machinery for granin sorting to this biogenesis pathway is largely unknown. The mu mutant mouse, which carries a spontaneous null mutation on the Muted gene (also known as Bloc1s5), which encodes a subunit of the biogenesis of lysosome-related organelles complex-1 (BLOC-1), is a mouse model of Hermansky-Pudlak syndrome. Here, we found that LDCVs were enlarged in mu adrenal chromaffin cells. Chromogranin A (CgA, also known as CHGA) was increased in mu adrenals and muted-knockdown cells. The increased CgA in mu mice was likely due a failure to export this molecule out of immature LDCVs, which impairs LDCV maturation and docking. In mu chromaffin cells, the size of readily releasable pool and the vesicle release frequency were reduced. Our studies suggest that the muted protein is involved in the selective export of CgA during the biogenesis of LDCVs.

  9. Dearth and Delayed Maturation of Testicular Germ Cells in Fanconi Anemia E Mutant Male Mice

    Science.gov (United States)

    Fu, Chun; Begum, Khurshida; Jordan, Philip W.; He, Yan; Overbeek, Paul A.

    2016-01-01

    After using a self-inactivating lentivirus for non-targeted insertional mutagenesis in mice, we identified a transgenic family with a recessive mutation that resulted in reduced fertility in homozygous transgenic mice. The lentiviral integration site was amplified by inverse PCR. Sequencing revealed that integration had occurred in intron 8 of the mouse Fance gene, which encodes the Fanconi anemia E (Fance) protein. Fanconi anemia (FA) proteins play pivotal roles in cellular responses to DNA damage and Fance acts as a molecular bridge between the FA core complex and Fancd2. To investigate the reduced fertility in the mutant males, we analyzed postnatal development of testicular germ cells. At one week after birth, most tubules in the mutant testes contained few or no germ cells. Over the next 2–3 weeks, germ cells accumulated in a limited number of tubules, so that some tubules contained germ cells around the full periphery of the tubule. Once sufficient numbers of germ cells had accumulated, they began to undergo the later stages of spermatogenesis. Immunoassays revealed that the Fancd2 protein accumulated around the periphery of the nucleus in normal developing spermatocytes, but we did not detect a similar localization of Fancd2 in the Fance mutant testes. Our assays indicate that although Fance mutant males are germ cell deficient at birth, the extant germ cells can proliferate and, if they reach a threshold density, can differentiate into mature sperm. Analogous to previous studies of FA genes in mice, our results show that the Fance protein plays an important, but not absolutely essential, role in the initial developmental expansion of the male germ line. PMID:27486799

  10. Mature red blood cells: from optical model to inverse light-scattering problem.

    Science.gov (United States)

    Gilev, Konstantin V; Yurkin, Maxim A; Chernyshova, Ekaterina S; Strokotov, Dmitry I; Chernyshev, Andrei V; Maltsev, Valeri P

    2016-04-01

    We propose a method for characterization of mature red blood cells (RBCs) morphology, based on measurement of light-scattering patterns (LSPs) of individual RBCs with the scanning flow cytometer and on solution of the inverse light-scattering (ILS) problem for each LSP. We considered a RBC shape model, corresponding to the minimal bending energy of the membrane with isotropic elasticity, and constructed an analytical approximation, which allows rapid simulation of the shape, given the diameter and minimal and maximal thicknesses. The ILS problem was solved by the nearest-neighbor interpolation using a preliminary calculated database of 250,000 theoretical LSPs. For each RBC in blood sample we determined three abovementioned shape characteristics and refractive index, which also allows us to calculate volume, surface area, sphericity index, spontaneous curvature, hemoglobin concentration and content.

  11. Oral traumatic neuroma with mature ganglion cells: A case report and review of the literature

    Directory of Open Access Journals (Sweden)

    Lopes Marcio

    2009-01-01

    Full Text Available Traumatic neuromas are characterized by the presence of pain, burning, or paresthesia, associated with a history of trauma, normally surgery, in the same site. In the oral cavity, the most commonly affected sites are the lip, tongue, and mental nerve area. Pressure on the suspected area usually provokes pain. They microscopically consist of a proliferation of nerve fascicles embedded in a background of collagen. We present a case of a 42-year-old Latin American female patient complaining of a painful solitary nodule erupting on the lingual surface of the mandibular body. Histopathological analysis showed a traumatic neuroma associated with mature ganglion cells, which is an extremely unusual finding. After complete removal of the lesion the symptoms disappeared. To the best of our knowledge, this is the first case of a unique lesion with unusual clinical and histopathological features reported in the English language literature.

  12. mRNA translation during oocyte maturation plays a key role in development of primordial germ cells in Xenopus embryos

    Indian Academy of Sciences (India)

    Bahman Zeynali; Keith E Dixon

    2004-09-01

    It is believed that cytoplasmic localization in the egg is necessary for development of primordial germ cells (PGCs) in Xenopus embryos. In this study, we sought to determine if translation of maternal mRNA during oocyte maturation is involved in the development of PGCs. Donor oocytes were collected from both stimulated (those who receive gonadotropin) and unstimulated females, artificially matured and fertilized using a host transfer technique. Using chloramphenicol (50 M and 500 M RNA), RNA translation was inhibited during oocyte maturation. Our results showed that in unstimulated embryos treated with 50 M chloramphenicol, there was a significant reduction in the number of PGCs reaching genital ridges. In stimulated embryos, however, the number of PGCs was unchanged unless a higher concentration (500 M) of chloramphenicol was used. From these results it is suggested that maternal mRNA translation during oocyte maturation plays a key role in development of PGCs.

  13. Lamins regulate cell trafficking and lineage maturation of adult human hematopoietic cells

    OpenAIRE

    Shin, Jae-Won; Spinler, Kyle R.; Swift, Joe; Chasis, Joel A.; Mohandas, Narla; Discher, Dennis E.

    2013-01-01

    Comparing human blood cell types, nuclear diversity is visually striking but unexplained: quasi-spherical nuclei in stem/progenitor cells and T cells contrast with multilobed nuclei in neutrophils, giant nuclei in megakaryocytes, and anuclear erythrocytes. We hypothesized broad roles for the major nuclear structure proteins—lamins—and developed mass spectrometry-calibrated intracellular flow cytometry to quantify lamin-A:B ratios. This ratio controls both nuclear viscoelasticity and cell traf...

  14. Direct hepatic differentiation of mouse embryonic stem cells induced by valproic acid and cytokines

    Institute of Scientific and Technical Information of China (English)

    Xue-Jun Dong; Guo-Rong Zhang; Qing-Jun Zhou; Ruo-Lang Pan; Ye Chen; Li-Xin Xiang; Jian-Zhong Shao

    2009-01-01

    AIM: To develop a protocol for direct hepatic lineage differentiation from early developmental progenitors to a population of mature hepatocytes. METHODS: Hepatic progenitor cells and then mature hepatocytes from mouse embryonic stem (ES) cells were obtained in a sequential manner, induced by valproic acid (VPA) and cytokines (hepatocyte growth factor, epidermal growth factor and insulin). Morphological changes of the differentiated cells were examined by phase-contrast microscopy and electron microscopy. Reverse transcription polymerase chain reaction and immunocytochemical analyses were used to evaluate the gene expression profiles of the VPA-induced hepatic progenitors and the hepatic progenitor-derived hepatocytes. Glycogen storage, cytochrome P450 activity, transplantation assay, differentiation of bile duct-like structures and tumorigenic analyses were performed for the functional identification of the differentiated cells. Furthermore, FACS and electron microscopy were used for the analyses of cell cycle profile and apoptosis in VPA-induced hepatic differentiated cells. RESULTS: Based on the combination of VPA and cytokines, mouse ES cells differentiated into a uniform and homogeneous cell population of hepatic progenitor cells and then matured into functional hepatocytes. The progenitor population shared several characteristics with ES cells and hepatic stem/progenitor cells, and represented a novel progenitor cell between ES and hepatic oval cells in embryonic development. The differentiated hepatocytes from progenitor cells shared typical characteristics with mature hepatocytes, including the patterns of gene expression, immunological markers, in vitro hepatocyte functions and in vivo capacity to restore acute-damaged liver function. In addition, the differentiation of hepatic progenitor cells from ES cells was accompanied by significant cell cycle arrest and selective survival of differentiating cells towards hepatic lineages. CONCLUSION: Hepatic cells

  15. Natural Killer Cells Are Activated by Lactic Acid Bacteria-Matured Dendritic Cells

    DEFF Research Database (Denmark)

    Fink, Lisbeth Nielsen; Christensen, Hanne Risager; Frøkiær, Hanne

    of certain lactic acid bacteria has been shown to increase in vivo NK cytotoxicity. Here, we investigated how human gut flora-derived lactobacilli affect NK cells in vitro, by measuring proliferation and IFN-gamma production of human NK cells upon bacterial stimulation. Human peripheral blood NK cells were...... with lactic acid bacteria will affect NK cell activation. Such activation of NK cells may potentially skew an on-going or subsequent immune response towards a Th1 response....... incubated with 10 microg/ml UV-inactivated bacteria or 10 microg/ml phytohemagglutinin (PHA) for four days. Proliferation was assessed by incorporation of radioactive thymidine into NK cell DNA. The IFN-gamma concentration was measured by ELISA. Incubation of NK cells with a Lactobacillus acidophilus strain...

  16. Dietary restriction and fasting arrest B and T cell development and increase mature B and T cell numbers in bone marrow.

    Science.gov (United States)

    Shushimita, Shushimita; de Bruijn, Marjolein J W; de Bruin, Ron W F; IJzermans, Jan N M; Hendriks, Rudi W; Dor, Frank J M F

    2014-01-01

    Dietary restriction (DR) delays ageing and extends life span. Both long- and short-term DR, as well as short-term fasting provide robust protection against many "neuronal and surgery related damaging phenomena" such as Parkinson's disease and ischemia-reperfusion injury. The exact mechanism behind this phenomenon has not yet been elucidated. Its anti-inflammatory actions prompted us to thoroughly investigate the consequences of DR and fasting on B and T cell compartments in primary and secondary lymphoid organs of male C57Bl/6 mice. In BM we found that DR and fasting cause a decrease in the total B cell population and arrest early B cell development, while increasing the number of recirculating mature B cells. In the fasting group, a significant reduction in peripheral B cell counts was observed in both spleen and mesenteric lymph nodes (mLN). Thymopoiesis was arrested significantly at double negative DN2 stage due to fasting, whereas DR resulted in a partial arrest of thymocyte development at the DN4 stage. Mature CD3(+) T cell populations were increased in BM and decreased in both spleen and mLN. Thus, DR arrests B cell development in the BM but increases the number of recirculating mature B cells. DR also arrests maturation of T cells in thymus, resulting in depletion of mature T cells from spleen and mLN while recruiting them to the BM. The functional relevance in relation to protection against organ damage needs to be determined.

  17. Dietary restriction and fasting arrest B and T cell development and increase mature B and T cell numbers in bone marrow.

    Directory of Open Access Journals (Sweden)

    Shushimita Shushimita

    Full Text Available Dietary restriction (DR delays ageing and extends life span. Both long- and short-term DR, as well as short-term fasting provide robust protection against many "neuronal and surgery related damaging phenomena" such as Parkinson's disease and ischemia-reperfusion injury. The exact mechanism behind this phenomenon has not yet been elucidated. Its anti-inflammatory actions prompted us to thoroughly investigate the consequences of DR and fasting on B and T cell compartments in primary and secondary lymphoid organs of male C57Bl/6 mice. In BM we found that DR and fasting cause a decrease in the total B cell population and arrest early B cell development, while increasing the number of recirculating mature B cells. In the fasting group, a significant reduction in peripheral B cell counts was observed in both spleen and mesenteric lymph nodes (mLN. Thymopoiesis was arrested significantly at double negative DN2 stage due to fasting, whereas DR resulted in a partial arrest of thymocyte development at the DN4 stage. Mature CD3(+ T cell populations were increased in BM and decreased in both spleen and mLN. Thus, DR arrests B cell development in the BM but increases the number of recirculating mature B cells. DR also arrests maturation of T cells in thymus, resulting in depletion of mature T cells from spleen and mLN while recruiting them to the BM. The functional relevance in relation to protection against organ damage needs to be determined.

  18. Experimental investigation of changes in methane adsorption of bitumen-free Woodford Shale with thermal maturation induced by hydrous pyrolysis

    Science.gov (United States)

    Hu, Haiyan; Zhang, Tongwei; Wiggins-Camacho, Jaclyn D.; Ellis, Geoffrey S.; Lewan, Michael D.; Zhang, Xiayong

    2014-01-01

    This study quantifies the effects of organic-matter (OM) thermal maturity on methane (CH4) sorption, on the basis of five samples that were artificially matured through hydrous pyrolysis achieved by heating samples of immature Woodford Shale under five different time–temperature conditions. CH4-sorption isotherms at 35 °C, 50 °C, and 65 °C, and pressures up to 14 MPa on dry, solvent-extracted samples of the artificially matured Woodford Shale were measured. The results showed that CH4-sorption capacity, normalized to TOC, varied with thermal maturity, following the trend: maximum oil (367 °C) > oil cracking (400 °C) > maximum bitumen/early oil (333 °C) > early bitumen (300 °C) > immature stage (130 °C). The Langmuir constants for the samples at maximum-oil and oil-cracking stages are larger than the values for the bitumen-forming stages. The total pore volume, determined by N2 physisorption at 77 K, increases with increased maturation: mesopores, 2–50 nm in width, were created during the thermal conversion of organic-matter and a dramatic increase in porosity appeared when maximum-bitumen and maximum-oil generation stages were reached. A linear relationship between thermal maturity and Brunauer–Emmett–Teller (BET) surface area suggests that the observed increase in CH4-sorption capacity may be the result of mesopores produced during OM conversion. No obvious difference is observed in pore-size distribution and pore volume for samples with pores 2 physisorption at 273 K. The isosteric heat of adsorption and the standard entropy for artificially matured samples ranged from 17.9 kJ mol−1 to 21.9 kJ mol−1 and from −85.4 J mol−1 K−1 to −101.8 J mol−1 K−1, respectively. These values are similar to the values of immature Woodford kerogen concentrate previously observed, but are larger than naturally matured organic-rich shales. High-temperature hydrous pyrolysis might have induced Lewis acid sites on both organic and mineral surfaces

  19. T cells expressing an anti-B-cell maturation antigen chimeric antigen receptor cause remissions of multiple myeloma.

    Science.gov (United States)

    Ali, Syed Abbas; Shi, Victoria; Maric, Irina; Wang, Michael; Stroncek, David F; Rose, Jeremy J; Brudno, Jennifer N; Stetler-Stevenson, Maryalice; Feldman, Steven A; Hansen, Brenna G; Fellowes, Vicki S; Hakim, Frances T; Gress, Ronald E; Kochenderfer, James N

    2016-09-29

    Therapies with novel mechanisms of action are needed for multiple myeloma (MM). B-cell maturation antigen (BCMA) is expressed in most cases of MM. We conducted the first-in-humans clinical trial of chimeric antigen receptor (CAR) T cells targeting BCMA. T cells expressing the CAR used in this work (CAR-BCMA) specifically recognized BCMA-expressing cells. Twelve patients received CAR-BCMA T cells in this dose-escalation trial. Among the 6 patients treated on the lowest 2 dose levels, limited antimyeloma activity and mild toxicity occurred. On the third dose level, 1 patient obtained a very good partial remission. Two patients were treated on the fourth dose level of 9 × 10(6) CAR(+) T cells/kg body weight. Before treatment, the first patient on the fourth dose level had chemotherapy-resistant MM, making up 90% of bone marrow cells. After treatment, bone marrow plasma cells became undetectable by flow cytometry, and the patient's MM entered a stringent complete remission that lasted for 17 weeks before relapse. The second patient on the fourth dose level had chemotherapy-resistant MM making up 80% of bone marrow cells before treatment. Twenty-eight weeks after this patient received CAR-BCMA T cells, bone marrow plasma cells were undetectable by flow cytometry, and the serum monoclonal protein had decreased by >95%. This patient is in an ongoing very good partial remission. Both patients treated on the fourth dose level had toxicity consistent with cytokine-release syndrome including fever, hypotension, and dyspnea. Both patients had prolonged cytopenias. Our findings demonstrate antimyeloma activity of CAR-BCMA T cells. This trial was registered to www.clinicaltrials.gov as #NCT02215967.

  20. Accelerated Maturation of Human Stem Cell-Derived Pancreatic Progenitor Cells into Insulin-Secreting Cells in Immunodeficient Rats Relative to Mice

    Directory of Open Access Journals (Sweden)

    Jennifer E. Bruin

    2015-12-01

    Full Text Available Pluripotent human embryonic stem cells (hESCs are a potential source of transplantable cells for treating patients with diabetes. To investigate the impact of the host recipient on hESC-derived pancreatic progenitor cell maturation, cells were transplanted into immunodeficient SCID-beige mice or nude rats. Following the transplant, basal human C-peptide levels were consistently higher in mice compared with rats, but only rats showed robust meal- and glucose-responsive human C-peptide secretion by 19–21 weeks. Grafts from rats contained a higher proportion of insulin:glucagon immunoreactivity, fewer exocrine cells, and improved expression of mature β cell markers compared with mice. Moreover, ECM-related genes were enriched, the collagen network was denser, and blood vessels were more intricately integrated into the engrafted endocrine tissue in rats relative to mice. Overall, hESC-derived pancreatic progenitor cells matured faster in nude rats compared with SCID-beige mice, indicating that the host recipient can greatly influence the fate of immature pancreatic progenitor cells post-transplantation.

  1. In vitro large scale production of human mature red blood cells from hematopoietic stem cells by coculturing with human fetal liver stromal cells.

    Science.gov (United States)

    Xi, Jiafei; Li, Yanhua; Wang, Ruoyong; Wang, Yunfang; Nan, Xue; He, Lijuan; Zhang, Peng; Chen, Lin; Yue, Wen; Pei, Xuetao

    2013-01-01

    In vitro models of human erythropoiesis are useful in studying the mechanisms of erythroid differentiation in normal and pathological conditions. Here we describe an erythroid liquid culture system starting from cord blood derived hematopoietic stem cells (HSCs). HSCs were cultured for more than 50 days in erythroid differentiation conditions and resulted in a more than 10(9)-fold expansion within 50 days under optimal conditions. Homogeneous erythroid cells were characterized by cell morphology, flow cytometry, and hematopoietic colony assays. Furthermore, terminal erythroid maturation was improved by cosculturing with human fetal liver stromal cells. Cocultured erythroid cells underwent multiple maturation events, including decrease in size, increase in glycophorin A expression, and nuclear condensation. This process resulted in extrusion of the pycnotic nuclei in up to 80% of the cells. Importantly, they possessed the capacity to express the adult definitive β -globin chain upon further maturation. We also show that the oxygen equilibrium curves of the cord blood-differentiated red blood cells (RBCs) are comparable to normal RBCs. The large number and purity of erythroid cells and RBCs produced from cord blood make this method useful for fundamental research in erythroid development, and they also provide a basis for future production of available RBCs for transfusion.

  2. Prolactin/Jak2 directs apical/basal polarization and luminal linage maturation of mammary epithelial cells through regulation of the Erk1/2 pathway

    Directory of Open Access Journals (Sweden)

    Fengming Liu

    2015-09-01

    Full Text Available Tissue development/remodeling requires modulations in both cellular architecture and phenotype. Aberration in these processes leads to tumorigenesis. During the pregnancy/lactation cycle the mammary epithelial cells undergo complex morphological and phenotypic programs resulting in the acquisition of apical/basal (A/B polarization and cellular maturation necessary for proper lactation. Still the hormonal regulations and cellular mechanisms controlling these events are not entirely elucidated. Here we show that prolactin (PRL/Jak2 pathway in mammary epithelial cells uniquely signals to establish A/B polarity as determined by the apical localization of the tight junction protein zona occludens 1 (ZO-1 and the basal/lateral localization of E-cadherin, and the apical trafficking of lipid droplets. As well, our results indicate that this pathway regulates mammary stem cell hierarchy by inducing the differentiation of luminal progenitor (EpCAMhi/CD49fhi cells to mature luminal (EpCAMhi/CD49flow cells. Moreover, our data indicate that PRL/Jak2 coordinates both of these cellular events through limiting the mitogen activated protein kinase (Erk1/2 pathway. Together our findings define a novel unifying mechanism coupling mammary epithelial cell A/B polarization and terminal differentiation.

  3. Mesenchymal Stem Cells Inhibit Dendritic Cell Maturation and Their Allosti mulatory Capacity

    Institute of Scientific and Technical Information of China (English)

    Sophie; PACZESNY; Veronique; LATGER; CANNARD; Luc; MARCHAL; Bernard; FOLLIGUET; Jean-Franéois; STOLTZ; Assia; ELJAAFARI

    2005-01-01

    1 IntroductionDendritic cells (DC) are the most potent antigen-presenting cells. They play an important role in both initiation of immunity and maintenance of immune tolerance. In the recent years, they have been used in humans for the treatment of tumors. DCs are very poor in blood; however, they can be generated in vitro from either CD34~+ hematopoietic stem cell precursors or peripheral blood monocytes, by using appropriate cytokines~([1]). However, the microenvironment can influence their differentiatio...

  4. Acute steroid-induced myopathy during lung Maturation in a patient with threatened preterm Delivery

    OpenAIRE

    Rojas-Suarez José Antonio; Arrieta-López Elizabeth; Miranda-Quintero Jesid E; Fernández-Mercado Juan Carlos; De la Ossa-Mercado Olga

    2010-01-01

    Myopathy caused by steroids is a serious and unusual side effect related with administration of high doses of steroids. In this article we had reported a possible deleterious and serious effect on the mother health when steroids are used in the treatment of preterm childbirth. The case of a pregnant patient of 28 gestational week that consults to the emergency department with signs of preterm birth, receives tocolysis and lung maturation with Betametasona (12 mg the first day and 12 mg at 24 ...

  5. Inducible T-cell receptor expression in precursor T-cells for leukemia control

    Science.gov (United States)

    Hoseini, Shahabuddin S; Hapke, Martin; Herbst, Jessica; Wedekind, Dirk; Baumann, Rolf; Heinz, Niels; Schiedlmeier, Bernhard; Vignali, Dario AA; van den Brink, Marcel R.M.; Schambach, Axel; Blazar, Bruce R.; Sauer, Martin G.

    2015-01-01

    Co-transplantation of hematopoietic stem cells with those engineered to express leukemia-reactive T cell receptors (TCRs) and differentiated ex vivo into precursor T cells (preTs) may reduce the risk of leukemia relapse. Since expression of potentially self-(leukemia-) reactive TCRs will lead to negative selection or provoke autoimmunity upon thymic maturation, we investigated a novel concept whereby TCR expression set under the control of an inducible promoter would allow timely controlled TCR expression. After in vivo maturation and gene induction, preTs developed potent anti-leukemia effects. Engineered preTs provided protection even after repeated leukemia challenges by giving rise to effector and central memory cells. Importantly, adoptive transfer of TCR-transduced allogeneic preTs mediated anti-leukemia effect without evoking graft-versus-host disease (GVHD). Earlier transgene induction forced CD8+ T cell development, was required to obtain a mature T cell subset of targeted specificity, allowed engineered T cells to efficiently pass positive selection and abrogated the endogenous T cell repertoire. Later induction favored CD4 differentiation and failed to produce a leukemia-reactive population emphasizing the dominant role of positive selection. Taken together, we provide new functional insights for the employment of TCR-engineered precursor cells as a controllable immunotherapeutic modality with significant anti-leukemia activity. PMID:25652739

  6. Developmental propagation of V(D)J recombination-associated DNA breaks and translocations in mature B cells via dicentric chromosomes.

    Science.gov (United States)

    Hu, Jiazhi; Tepsuporn, Suprawee; Meyers, Robin M; Gostissa, Monica; Alt, Frederick W

    2014-07-15

    Mature IgM(+) B-cell lymphomas that arise in certain ataxia telangiectasia-mutated (ATM)-deficient compound mutant mice harbor translocations that fuse V(D)J recombination-initiated IgH double-strand breaks (DSBs) on chromosome 12 to sequences downstream of c-myc on chromosome 15, generating dicentric chromosomes and c-myc amplification via a breakage-fusion-bridge mechanism. As V(D)J recombination DSBs occur in developing progenitor B cells in the bone marrow, we sought to elucidate a mechanism by which such DSBs contribute to oncogenic translocations/amplifications in mature B cells. For this purpose, we applied high-throughput genome-wide translocation sequencing to study the fate of introduced c-myc DSBs in splenic IgM(+) B cells stimulated for activation-induced cytidine deaminase (AID)-dependent IgH class switch recombination (CSR). We found frequent translocations of c-myc DSBs to AID-initiated DSBs in IgH switch regions in wild-type and ATM-deficient B cells. However, c-myc also translocated frequently to newly generated DSBs within a 35-Mb region downstream of IgH in ATM-deficient, but not wild-type, CSR-activated B cells. Moreover, we found such DSBs and translocations in activated B cells that did not express AID or undergo CSR. Our findings indicate that ATM deficiency leads to formation of chromosome 12 dicentrics via recombination-activating gene-initiated IgH DSBs in progenitor B cells and that these dicentrics can be propagated developmentally into mature B cells where they generate new DSBs downstream of IgH via breakage-fusion-bridge cycles. We propose that dicentrics formed by joining V(D)J recombination-associated IgH DSBs to DSBs downstream of c-myc in ATM-deficient B lineage cells similarly contribute to c-myc amplification and mature B-cell lymphomas.

  7. Effect of Somatic Cell Types and Culture Medium on in vitro Maturation, Fertilization and Early Development Capability of Buffalo Oocytes

    Directory of Open Access Journals (Sweden)

    H. Jamil*, H. A. Samad, N. Rehman, Z. I. Qureshi and L. A. Lodhi

    2011-04-01

    Full Text Available This study was designed to evaluate the efficacy of different somatic cell types and media in supporting in vitro maturation (IVM, in vitro fertilization (IVF and early embryonic development competence of buffalo follicular oocytes. Cumulus oocyte complexes were collected for maturation from follicles (>6mm of buffalo ovaries collected at the local abattoir. Oocytes were co-cultured in tissue culture medium (TCM-199 with either granulosa cells, cumulus cells, or buffalo oviductal epithelial cells (BOEC @ 3x106 cells/ml or in TCM-199 without helper cells (control at 39°C and 5%CO2 in humidified air. Fresh semen was prepared in modified Ca++ free Tyrode medium. Fertilization was carried out in four types of media: i Tyrode lactate albumin pyruvate (TALP, ii TALP+BOEC, iii modified Ca++ free Tyrode and iv modified Ca++ free Tyrode+BOEC. Fertilized oocytes were cultured for early embryonic development in TCM-199 with and without BOEC. Higher maturation rates were observed in the granulosa (84.24% and cumulus cells (83.44% than BOEC co culture system (73.37%. Highest fertilization rate was obtained in modified Ca++ free Tyrode with BOEC co culture (70.42%, followed by modified Ca++ free Tyrode alone (63.77%, TALP with BOEC (36.92% and TALP alone (10.94%. Development of early embryos (8-cell stage improved in TCM-199 with BOEC co culture than TCM-199 alone. From the results of this study, it can be concluded that addition of somatic cells (granulosa cells, cumulus cells results in higher maturation rates of buffalo follicular oocytes than BOEC co culture system, while fertilization rate improved in modified Ca++ free Tyrode with and without BOEC. Addition of BOEC to TCM-199 improved the developmental capacity of early embryo.

  8. Selective ablation of the androgen receptor in mouse sertoli cells affects sertoli cell maturation, barrier formation and cytoskeletal development.

    Directory of Open Access Journals (Sweden)

    Ariane Willems

    Full Text Available The observation that mice with a selective ablation of the androgen receptor (AR in Sertoli cells (SC (SCARKO mice display a complete block in meiosis supports the contention that SC play a pivotal role in the control of germ cell development by androgens. To delineate the physiological and molecular mechanism responsible for this control, we compared tubular development in pubertal SCARKO mice and littermate controls. Particular attention was paid to differences in SC maturation, SC barrier formation and cytoskeletal organization and to the molecular mediators potentially involved. Functional analysis of SC barrier development by hypertonic perfusion and lanthanum permeation techniques and immunohistochemical analysis of junction formation showed that SCARKO mice still attempt to produce a barrier separating basal and adluminal compartment but that barrier formation is delayed and defective. Defective barrier formation was accompanied by disturbances in SC nuclear maturation (immature shape, absence of prominent, tripartite nucleoli and SC polarization (aberrant positioning of SC nuclei and cytoskeletal elements such as vimentin. Quantitative RT-PCR was used to study the transcript levels of genes potentially related to the described phenomena between day 8 and 35. Differences in the expression of SC genes known to play a role in junction formation could be shown from day 8 for Cldn11, from day 15 for Cldn3 and Espn, from day 20 for Cdh2 and Jam3 and from day 35 for ZO-1. Marked differences were also noted in the transcript levels of several genes that are also related to cell adhesion and cytoskeletal dynamics but that have not yet been studied in SC (Actn3, Ank3, Anxa9, Scin, Emb, Mpzl2. It is concluded that absence of a functional AR in SC impedes the remodeling of testicular tubules expected at the onset of spermatogenesis and interferes with the creation of the specific environment needed for germ cell development.

  9. AID activity in B cells strongly correlates with polyclonal antibody affinity maturation in-vivo following pandemic 2009-H1N1 vaccination in humans.

    Science.gov (United States)

    Khurana, Surender; Frasca, Daniela; Blomberg, Bonnie; Golding, Hana

    2012-09-01

    The role of Activation-Induced Cytidine Deaminase (AID) in somatic hypermutation and polyclonal antibody affinity maturation has not been shown for polyclonal responses in humans. We investigated whether AID induction in human B cells following H1N1pdm09 vaccination correlated with in-vivo antibody affinity maturation against hemagglutinin domains in plasma of young and elderly individuals. AID was measured by qPCR in B cells from individuals of different ages immunized with the H1N1pdm09 influenza vaccine. Polyclonal antibody affinity in human plasma for the HA1 and HA2 domains of the H1N1pdm09 hemagglutinin was measured by antibody-antigen complex dissociation rates using real time kinetics in Surface Plasmon Resonance. Results show an age-related decrease in AID induction in B cells following H1N1pdm09 vaccination. Levels of AID mRNA before vaccination and fold-increase of AID mRNA expression after H1N1pdm09 vaccination directly correlated with increase in polyclonal antibody affinity to the HA1 globular domain (but not to the conserved HA2 stalk). In the younger population, significant affinity maturation to the HA1 globular domain was observed, which associated with initial levels of AID and fold-increase in AID after vaccination. In some older individuals (>65 yr), higher affinity to the HA1 domain was observed before vaccination and H1N1pdm09 vaccination resulted in minimal change in antibody affinity, which correlated with low AID induction in this age group. These findings demonstrate for the first time a strong correlation between AID induction and in-vivo antibody affinity maturation in humans. The ability to generate high affinity antibodies could have significant impact on the elucidation of age-specific antibody responses following vaccination and eventual clinical efficacy and disease outcome.

  10. DNA methylation imprinting errors in spermatogenic cells from maturation arrest azoospermic patients.

    Science.gov (United States)

    Marques, P I; Fernandes, S; Carvalho, F; Barros, A; Sousa, M; Marques, C J

    2017-03-10

    Imprinting errors have been described in spermatozoa from infertile patients with oligozoospermia and azoospermia. However, little is known about methylation of imprinted genes in other spermatogenic cells from azoospermic patients. Therefore, we aimed to evaluate the methylation status of single CpGs located in the differentially methylated regions (DMRs) of two imprinted genes, one paternally (H19) and one maternally (MEST) methylated, in primary spermatocytes of azoospermic patients presenting complete (MAc, n = 7) and incomplete (MAi, n = 8) maturation arrest, as well as in other spermatogenic cells from MAi patients that presented focus of complete spermatogenesis in some seminiferous tubules. We observed H19 imprinting errors in primary spermatocytes from one MAi patient and MEST imprinting errors in one MAi and two MAc patients. Additionally, H19 imprinting errors were observed in elongated spermatids/spermatozoa from one MAi patient. Nevertheless, no statistical differences were found for H19 and MEST global methylation levels (percentage of methylated and unmethylated CpGs, respectively) between patients with complete and incomplete MA and also between MA groups and a control group. These results provide further evidence that imprinting errors occur in spermatogenic cells from patients presenting impaired spermatogenesis, as we and others have previously described in ejaculated and testicular spermatozoa. As paternal imprinting errors can be transmitted to the embryo by the sperm cell, they can provide a possible explanation for poor embryo development and/or low pregnancy rates as correct expression of imprinted genes is crucial for embryo and placental development and function. Therefore, in cases with male factor infertility where unsuccessful in vitro fertilization (IVF) treatments are recurrent, analysis of imprinting marks in spermatozoa might be a useful diagnostic tool.

  11. Cellular maturation defects in Bruton's tyrosine kinase-deficient immature B cells are amplified by premature B cell receptor expression and reduced by receptor editing

    NARCIS (Netherlands)

    S. Middendorp; R.W. Hendriks (Rudi)

    2004-01-01

    textabstractIn the mouse, Bruton's tyrosine kinase (Btk) is essential for efficient developmental progression of CD43(+)CD2(-) large cycling into CD43(-)CD2(+) small resting pre-B cells in the bone marrow and of IgM(high) transitional type 2 B cells into IgM(low) mature B cells in

  12. CD69 modulates sphingosine-1-phosphate-induced migration of skin dendritic cells

    OpenAIRE

    Lamana, Amalia; Martin, Pilar; de la Fuente, Hortensia; Martinez-Muñoz, Laura; Cruz-Adalia, Aranzazu; Ramirez-Huesca, Marta; Escribano, Cristina; Gollmer, Kathrin; Mellado, Mario; Stein, Jens V.; Rodriguez-Fernandez, Jose Luis; Sanchez-Madrid, Francisco; del Hoyo, Gloria Martinez

    2011-01-01

    In this study, we have investigated the role of CD69, an early inducible leukocyte activation receptor, in murine dendritic cell (DC) differentiation, maturation, and migration. Skin DCs and DC subsets present in mouse lymphoid organs express CD69 in response to maturation stimuli. Using a contact sensitization model, we show that skin DCs migrated more efficiently to draining lymph nodes (LNs) in the absence of CD69. This was confirmed by subcutaneous transfer of CD69–/– DCs, which presented...

  13. Human fetal striatum-derived neural stem (NS) cells differentiate to mature neurons in vitro and in vivo.

    Science.gov (United States)

    Monni, Emanuela; Cusulin, Carlo; Cavallaro, Maurizio; Lindvall, Olle; Kokaia, Zaal

    2014-01-01

    Clonogenic neural stem (NS) cell lines grown in adherent cultures have previously been established from embryonic stem cells and fetal and adult CNS in rodents and from human fetal brain and spinal cord. Here we describe the isolation of a new cell line from human fetal striatum (hNS cells). These cells showed properties of NS cells in vitro such as monolayer growth, high proliferation rate and expression of radial glia markers. The hNS cells expressed an early neuronal marker while being in the proliferative state. Under appropriate conditions, the hNS cells were efficiently differentiated to neurons, and after 4 weeks about 50% of the cells were βIII tubulin positive. They also expressed the mature neuronal marker NeuN and markers of neuronal subtypes, GABA, calbindin, and DARPP32. After intrastriatal implantation into newborn rats, the hNS cells survived and many of them migrated outside the transplant core into the surrounding tissue. A high percentage of cells in the grafts expressed the neuroblast marker DCX, indicating their neurogenic potential, and some of the cells differentiated to NeuN+ mature neurons. The human fetal striatum-derived NS cell line described here should be a useful tool for studies on cell replacement strategies in models of the striatal neuronal loss occurring in Huntington's disease and stroke.

  14. Expression of mixed lineage kinase-1 in pancreatic beta-cell lines at different stages of maturation and during embryonic pancreas development.

    Science.gov (United States)

    DeAizpurua, H J; Cram, D S; Naselli, G; Devereux, L; Dorow, D S

    1997-06-27

    Events controlling differentiation to insulin-secreting beta-cells in the pancreas are not well understood, although beta-cells are thought to arise from pluripotent ductal precursor cells. To search for signaling proteins that might be involved in beta-cell maturation, we analyzed protein kinase expression in two developmentally and functionally distinct pancreatic beta-cell lines, RIN-5AH and RIN-A12, by reverse transcriptase polymerase chain reaction. A number of tyrosine and serine/threonine kinases were identified in both lines. One protein kinase, mixed lineage kinase-1 (MLK-1), was expressed at both the RNA and protein levels in RIN-5AH cells, which display an immature beta-cell phenotype, but was not detected in the more mature RIN-A12 cells. Furthermore, levels of MLK-1 mRNA and protein were increased after brief stimulation of RIN-5AH cells with either the differentiation inducer, sodium butyrate, or with serum after serum starvation. These increases in expression were independent of phenotypic markers such as insulin secretion or surface expression of major histocompatibility class I- and A2B5-reactive ganglioside. In addition, increases in MLK-1 expression in the stimulated RIN-5AH cells were accompanied by phosphorylation of MLK-1 on serine but not tyrosine. Antisense oligonucleotides to two distinct regions of MLK-1 caused RIN-5AH cells, but not RIN-A12 cells, to adopt a highly undifferentiated morphology, with a reduction in DNA synthesis and MLK-1 protein levels and elevated glucagon mRNA levels, but with no effect on insulin mRNA. In an immunohistochemical survey of embryonic mouse tissues, we found that temporal expression of MLK-1 was regulated in a tissue-specific manner. In the embryonic pancreas, MLK-1 expression was evident in ductal cells from day 13 to 16 but was not detected in late stage gestation or neonatal pancreas. These data suggest that MLK-1 is regulated in immature pancreatic beta-cells and their ductal precursors at the level of

  15. Mycobacterium tuberculosis infection induces non-apoptotic cell death of human dendritic cells

    LENUS (Irish Health Repository)

    Ryan, Ruth CM

    2011-10-24

    Abstract Background Dendritic cells (DCs) connect innate and adaptive immunity, and are necessary for an efficient CD4+ and CD8+ T cell response after infection with Mycobacterium tuberculosis (Mtb). We previously described the macrophage cell death response to Mtb infection. To investigate the effect of Mtb infection on human DC viability, we infected these phagocytes with different strains of Mtb and assessed viability, as well as DNA fragmentation and caspase activity. In parallel studies, we assessed the impact of infection on DC maturation, cytokine production and bacillary survival. Results Infection of DCs with live Mtb (H37Ra or H37Rv) led to cell death. This cell death proceeded in a caspase-independent manner, and without nuclear fragmentation. In fact, substrate assays demonstrated that Mtb H37Ra-induced cell death progressed without the activation of the executioner caspases, 3\\/7. Although the death pathway was triggered after infection, the DCs successfully underwent maturation and produced a host-protective cytokine profile. Finally, dying infected DCs were permissive for Mtb H37Ra growth. Conclusions Human DCs undergo cell death after infection with live Mtb, in a manner that does not involve executioner caspases, and results in no mycobactericidal effect. Nonetheless, the DC maturation and cytokine profile observed suggests that the infected cells can still contribute to TB immunity.

  16. In Vitro Differentiation and Maturation of Human Embryonic Stem Cell into Multipotent Cells

    Directory of Open Access Journals (Sweden)

    Amer Mahmood

    2011-01-01

    Full Text Available Human embryonic stem cells (hESCs, which have the potential to generate virtually any differentiated progeny, are an attractive cell source for transplantation therapy, regenerative medicine, and tissue engineering. To realize this potential, it is essential to be able to control ESC differentiation and to direct the development of these cells along specific pathways. Basic science in the field of embryonic development, stem cell differentiation, and tissue engineering has offered important insights into key pathways and scaffolds that regulate hESC differentiation, which have produced advances in modeling gastrulation in culture and in the efficient induction of endoderm, mesoderm, ectoderm, and many of their downstream derivatives. These findings have lead to identification of several pathways controlling the differentiation of hESCs into mesodermal derivatives such as myoblasts, mesenchymal cells, osteoblasts, chondrocytes, adipocytes, as well as hemangioblastic derivatives. The next challenge will be to demonstrate the functional utility of these cells, both in vitro and in preclinical models of bone and vascular diseases.

  17. Generation of functional podocytes from human induced pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Osele Ciampi

    2016-07-01

    Full Text Available Generating human podocytes in vitro could offer a unique opportunity to study human diseases. Here, we describe a simple and efficient protocol for obtaining functional podocytes in vitro from human induced pluripotent stem cells. Cells were exposed to a three-step protocol, which induced their differentiation into intermediate mesoderm, then into nephron progenitors and, finally, into mature podocytes. After differentiation, cells expressed the main podocyte markers, such as synaptopodin, WT1, α-Actinin-4, P-cadherin and nephrin at the protein and mRNA level, and showed the low proliferation rate typical of mature podocytes. Exposure to Angiotensin II significantly decreased the expression of podocyte genes and cells underwent cytoskeleton rearrangement. Cells were able to internalize albumin and self-assembled into chimeric 3D structures in combination with dissociated embryonic mouse kidney cells. Overall, these findings demonstrate the establishment of a robust protocol that, mimicking developmental stages, makes it possible to derive functional podocytes in vitro.

  18. cFos Mediates cAMP-Dependent Generation of ROS and Rescue of Maturation Program in Retinoid-Resistant Acute Promyelocytic Leukemia Cell Line NB4-LR1

    Science.gov (United States)

    Carrier, Jean-Luc; Javadi, Pasha; Bourrier, Emilie; Camus, Céline; Ségal-Bendirdjian, Evelyne; Karniguian, Aïda

    2012-01-01

    A determining role has been assigned to cAMP in the signaling pathways that relieve resistance to anti-leukemia differentiation therapy. However, the underlying mechanisms have not been elucidated yet. Here, we identify cFos as a critical cAMP effector, able to regulate the re-expression and splicing of epigenetically silenced genes associated with maturation (CD44) in retinoid-resistant NB4-LR1 leukemia cells. Furthermore, using RNA interference approach, we show that cFos mediates cAMP-induced ROS generation, a critical mediator of neutrophil maturation, and in fine differentiation. This study highlights some of the mechanisms by which cAMP acts to overcome resistance, and reveals a new alternative cFos-dependent pathway which, though nonexistent in retinoid-sensitive NB4 cells, is essential to rescue the maturation program of resistant cells. PMID:23209736

  19. cFos mediates cAMP-dependent generation of ROS and rescue of maturation program in retinoid-resistant acute promyelocytic leukemia cell line NB4-LR1.

    Directory of Open Access Journals (Sweden)

    Jean-Luc Carrier

    Full Text Available A determining role has been assigned to cAMP in the signaling pathways that relieve resistance to anti-leukemia differentiation therapy. However, the underlying mechanisms have not been elucidated yet. Here, we identify cFos as a critical cAMP effector, able to regulate the re-expression and splicing of epigenetically silenced genes associated with maturation (CD44 in retinoid-resistant NB4-LR1 leukemia cells. Furthermore, using RNA interference approach, we show that cFos mediates cAMP-induced ROS generation, a critical mediator of neutrophil maturation, and in fine differentiation. This study highlights some of the mechanisms by which cAMP acts to overcome resistance, and reveals a new alternative cFos-dependent pathway which, though nonexistent in retinoid-sensitive NB4 cells, is essential to rescue the maturation program of resistant cells.

  20. During seminiferous tubule maturation testosterone and synergistic action of FSH with estradiol support germ cell survival while estradiol alone has pro-apoptotic effect.

    Directory of Open Access Journals (Sweden)

    Katarzyna Marchlewsk

    2008-04-01

    Testosterone eliminates estradiol--induced germ cell apoptosis when both hormones act in concert. 4 FSH in concert with estradiol, but neither one of the hormone alone, accelerate Sertoli cell differentiation and effectively inhibit germ cell apoptosis. 5 During seminiferous tubule maturation testosterone and the synergistic action of FSH with estradiol support germ cell survival while estradiol alone has an inhibitory, pro-apoptotic effect.

  1. Concepts in mature T-cell lymphomas - highlights from an international joint symposium on T-cell immunology and oncology().

    Science.gov (United States)

    Herling, Marco; Rengstl, Benjamin; Scholtysik, René; Hartmann, Sylvia; Küppers, Ralf; Hansmann, Martin-Leo; Diebner, Hans H; Roeder, Ingo; Abken, Hinrich; Newrzela, Sebastian; Kirberg, Jörg

    2017-04-01

    Growing attention in mature T-cell lymphomas/leukemias (MTCL) is committed to more accurate and meaningful classifications, improved pathogenetic concepts and expanded therapeutic options. This requires considerations of the immunologic concepts of T-cell homeostasis and the specifics of T-cell receptor (TCR) affinities and signaling. Scientists from various disciplines established the CONTROL-T research unit and in an international conference on MTCL they brought together experts from T-cell immunity, oncology, immunotherapy and systems biology. We report here meeting highlights on the covered topics of diagnostic pitfalls, implications by the new WHO classification, insights from discovered genomic lesions as well as TCR-centric concepts of cellular dynamics in host defense, auto-immunity and tumorigenic clonal escape, including predictions to be derived from in vivo imaging and mathematical modeling. Presentations on novel treatment approaches were supplemented by strategies of optimizing T-cell immunotherapies. Work packages, that in joint efforts would advance the field of MTCL more efficiently, are identified.

  2. Mouse cumulus-denuded oocytes restore developmental capacity completely when matured with optimal supplementation of cysteamine, cystine, and cumulus cells.

    Science.gov (United States)

    Zhou, Ping; Wu, Yan-Guang; Wei, De-Li; Li, Qing; Wang, Gang; Zhang, Jie; Luo, Ming-Jiu; Tan, Jing-He

    2010-04-01

    Our objectives were to study how cysteamine, cystine, and cumulus cells (CCs), as well as oocytes interact to increase oocyte intracellular glutathione (GSH) and thereby to establish an efficient in vitro maturation system for cumulus-denuded oocytes (DOs). Using M16 that contained no thiol as maturation medium, we showed that when supplemented alone, neither cystine nor cysteamine promoted GSH synthesis of mouse DOs, but they did when used together. Although goat CCs required either cysteamine or cystine to promote GSH synthesis, mouse CCs required both. In the presence of cystine, goat CCs produced cysteine but mouse CCs did not. Cysteamine reduced cystine to cysteine in cell-free M16. When TCM-199 that contained 83 microM cystine was used as maturation medium, supplementation with cysteamine alone had no effect, but supplementation with 100 microM cysteamine and 200 microM cystine increased blastulation of DOs matured with CC coculture to a level as high as achieved in cumulus-surrounded oocytes (COCs). Similar numbers of young were produced after two-cell embryos from mouse COCs or CC-cocultured DOs matured with optimal thiol supplementation were transferred to pseudopregnant recipients. It is concluded that 1) mouse CCs can use neither cysteamine nor cystine to promote GSH synthesis, but goat CCs can use either one; 2) goat CCs promote mouse oocyte GSH synthesis by reducing cystine to cysteine, but how they use cysteamine requires further investigation; and 3) mouse DOs can use neither cystine nor cysteamine for GSH synthesis, but they restore developmental capacity completely when matured in the presence of optimum supplementation of cysteamine, cystine, and CCs.

  3. Graphene derivative scaffolds facilitate in vitro cell survival and maturation of dopaminergic SN4741 cells

    OpenAIRE

    Rodriguez-Losada, Noela; Wendelbo, Rune; Garcia-fernandez, Maria; Pavia, Jose; Martinez-Montañez, Elisa; Lara Muñoz, José Pablo; Arenas, Ernest; Aguirre, José A.

    2014-01-01

    The emerging carbon nanomaterial Graphene (G), in the form of scaffold structure, has an efficient bioconjugation with common biomolecules and activates cell differentiation of neuronal stem cells, providing a promising approach for neural regeneration. We propose the use of G as a scaffold to re-address the dopaminergic (DA) neurons and the residual axons from dead or apoptotic DA neurons in Parkinson´s disease (PD). G could act as a physical support to promote the axonal sprout as a “decele...

  4. [In vitro maturation of the oocytes of the common frog with and without follicular envelopes as affected by cholesterol].

    Science.gov (United States)

    Skoblina, M N; Kondrat'eva, O T

    1983-01-01

    Cholesterol was shown to induce in vitro maturation of the Rana temporaria oocytes both in the presence and in the absence of the follicle cells. The maturation of the denuded oocytes required their much shorter treatment with cholesterol. Ethidium bromide inhibited the cholesterol-induced maturation of the oocytes with follicle envelopes and either did not affect the oocyte maturation after the follicle envelope removal or stimulated it. Amino-gluthetimide inhibited the cholesterol-induced oocyte maturation irrespective of the presence of follicle cells. The mechanism inducing effect of cholesterol on follicle and oocyte is discussed.

  5. A Pathway Switch Directs BAFF Signaling to Distinct NFκB Transcription Factors in Maturing and Proliferating B Cells

    Directory of Open Access Journals (Sweden)

    Jonathan V. Almaden

    2014-12-01

    Full Text Available BAFF, an activator of the noncanonical NFκB pathway, provides critical survival signals during B cell maturation and contributes to B cell proliferation. We found that the NFκB family member RelB is required ex vivo for B cell maturation, but cRel is required for proliferation. Combined molecular network modeling and experimentation revealed Nfkb2 p100 as a pathway switch; at moderate p100 synthesis rates in maturing B cells, BAFF fully utilizes p100 to generate the RelB:p52 dimer, whereas at high synthesis rates, p100 assembles into multimeric IκBsome complexes, which BAFF neutralizes in order to potentiate cRel activity and B cell expansion. Indeed, moderation of p100 expression or disruption of IκBsome assembly circumvented the BAFF requirement for full B cell expansion. Our studies emphasize the importance of p100 in determining distinct NFκB network states during B cell biology, which causes BAFF to have context-dependent functional consequences.

  6. Role of dystroglycan in limiting contraction-induced injury to the sarcomeric cytoskeleton of mature skeletal muscle.

    Science.gov (United States)

    Rader, Erik P; Turk, Rolf; Willer, Tobias; Beltrán, Daniel; Inamori, Kei-Ichiro; Peterson, Taylor A; Engle, Jeffrey; Prouty, Sally; Matsumura, Kiichiro; Saito, Fumiaki; Anderson, Mary E; Campbell, Kevin P

    2016-09-27

    Dystroglycan (DG) is a highly expressed extracellular matrix receptor that is linked to the cytoskeleton in skeletal muscle. DG is critical for the function of skeletal muscle, and muscle with primary defects in the expression and/or function of DG throughout development has many pathological features and a severe muscular dystrophy phenotype. In addition, reduction in DG at the sarcolemma is a common feature in muscle biopsies from patients with various types of muscular dystrophy. However, the consequence of disrupting DG in mature muscle is not known. Here, we investigated muscles of transgenic mice several months after genetic knockdown of DG at maturity. In our study, an increase in susceptibility to contraction-induced injury was the first pathological feature observed after the levels of DG at the sarcolemma were reduced. The contraction-induced injury was not accompanied by increased necrosis, excitation-contraction uncoupling, or fragility of the sarcolemma. Rather, disruption of the sarcomeric cytoskeleton was evident as reduced passive tension and decreased titin immunostaining. These results reveal a role for DG in maintaining the stability of the sarcomeric cytoskeleton during contraction and provide mechanistic insight into the cause of the reduction in strength that occurs in muscular dystrophy after lengthening contractions.

  7. Conditional induction of Math1 specifies embryonic stem cells to cerebellar granule neuron lineage and promotes differentiation into mature granule neurons.

    Science.gov (United States)

    Srivastava, Rupali; Kumar, Manoj; Peineau, Stéphane; Csaba, Zsolt; Mani, Shyamala; Gressens, Pierre; El Ghouzzi, Vincent

    2013-04-01

    Directing differentiation of embryonic stem cells (ESCs) to specific neuronal subtype is critical for modeling disease pathology in vitro. An attractive means of action would be to combine regulatory differentiation factors and extrinsic inductive signals added to the culture medium. In this study, we have generated mature cerebellar granule neurons by combining a temporally controlled transient expression of Math1, a master gene in granule neuron differentiation, with inductive extrinsic factors involved in cerebellar development. Using a Tetracyclin-On transactivation system, we overexpressed Math1 at various stages of ESCs differentiation and found that the yield of progenitors was considerably increased when Math1 was induced during embryonic body stage. Math1 triggered expression of Mbh1 and Mbh2, two target genes directly involved in granule neuron precursor formation and strong expression of early cerebellar territory markers En1 and NeuroD1. Three weeks after induction, we observed a decrease in the number of glial cells and an increase in that of neurons albeit still immature. Combining Math1 induction with extrinsic factors specifically increased the number of neurons that expressed Pde1c, Zic1, and GABAα6R characteristic of mature granule neurons, formed "T-shaped" axons typical of granule neurons, and generated synaptic contacts and action potentials in vitro. Finally, in vivo implantation of Math1-induced progenitors into young adult mice resulted in cell migration and settling of newly generated neurons in the cerebellum. These results show that conditional induction of Math1 drives ESCs toward the cerebellar fate and indicate that acting on both intrinsic and extrinsic factors is a powerful means to modulate ESCs differentiation and maturation into a specific neuronal lineage.

  8. Maturation of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in 3D collagen matrix: Effects of niche cell supplementation and mechanical stimulation.

    Science.gov (United States)

    Zhang, W; Kong, C W; Tong, M H; Chooi, W H; Huang, N; Li, R A; Chan, B P

    2017-02-01

    Cardiomyocytes derived from human embryonic stem cells (hESC-CMs) are regarded as a promising source for regenerative medicine, drug testing and disease modeling. Nevertheless, cardiomyocytes are immature in terms of their contractile structure, metabolism and electrophysiological properties. Here, we fabricate cardiac muscle strips by encapsulating hESC-CMs in collagen-based biomaterials. Supplementation of niche cells at 3% to the number of hESC-CMs enhance the maturation of the hESC-CMs in 3D tissue matrix. The benefits of adding mesenchymal stem cells (MSCs) are comparable to that of adding fibroblasts. These two cell types demonstrate similar effects in promoting the compaction and cell spreading, as well as expression of maturation markers at both gene and protein levels. Mechanical loading, particularly cyclic stretch, produces engineered cardiac tissues with higher maturity in terms of twitch force, elastic modulus, sarcomere length and molecular signature, when comparing to static stretch or non-stretched controls. The current study demonstrates that the application of niche cells and mechanical stretch both stimulate the maturation of hESC-CMs in 3D architecture. Our results therefore suggest that this 3D model can be used for in vitro cardiac maturation study.

  9. Induced pluripotent stem cell - derived neurons for the study of spinocerebellar ataxia type 3

    DEFF Research Database (Denmark)

    Hansen, Susanne K; Stummann, Tina C; Borland, Helena;

    2016-01-01

    The neurodegenerative disease spinocerebellar ataxia type 3 (SCA3) is caused by a CAG-repeat expansion in the ATXN3 gene. In this study, induced pluripotent stem cell (iPSC) lines were established from two SCA3 patients. Dermal fibroblasts were reprogrammed using an integration-free method...... displaying synchronized spontaneous calcium oscillations within 28days of maturation, and expressed the mature neuronal markers NeuN and Synapsin 1 implying a relatively advanced state of maturity, although not comparable to that of the adult human brain. Interestingly, we were not able to recapitulate...

  10. CD103 or LFA-1 engagement at the immune synapse between cytotoxic T cells and tumor cells promotes maturation and regulates T-cell effector functions.

    Science.gov (United States)

    Franciszkiewicz, Katarzyna; Le Floc'h, Audrey; Boutet, Marie; Vergnon, Isabelle; Schmitt, Alain; Mami-Chouaib, Fathia

    2013-01-15

    T-cell adhesion/costimulatory molecules and their cognate receptors on target cells play a major role in T-cell receptor (TCR)-mediated activities. Here, we compared the involvement of CD103 and LFA-1, and their respective ligands, in the maturation of the cytotoxic immune synapse (cIS) and in the activation of CTL effector functions. Our results indicate that cytotoxicity toward cancer cells and, to a lesser extent, cytokine production by specific CTL require, together with TCR engagement, the interaction of either CD103 with E-cadherin or LFA-1 with ICAM-1. Flow-based adhesion assay showed that engagement of CD103 or LFA-1, together with TCR, enhances the strength of the T-cell/target cell interaction. Moreover, electron microscopic analyses showed that integrin-dependent mature cIS (mcIS) displays a cohesive ultrastructure, with tight membrane contacts separated by extensive clefts. In contrast, immature cIS (icIS), which is unable to trigger target cell lysis, is loose, with multiple protrusions in the effector cell membrane. Experiments using confocal microscopy revealed polarized cytokine release and degranulation at the mcIS associated with target cell killing, whereas icIS is characterized by failure of IFN-γ and granzyme B relocalization. Thus, interactive forces between CTL and epithelial tumor cells, mainly regulated by integrin engagement, correlate with maturity and the ultrastructure of the cIS and influence CTL effector functions. These results provide new insights into molecular mechanisms regulating antitumor CTL responses and may lead to the development of more efficient cancer immunotherapy strategies.

  11. Genotyping of Tunisian azoospermic men with Sertoli cell-only and maturation arrest.

    Science.gov (United States)

    Hadjkacem-Loukil, Lobna; Hadj-Kacem, Hassen; Hadj Salem, Ikhlass; Bahloul, Ali; Fakhfakh, Faiza; Ayadi, Hammadi

    2011-07-06

    Azoospermia factor (AZF) deletions were associated with severe oligospermia and azoospermia with testicular histologies varying from maturation arrest (MA) to Sertoli cell-only (SCO) phenotypes. Abnormal androgen receptor (AR) structure or function has also been implicated in male infertility. To assess the contribution of these genetic defects to azoospermic patients, 19 Tunisian men with SCO (n = 13) or MA (n = 6) were enrolled in this study. Using immunohistochemistry method, we evaluated the expression of AR in testicular biopsy for the two phenotypes. PCR with primers flanking the AR-(CAG)n region and direct sequencing were used to determine AR-(CAG)n length. And PCR amplification of 14 sequence-tagged sites (STSs) located at Yq was used to determine the rate and extent of Y microdeletions. We found a significant difference of the AR expression between SCO and MA cases. Hence, this expression in the testis depends on the status of spermatogenesis. However, we did not find any relationship between the (CAG) repeat and the testicular histology (neither for SCO nor MA). On the other hand, we found a high frequency of AZF deletions (46.2%) in SCOS and in MA (50%). The present results also suggest the contribution of Y chromosome microdeletions in SCO and MA pathogenesis.

  12. Aflatoxins of type B and G affect porcine dendritic cell maturation in vitro.

    Science.gov (United States)

    Mehrzad, Jalil; Devriendt, Bert; Baert, Kim; Cox, Eric

    2015-01-01

    The toxic effects of highly carcinogenic mycotoxins, especially aflatoxins (AF), on key antigen-presenting cells, such as dendritic cells (DC), are largely unknown. To elucidate the effect of AF on DC function, porcine monocyte-derived DC (MoDC) were treated with a mixture of several AF (i.e., AFB1, AFB2, AFG1, and AFG2) and the phagocytic capacity, the membrane expression level of several DC activation markers, the T-cell proliferation-inducing capacity, and the cytokine secretion pattern were assessed. As compared to untreated MoDC, AF significantly up-regulated the expression of the co-stimulatory molecules CD25 and CD80/86. However, the phagocytic activity of MoDC was not affected by AF treatment. While the cytokine secretion pattern of AF-treated MoDC was similar to control MoDC, the T-cell proliferation-inducing capacity of MoDC was increased upon aflatoxin treatment. The results indicate that a mixture of naturally occurring AF enhances the antigen-presenting capacity of DC, which could explain the observed immunotoxicity of AF by breaking down tolerance and further emphasizes the need to reduce the admissible level of AF in agricultural commodities.

  13. Probiotic metabolites from Bacillus coagulans GanedenBC30TM support maturation of antigen-presenting cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Kathleen F Benson; Kimberlee A Redman; Steve G Carter; David Keller; Sean Farmer; John R Endres; Gitte S Jensen

    2012-01-01

    AIM:To study the effects of probiotic metabolites on maturation stage of antigen-presenting immune cells.METHODS:Ganeden Bacillus coagulans 30 (GBC30)bacterial cultures in log phase were used to isolate the secreted metabolite (MET) fraction.A second fraction was made to generate a crude cell-wall-enriched fraction,by centrifugation and lysis,followed by washing.A preparation of MET was subjected to size exclusion centrifugation,generating three fractions:< 3 kDa,3-30 kDa,and 30-200 kDa and activities were tested in comparison to crude MET and cell wall in primary cultures of human peripheral blood mononuclear cell (PBMC) as a source of antigen-presenting mononuclear phagocytes.The maturation status of mononudear phagocytes was evaluated by staining with monoclonal antibodies towards CD14,CD16,CD80 and CD86 and analyzed by flow cytometry.RESULTS:Treatment of PBMC with MET supported maturation of mononuclear phagocytes toward both macrophage and dendritic cell phenotypes.The biological activity unique to the metabolites included a reduction of CD14+ CD16+ pro-inflammatory ceils,and this property was associated with the high molecular weight metabolite fraction.Changes were also seen for the dendritic cell maturation markers CD80 and CD86.On CD14dim cells,an increase in both CD80 and CD86 expression was seen,in contrast to a selective increase in CD86 expression on CD14bright cells.The co-expression of CD80 and CD86 indicates effective antigen presentation to T cells and support of T helper cell differentiation.The selective expression of CD86 in the absence of CD80 points to a role in generating T regulatory cells.CONCLUSION:The data show that a primary mechanism of action of GBC30 metabolites involves support of more mature phenotypes of antigen-presenting cells,important for immunological decision-making.

  14. Mechanisms of ethanol-induced death of cerebellar granule cells.

    Science.gov (United States)

    Luo, Jia

    2012-03-01

    Maternal ethanol exposure during pregnancy may cause fetal alcohol spectrum disorders (FASD). FASD is the leading cause of mental retardation. The most deleterious effect of fetal alcohol exposure is inducing neuroapoptosis in the developing brain. Ethanol-induced loss of neurons in the central nervous system underlies many of the behavioral deficits observed in FASD. The cerebellum is one of the brain areas that are most susceptible to ethanol during development. Ethanol exposure causes a loss of both cerebellar Purkinje cells and granule cells. This review focuses on the toxic effect of ethanol on cerebellar granule cells (CGC) and the underlying mechanisms. Both in vitro and in vivo studies indicate that ethanol induces apoptotic death of CGC. The vulnerability of CGC to ethanol-induced death diminishes over time as neurons mature. Several mechanisms for ethanol-induced apoptosis of CGC have been suggested. These include inhibition of N-methyl-D-aspartate receptors, interference with signaling by neurotrophic factors, induction of oxidative stress, modulation of retinoid acid signaling, disturbance of potassium channel currents, thiamine deficiency, and disruption of translational regulation. Cultures of CGC provide an excellent system to investigate cellular/molecular mechanisms of ethanol-induced neurodegeneration and to evaluate interventional strategies. This review will also discuss the approaches leading to neuroprotection against ethanol-induced neuroapoptosis.

  15. HIV-1 alters neural and glial progenitor cell dynamics in the central nervous system: coordinated response to opiates during maturation.

    Science.gov (United States)

    Hahn, Yun Kyung; Podhaizer, Elizabeth M; Hauser, Kurt F; Knapp, Pamela E

    2012-12-01

    HIV-associated neurocognitive disorders (HANDs) are common sequelae of human immunodeficiency virus (HIV) infection, even when viral titers are well controlled by antiretroviral therapy. Evidence in patients and animal models suggests that neurologic deficits are increased during chronic opiate exposure. We have hypothesized that central nervous system (CNS) progenitor cells in both adult and developing CNS are affected by HIV infection and that opiates exacerbate these effects. To examine this question, neural progenitors were exposed to HIV-1 Tat(1-86) in the developing brain of inducible transgenic mice and in vitro. We examined whether Tat affected the proliferation or balance of progenitor populations expressing nestin, Sox2, and Olig2. Disease relevance was further tested by exposing human-derived progenitors to supernatant from HIV-1 infected monocytes. Studies concentrated on striatum, a region preferentially targeted by HIV and opiates. Results were similar among experimental paradigms. Tat or HIV exposure reduced the proliferation of undifferentiated (Sox2(+)) progenitors and oligodendroglial (Olig2(+)) progenitors. Coexposure to morphine exacerbated the effects of Tat or HIV-1(SF162) supernatant, but partially reversed HIV-1(IIIB) supernatant effects. Populations of Sox2(+) and Olig2(+) cells were also reduced by Tat exposure, although progenitor survival was unaffected. In rare instances, p24 immunolabeling was detected in viable human progenitors by confocal imaging. The vulnerability of progenitors is likely to distort the dynamic balance among neuron/glial populations as the brain matures, perhaps contributing to reports that neurologic disease is especially prevalent in pediatric HIV patients. Pediatric disease is atypical in developed regions but remains a serious concern in resource-limited areas where infection occurs commonly at birth and through breast feeding.

  16. Transcriptional Reprogramming of Mature CD4+ T helper Cells generates distinct MHC class II-restricted Cytotoxic T Lymphocytes

    Science.gov (United States)

    Mucida, Daniel; Husain, Mohammad Mushtaq; Muroi, Sawako; van Wijk, Femke; Shinnakasu, Ryo; Naoe, Yoshinori; Reis, Bernardo Sgarbi; Huang, Yujun; Lambolez, Florence; Docherty, Michael; Attinger, Antoine; Shui, Jr-Wen; Kim, Gisen; Lena, Christopher J.; Sakaguchi, Shinya; Miyamoto, Chizuko; Wang, Peng; Atarashi, Koji; Park, Yunji; Nakayama, Toshinori; Honda, Kenya; Ellmeier, Wilfried; Kronenberg, Mitchell; Taniuchi, Ichiro; Cheroutre, Hilde

    2013-01-01

    TCRαβ thymocytes differentiate to either CD8αβ cytotoxic T lymphocytes or CD4+ T helper cells. This functional dichotomy is controlled by key transcription factors, including the T helper master regulator, ThPOK, which suppresses the cytolytic program in MHC class II-restricted CD4+ thymocytes. ThPOK continues to repress CD8-lineage genes in mature CD4+ T cells, even as they differentiate to T helper effector subsets. Here we show that the T helper-fate was not fixed and that mature antigen-stimulated CD4+ T cells could terminate Thpok expression and reactivate CD8-lineage genes. This unexpected plasticity resulted in the post-thymic termination of the T helper-program and the functional differentiation of distinct MHC class II-restricted CD4+ cytotoxic T lymphocytes. PMID:23334788

  17. Cell therapy using induced pluripotent stem cells or somatic stem cells: this is the question.

    Science.gov (United States)

    Somoza, Rodrigo A; Rubio, Francisco J

    2012-05-01

    A lot of effort has been developed to bypass the use of embryonic stem cells (ES) in human therapies, because of several concerns and ethical issues. Some unsolved problems of using stem cells for human therapies, excluding the human embryonic origin, are: how to regulate cell plasticity and proliferation, immunological compatibility, potential adverse side-effects when stem cells are systemically administrated, and the in vivo signals to rule out a specific cell fate after transplantation. Currently, it is known that almost all tissues of an adult organism have somatic stem cells (SSC). Whereas ES are primary involved in the genesis of new tissues and organs, SSC are involved in regeneration processes, immuno-regulatory and homeostasis mechanisms. Although the differentiating potential of ES is higher than SSC, several studies suggest that some types of SSC, such as mesenchymal stem cells (MSC), can be induced epigenetically to differentiate into tissue-specific cells of different lineages. This unexpected pluripotency and the variety of sources that they come from, can make MSC-like cells suitable for the treatment of diverse pathologies and injuries. New hopes for cell therapy came from somatic/mature cells and the discovery that could be reprogrammed to a pluripotent stage similar to ES, thus generating induced pluripotent stem cells (iPS). For this, it is necessary to overexpress four main reprogramming factors, Sox2, Oct4, Klf4 and c-Myc. The aim of this review is to analyze the potential and requirements of cellular based tools in human therapy strategies, focusing on the advantage of using MSC over iPS.

  18. Notch signaling differentially regulates the cell fate of early endocrine precursor cells and their maturing descendants in the mouse pancreas and intestine.

    Science.gov (United States)

    Li, Hui Joyce; Kapoor, Archana; Giel-Moloney, Maryann; Rindi, Guido; Leiter, Andrew B

    2012-11-15

    Notch signaling inhibits differentiation of endocrine cells in the pancreas and intestine. In a number of cases, the observed inhibition occurred with Notch activation in multipotential cells, prior to the initiation of endocrine differentiation. It has not been established how direct activation of Notch in endocrine precursor cells affects their subsequent cell fate. Using conditional activation of Notch in cells expressing Neurogenin3 or NeuroD1, we examined the effects of Notch in both organs, on cell fate of early endocrine precursors and maturing endocrine-restricted cells, respectively. Notch did not preclude the differentiation of a limited number of endocrine cells in either organ when activated in Ngn3(+) precursor cells. In addition, in the pancreas most Ngn3(+) cells adopted a duct but not acinar cell fate; whereas in intestinal Ngn3(+) cells, Notch favored enterocyte and goblet cell fates, while selecting against endocrine and Paneth cell differentiation. A small fraction of NeuroD1(+) cells in the pancreas retain plasticity to respond to Notch, giving rise to intraislet ductules as well as cells with no detectable pancreatic lineage markers that appear to have limited ultrastructural features of both endocrine and duct cells. These results suggest that Notch directly regulates cell fate decisions in multipotential early endocrine precursor cells. Some maturing endocrine-restricted NeuroD1(+) cells in the pancreas switch to the duct lineage in response to Notch, indicating previously unappreciated plasticity at such a late stage of endocrine differentiation.

  19. A potential role of karyopherin a2 in the impaired maturation of dendritic cells observed in glioblastoma patients

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    Konstantinos Gousias

    2015-03-01

    Full Text Available Aim: Patients with glioblastomas demonstrate well-documented immunological impairments including decreased numbers of mature dendritic cells (DCs. Recent data identified karyopherin a2 (KPNA2, a nucleocytoplasmic shuttling receptor, as diagnostic and prognostic biomarker for gliomas. The aim of this ongoing study is to correlate parameters of immunity and nucleocytoplasmic transport in glioblastoma patients. Methods: We preoperatively collected serum from 17 patients with glioblastomas and determined DC subsets (HLA DR+ Lin-, CD34-, CD45+, CD123+, CD11+ were analyzed using a 6-color flow cytometry panel. Expression levels of KPNA2 and nuclear accumulation of p53 were evaluated semi-quantitatively by immunohistochemistry. O6-methylguanine DNA methyltransferase (MGMT and isocitrate dehydrogenase-1 (IDH-1 status were assessed by pyrosequencing and immunohistochemistry, respectively. Results: Median expression levels for both KPNA2 and p53 were 5-10%. IDH-1-R132H mutation and MGMT promoter hypermethylation was detected in 3/16 and 1/9 patients, respectively. Mean counts of total mature DCs, myeloid DCs and plasmacytoid DCs were 9.6, 2.1, 3.4 cells/μL. A preliminary analysis suggests an association between low KPNA2 nuclear expression and increased numbers of mature DCs. However, this correlation did not reach statistical significance so far (P = 0.077. Conclusion: Our preliminary data may indicate a role of KPNA2 in the impaired maturation of DCs observed in glioblastoma patients.

  20. Interleukin-10 inhibits lipopolysaccharide induced miR-155 precursor stability and maturation.

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    Sylvia T Cheung

    Full Text Available The anti-inflammatory cytokine interleukin-10 (IL-10 is essential for attenuating the inflammatory response, which includes reducing the expression of pro-inflammatory microRNA-155 (miR-155 in lipopolysaccharide (LPS activated macrophages. miR-155 enhances the expression of pro-inflammatory cytokines such as TNFα and suppresses expression of anti-inflammatory molecules such as SOCS1. Therefore, we examined the mechanism by which IL-10 inhibits miR-155. We found that IL-10 treatment did not affect the transcription of the miR-155 host gene nor the nuclear export of pre-miR-155, but rather destabilized both pri-miR-155 and pre-miR-155 transcripts, as well as interfered with the final maturation of miR-155. This inhibitory effect of IL-10 on miR-155 expression involved the contribution of both the STAT3 transcription factor and the phosphoinositol phosphatase SHIP1. This is the first report showing evidence that IL-10 regulates miRNA expression post-transcriptionally.

  1. Basal forebrain cholinergic input is not essential for lesion-induced plasticity in mature auditory cortex.

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    Kamke, Marc R; Brown, Mel; Irvine, Dexter R F

    2005-11-23

    The putative role of the basal forebrain cholinergic system in mediating lesion-induced plasticity in topographic cortical representations was investigated. Cholinergic immunolesions were combined with unilateral restricted cochlear lesions in adult cats, demonstrating the consequence of cholinergic depletion on lesion-induced plasticity in primary auditory cortex (AI). Immunolesions almost eliminated the cholinergic input to AI, while cochlear lesions produced broad high-frequency hearing losses. The results demonstrate that the near elimination of cholinergic input does not disrupt reorganization of the tonotopic representation of the lesioned (contralateral) cochlea in AI and does not affect the normal representation of the unlesioned (ipsilateral) cochlea. It is concluded that cholinergic basal forebrain input to AI is not essential for the occurrence of lesion-induced plasticity in AI.

  2. Cumulus Cell Role on Mouse Germinal Vesicle Oocyte Maturation, Fertilization, and Subsequent Embryo Development to Blastocyst Stage In Vitro

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    Reza Mahmodi

    2009-01-01

    Full Text Available Objective: The purpose of this study is to investigate the effect of cumulus cells on maturation,fertilization and subsequent development of mouse germinal vesicle oocytes.Materials and Methods: A total of 470 germinal vesicle (GV oocytes were obtained from26 ovaries of 3- 4 week old ICR female mice 48 hours after injection of 5 IU pregnant mareserum gonadotropin (PMSG. Collected oocytes were divided into two groups; group I: GVoocytes without cumulus cells (denuded oocyte, group II: GV oocytes with cumulus cells(cumulus-oocyte complex. The oocytes in both groups were cultured in TCM-199 mediumsupplemented with 10% fetal bovine serum (FBS for 22- 24 hours in a humidified atmosphereof 5% CO2 in air at 37°C. Oocyte maturation was scored under inverted microscope.To do in vitro fertilization, matured oocytes from each group were placed in T6 mediumand capacitated spermatozoa were added. Then the fertilized oocytes were cultured andassessed for cleavage to the 2-cell stage 24 hours and production of blastocyst 120 hoursafter fertilization. Data was analyzed by chi-square test and differences in the values wereconsiderable significant when p<0.05.Results: Maturation, fertilization, cleavage and blastocyst rates in denuded oocytes were:76.32%, 57.49%, 51.15% and 19.14% respectively. In the cumulus-oocyte complex rateswere: 89.41%, 80.76%, 75.58% and 45.62% respectively; all in the cumulus-oocyte complexwere significantly higher than those of denuded oocytes (p<0.05.Conclusion: The present study indicates that cumulus cells have important role duringmaturation, fertilization and subsequent embryo development to the blastocyst stage.

  3. Bordetella pertussis naturally occurring isolates with altered lipooligosaccharide structure fail to fully mature human dendritic cells.

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    Brummelman, Jolanda; Veerman, Rosanne E; Hamstra, Hendrik Jan; Deuss, Anna J M; Schuijt, Tim J; Sloots, Arjen; Kuipers, Betsy; van Els, Cécile A C M; van der Ley, Peter; Mooi, Frits R; Han, Wanda G H; Pinelli, Elena

    2015-01-01

    Bordetella pertussis is a Gram-negative bacterium and the causative agent of whooping cough. Despite high vaccination coverage, outbreaks are being increasingly reported worldwide. Possible explanations include adaptation of this pathogen, which may interfere with recognition by the innate immune system. Here, we describe innate immune recognition and responses to different B. pertussis clinical isolates. By using HEK-Blue cells transfected with different pattern recognition receptors, we found that 3 out of 19 clinical isolates failed to activate Toll-like receptor 4 (TLR4). These findings were confirmed by using the monocytic MM6 cell line. Although incubation with high concentrations of these 3 strains resulted in significant activation of the MM6 cells, it was found to occur mainly through interaction with TLR2 and not through TLR4. When using live bacteria, these 3 strains also failed to activate TLR4 on HEK-Blue cells, and activation of MM6 cells or human monocyte-derived dendritic cells was significantly lower than activation induced by the other 16 strains. Mass spectrum analysis of the lipid A moieties from these 3 strains indicated an altered structure of this molecule. Gene sequence analysis revealed mutations in genes involved in lipid A synthesis. Findings from this study indicate that B. pertussis isolates that do not activate TLR4 occur naturally and that this phenotype may give this bacterium an advantage in tempering the innate immune response and establishing infection. Knowledge on the strategies used by this pathogen in evading the host immune response is essential for the improvement of current vaccines or for the development of new ones.

  4. Hypertrophy of mature Xenopus muscle fibres in culture induced by synergy of albumin and insulin.

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    Jaspers, R T; van Beek-Harmsen, B J; Blankenstein, M A; Goldspink, G; Huijing, P A; van der Laarse, W J

    2008-10-01

    The aim of this study was to investigate effects of albumin and insulin separately as well as in combination on mature muscle fibres during long-term culture. Single muscle fibres were dissected from m. iliofibularis of Xenopus laevis and attached to a force transducer in a culture chamber. Fibres were cultured in a serum-free medium at slack length (mean sarcomere length 2.3 mum) for 8 to 22 days. The medium was supplemented with (final concentrations): (1) bovine insulin (6 nmol/L or 200-600 nmol/L), (2) 0.2% bovine albumin or (3) 0.2% bovine albumin in combination with insulin (120 nmol/L). In culture medium with insulin, 50% of the muscle fibres became in-excitable within 7-12 days, whereas the other 50% were stable. Caffeine contractures of in-excitable muscle fibres produced 80.4 +/- 2.4% of initial peak tetanic force, indicating impaired excitation-contraction (E-C) coupling in in-excitable fibres. In the presence of albumin, all cultured muscle fibres were stable for at least 10 days. Muscle fibres cultured in medium with insulin or albumin exclusively did not hypertrophy or change the number of sarcomeres in series. In contrast, muscle fibres cultured with both albumin and insulin showed an increase in tetanic force and fibre cross-sectional area of 19.6 +/- 2.8% and 32.5 +/- 4.9%, respectively, (means +/- SEM.; P = 0.007) after 16.3 +/- 1.7 days, whereas the number of sarcomeres in series remained unchanged. We conclude that albumin prevents muscle fibre damage and preserves E-C coupling in culture. Furthermore, albumin is important in regulating muscle fibre adaptation by a synergistic action with growth factors like insulin.

  5. Transplantation of mature adipocyte-derived dedifferentiated fat cells promotes locomotor functional recovery by remyelination and glial scar reduction after spinal cord injury in mice.

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    Yamada, Hiromi; Ito, Daisuke; Oki, Yoshinao; Kitagawa, Masato; Matsumoto, Taro; Watari, Tosihiro; Kano, Koichiro

    2014-11-14

    Mature adipocyte-derived dedifferentiated fat cells (DFAT) have a potential to be useful as new cell-source for cell-based therapy for spinal cord injury (SCI), but the mechanisms remain unclear. The objective of this study was to examine whether DFAT-induced functional recovery is achieved through remyelination and/or glial scar reduction in a mice model of SCI. To accomplish this we subjected adult female mice (n=22) to SCI. On the 8th day post-injury locomotor tests were performed, and the mice were randomly divided into two groups (control and DFAT). The DFAT group received stereotaxic injection of DFAT, while the controls received DMEM medium. Functional tests were conducted at repeated intervals, until the 36th day, and immunohistochemistry or staining was performed on the spinal cord sections. DFAT transplantation significantly improved locomotor function of their hindlimbs, and promoted remyelination and glial scar reduction, when compared to the controls. There were significant and positive correlations between promotion of remyelination or/and reduction of glial scar, and recovery of locomotor function. Furthermore, transplanted DFAT expressed markers for neuron, astrocyte, and oligodendrocyte, along with neurotrophic factors, within the injured spinal cord. In conclusion, DFAT-induced functional recovery in mice after SCI is probably mediated by both cell-autonomous and cell-non-autonomous effects on remyelination of the injured spinal cord.

  6. Reelin Regulates the Maturation of Dendritic Spines, Synaptogenesis and Glial Ensheathment of Newborn Granule Cells

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    Bosch, Carles; Masachs, Nuria; Exposito-Alonso, David; Martínez, Albert; Teixeira, Cátia M.; Fernaud, Isabel; Pujadas, Lluís; Ulloa, Fausto; Comella, Joan X.; DeFelipe, Javier; Merchán-Pérez, Angel; Soriano, Eduardo

    2016-01-01

    The Reelin pathway is essential for both neural migration and for the development and maturation of synaptic connections. However, its role in adult synaptic formation and remodeling is still being investigated. Here, we investigated the impact of the Reelin/Dab1 pathway on the synaptogenesis of newborn granule cells (GCs) in the young-adult mouse hippocampus. We show that neither Reelin overexpression nor the inactivation of its intracellular adapter, Dab1, substantially alters dendritic spine numbers in these neurons. In contrast, 3D-electron microscopy (focused ion beam milling/scanning electron microscope) revealed that dysregulation of the Reelin/Dab1 pathway leads to both transient and permanent changes in the types and morphology of dendritic spines, mainly altering mushroom, filopodial, and branched GC spines. We also found that the Reelin/Dab1 pathway controls synaptic configuration of presynaptic boutons in the dentate gyrus, with its dysregulation leading to a substantial decrease in multi-synaptic bouton innervation. Lastly, we show that the Reelin/Dab1 pat