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J Neurosci ; 38(19): 4598-4609, 2018 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-29661967


In the rodent olfactory system, neuroblasts produced in the ventricular-subventricular zone of the postnatal brain migrate tangentially in chain-like cell aggregates toward the olfactory bulb (OB) through the rostral migratory stream (RMS). After reaching the OB, the chains are dissociated and the neuroblasts migrate individually and radially toward their final destination. The cellular and molecular mechanisms controlling cell-cell adhesion during this detachment remain unclear. Here we report that Fyn, a nonreceptor tyrosine kinase, regulates the detachment of neuroblasts from chains in the male and female mouse OB. By performing chemical screening and in vivo loss-of-function and gain-of-function experiments, we found that Fyn promotes somal disengagement from the chains and is involved in neuronal migration from the RMS into the granule cell layer of the OB. Fyn knockdown or Dab1 (disabled-1) deficiency caused p120-catenin to accumulate and adherens junction-like structures to be sustained at the contact sites between neuroblasts. Moreover, a Fyn and N-cadherin double-knockdown experiment indicated that Fyn regulates the N-cadherin-mediated cell adhesion between neuroblasts. These results suggest that the Fyn-mediated control of cell-cell adhesion is critical for the detachment of chain-forming neuroblasts in the postnatal OB.SIGNIFICANCE STATEMENT In the postnatal brain, newly born neurons (neuroblasts) migrate in chain-like cell aggregates toward their destination, where they are dissociated into individual cells and mature. The cellular and molecular mechanisms controlling the detachment of neuroblasts from chains are not understood. Here we show that Fyn, a nonreceptor tyrosine kinase, promotes the somal detachment of neuroblasts from chains, and that this regulation is critical for the efficient migration of neuroblasts to their destination. We further show that Fyn and Dab1 (disabled-1) decrease the cell-cell adhesion between chain-forming neuroblasts, which involves adherens junction-like structures. Our results suggest that Fyn-mediated regulation of the cell-cell adhesion of neuroblasts is critical for their detachment from chains in the postnatal brain.

Microbiol Immunol ; 50(3): 235-42, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16547421


Following immunization with HIV-1 infected cells, a hybridoma cell line termed 9F11 was established from the P3U-1 myeloma line fused with lymphocytes from a trans-chromosome (TC) mouse, that harbors human chromosomes containing immunoglobulin genes. The 9F11 human IgM monoclonal antibody (9F11 Ab) reacts with HIV-1 infected MOLT4 cells but not with uninfected MOLT4 cells, and causes immune cytolysis with homologous human complement at a concentration as low as 0.4 microg/ml. This Ab was used to perform immunoscreening of a cDNA expression library derived from HIV-1 infected cells. All positive cDNA clones contained SWAP-70 cDNA. SWAP-70 RNA and protein expression are much stronger in HIV-1 infected cells. SWAP-70 was also detected on the surface of HIV-1 infected cells by flow cytometric analysis. The monocyte cell line U937 cells expresses SWAP-70 on its cell surface regardless of whether it was infected with HIV-1. Furthermore, among PBMCs surface expression of SWAP-70 was detected on CD21+, CD56+ and CD14+ cells. Although CD3+ cells scarcely express SWAP-70 on their surface, once activated, they become positive. SWAP-70 may therefore serve as a marker for T cell differentiation as well as for HIV-1 infection.

Anticorpos Monoclonais/imunologia , Proteínas de Ligação a DNA/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Infecções por HIV/diagnóstico , HIV-1/imunologia , Proteínas Nucleares/metabolismo , Linfócitos T/virologia , Animais , Diferenciação Celular , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/imunologia , Humanos , Hibridomas , Imunoglobulina M/imunologia , Leucócitos Mononucleares/virologia , Ativação Linfocitária , Camundongos , Antígenos de Histocompatibilidade Menor , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Linfócitos T/citologia , Células U937
Microbiol Immunol ; 49(5): 447-59, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15905607


The trans-chromosome (TC) mouse that we used harbors human chromosomes 2, 14 and/or 22, and has undergone knock-out of its endogeneous genes coding for mu-and kappa-chains of immunoglobulin. One of these TC mice was immunized with HIV-1-infected U937 cells, and spleen cells from the immunized animal were fused with the mouse myeloma cell line to generate hybridoma cells. We selected hybridomas that produce human IgM antibodies (Abs) reactive with HIV-1-infected MOLT4 cells but not with uninfected MOLT4 cells. Two hybridoma cell lines were established termed 9F11 and 2G9. Although 0.4 mug/ml of 9F11 was able to induce complement-mediated cytolysis of the infected cells in the presence of fresh human serum, 2G9 could not. There was no difference between the two monoclonal Abs in the base sequences of cDNAs coding for the constant regions of mu-and kappa-chains. Therefore, we speculate that the ability to activate complement on homologous cell membranes might reflect the structural presentation of antigenic molecules, which could facilitate the binding of an IgM Ab to multiple binding sites resulting in escape from restriction by species-specific inhibitors of complement such as DAF (CD55) and CD59. On the other hand, 2G9 induced apoptosis of HIV-1-infected cells, including latently infected OM10.1 cells, although the Ag for 2G9 remains to be identified. Since both of the Abs had reduced reactivity toward HIV-1-infected MOLT4 cells following cultivation in the presence of tunicamycin, the responsible antigens would involve a sugar moiety.

Anticorpos Monoclonais/imunologia , Apoptose , Proteínas do Sistema Complemento/imunologia , Citotoxicidade Imunológica , HIV-1/imunologia , Imunoglobulina M/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Linhagem Celular , Cromossomos Humanos/genética , Proteínas Inativadoras do Complemento/fisiologia , Humanos , Hibridomas/imunologia , Regiões Constantes de Imunoglobulina/genética , Imunoglobulina M/biossíntese , Cadeias kappa de Imunoglobulina/genética , Cadeias mu de Imunoglobulina/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Análise de Sequência de DNA
J Immunol ; 173(1): 205-13, 2004 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15210776


Two mouse tumor cell lines, Meth A (BALB/c mouse-derived fibrosarcoma) and MM46 (C3H/He mouse-derived mammary tumor), were shown to express high levels of complement receptor-related gene y/p65 (Crry/p65), a membrane-bound complement-regulatory protein. Inhibiting the complement-regulatory activity of Crry/p65 with mAb 5D5 induced high levels of C3 deposition on in vivo tumor-derived Meth A and MM46 cells. To determine the effect of Crry/p65 blockade and increased C3 deposition on in vivo tumor growth, Meth A and MM46 cells were treated with 5D5 mAb and injected into BALB/c and C3H/He mice, respectively. Pretreating MM46 cells with 5D5 mAb significantly suppressed their tumorigenicity when injected s.c. Pretreatment with 5D5 mAb had a modest effect on Meth A s.c. tumor growth. Because complement is involved in the induction of an immune response, we investigated the effect of Crry/p65 blockade and increased C3 deposition on the immunogenicity of the tumor cells in a vaccination protocol. Vaccination of mice with irradiated Meth A cells pretreated with 5D5 mAb protected mice from subsequent challenge. In contrast, vaccination with irradiated Meth A cells without pretreatment was not protective. Survival was correlated with a high titer IgM response and specific CTL activity. These data demonstrate that the functional inhibition of Crry/p65 on tumor cells affects tumor growth and immunogenicity, and that the complement deposition resulting from this inhibition can act in concert with antitumor effector mechanisms to elicit potent antitumor immunity in vivo.

Vacinas Anticâncer/imunologia , Neoplasias Experimentais/terapia , Receptores de Complemento/imunologia , Animais , Linhagem Celular Tumoral , Ativação do Complemento , Complemento C3/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Vacinação
Biochim Biophys Acta ; 1624(1-3): 54-9, 2003 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-14642813


We previously reported the development of a "cytomedicine" that consists of cells trapped in alginate-poly-L-lysine-alginate (APA) microcapsules and agarose microbeads. The functional cells that are entrapped in semipermeable polymer are completely isolated from cellular immune system. However, the ability of cytomedicine to isolate cells from the humoral immune system, which plays an essential role in xenograft rejection, is low. Therefore, the goal of the present study was to develop a novel cytomedicine that could protect the entrapped cells from injury of the complement system. We investigated the applicability of the complement regulatory protein (CRP), Crry, to cytomedicine. Crry-transfected cells entrapped within agarose microbeads resisted injury by complement to a degree, while entrapment of Crry transfected cells within agarose microbeads containing polyvinyl sulfate (PVS), a novel cytomedical device with anti-complement activity, clearly protected against complement attack. These data indicate that the combination of a CRP and a cytomedical device with anti-complement activity is a superior device for cytomedical therapy.

Terapia Baseada em Transplante de Células e Tecidos/métodos , Proteínas Inativadoras do Complemento/farmacologia , Polivinil/farmacologia , Receptores de Complemento/genética , Sefarose/farmacologia , Animais , Células CHO , Cricetinae , Imunossupressão , Transfecção