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I Motta

Publications and source records attributed to I Motta.

28 records · Page 2Linked to original sources
PubMedDate not supplied

Cell-based delivery of cytokines allows for the differentiation of a doxycycline inducible oligodendrocyte precursor cell line in vitro.

BACKGROUND: Stem cells, having the property of self renewal, offer the promise of lifelong repair of damaged tissue. However, somatic tissue-committed primary stem cells are rare and difficult to expand in vitro. Genetically modified stem-like cells with the ability to expand conditionally provide a valuable tool with which to study stem cell biology, especially the cellular events of proliferation and differentiation. In addition, stem cells may be appropriate candidates for therapeutic applications. METHODS: Double transgenic mice possesing SV40 T antigen (Tag) under the control of the reverse tetracycline-transactivator (rtTA) were used to establish cell lines. One brain cell line was partially characterized by DNA sequencing, morphology, antigen expression using flow cytometry, confocal microscopy, and electrophysiology using the patch clamp technique. Cell cycle analysis was performed using propidium iodide staining; cell viability and H3-thymidine incorporation assays. The ability of this cell line to differentiate was assessed by confocal microscopy following co-culture with stem cells secreting cytokines. RESULTS: We report here the establishment and partial characterization of a cell line derived from the brain tissue of rtTA-SV40 Tag transgenic mice. Analysis of the morphology and antigen markers has shown that this cell line mimics some aspects of primary glial precursors. The results of electrophysiology are consistent with this and suggest that the cell line is derived from O2A glial precursor cells. Cell cycle progression of this cell line is doxycycline-dependent. In the absence of doxycycline, cells become apoptotic. Differentiation into mature type 2 astrocytes and (precursor) oligodendrocytes can be induced upon withdrawal of doxycycline and addition of epithelial stem cells secreting cytokine, such as hIL3 (human Interleukine 3) or hIL6 to the culture. In contrast, co-culturing with hCNTF (human Ciliary NeuroTrophic Factor)-secreting epithelial stem cells did not induce them to mature into progeny cell types. CONCLUSION: The differentiation of this O2A glial precursor line does not occur automatically in culture. Additional external help is required from the cell-based delivery of appropriate transgenic cytokines. Withdrawal of doxycycline from the culture medium removes the proliferation signals and induces a fatal outcome.

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PubMedDate not supplied

Improvement of an ELISA bioassay for the routine titration of murine interferon-gamma.

We improved an ELISA bioassay for murine IFN-gamma (MuIFN-gamma) based on measurement of Ia antigen on P388D1, a mouse macrophagic tumour line. Cells were cultured in microtitre plates in medium containing dilutions of IFN-gamma source. They were then washed and stained with a rat anti-mouse I amAb followed by mouse anti-rat peroxidase-labelled antibody. After incubation with substrate, the OD was read directly from microtitre plates. Standard curves obtained with reference NIH MuIFN-gamma showed that this assay allowed for the definition of unit values (giving 50% of the maximal effect) comparable to NIH international units (IU). It detected as low as 0.2 IU/ml of MuIFN-gamma and, in contrast to antiviral assays, was insensitive to IFN-alpha/beta. We used a concanavalin A-conditioned supernatant, which is a mixed source of lymphokines, to assess the specificity of our assay. Indeed, Ia expression induced by ConA-conditioned supernatant was fully inhibited by preincubation with anti-MuIFN-gamma antibodies. Using a stable indicator cell line, the present cell surface assay is easier to perform than other ELISA using bone-marrow-derived macrophages, and does not require cell fixation; its high sensitivity and specificity are comparable to that of immunoradiometric assays. It is thus valuable for routine MuIFN-gamma quantitations in culture supernatant and biological fluids.

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PubMedDate not supplied

Opposite effects of interleukin-4 on memory T helper cell development depend on interleukin-2.

We previously reported that cyclosporin A (CSA) promotes the generation of T helper memory cells during antigenic priming of murine spleen cells in vitro. More recently, we have demonstrated that interleukin-2 (IL2) has a downmodulating effect on T helper memory cell generation. The present data address the role of the other T cell growth factor, IL4, upon induction of these cells. The data presented here show that IL4 can interfere with this process: addition of rIL4 to immunosuppressed priming cultures leads to a considerable decrease in the helper activity of the recovered cells. However, in standard cultures, in which IL2 is normally produced, no effect of IL4 on T helper memory cell generation was found. Addition of IL4 has important consequences for cytokines produced upon antigenic restimulation. In standard cultures, IL4 primes for cells expressing high levels of IL2 and IL4 mRNA. Strikingly, in immunosuppressed priming cultures, IL4 counterbalances the CSA-induced blockade of the IFN gamma gene. Taken together, our results suggest that the unique role of IL4 is to drive T helper memory precursors into an IL4 production differentiation pathway. However, IL4 has a downmodulating effect on memory T helper cell induction when IL2 is not produced. These results confirm that synergy between IL2 and IL4 is mandatory for the directive role of IL4 upon IL4-producing cells. Furthermore, the finding that IL4 promotes the induction of IFN gamma in a CSA-resistant pathway represents a new tool for analysis of regulation of the IFN gamma gene.

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PubMedDate not supplied

A new approach to immunological memory using thymus-independent antigens.

Contrary to what has been reported of thymus-independent antigens, we recently demonstrated that trinitrophenylated lipopolysaccharide (TNP-LPS), a class 1 thymus-independent antigen, elicited an anti-TNP anamnestic response in C57BL/6 mice. The question of whether or not class 2 thymus-independent antigens (DNP-Ficoll and DNP-dextran) could also induce immunological memory in this mouse strain was examined. Evidence induce immunological memory in this mouse strain was examined. Evidence is presented that priming with either of these class 2 thymus-independent antigens resulted in the induction of memory B lymphocytes. However, while the memory cells generated by these two antigens were able to be activated by TNP-LPS, they were not triggered by class 2 thymus-independent antigens. Genetic analysis of the capacity of different mouse strains to mount a secondary response to TNP-LPS revealed that major histo-compatibility-associated genes did not play an essential role, but that IgH-V or closely linked gene(s) controlled the immunological memory to TNP-LPS. These findings are discussed in terms of regulatory phenomena which govern the expression of memory response to thymus-independent antigens.

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PubMedDate not supplied

Mitogenic signals are not required to circumvent the cyclosporin-induced inhibition of TNP-specific B memory cell expression.

Previous studies have shown that TNP-LPS-induced B memory cells can be recalled by either TNP-LPS or DNP-Ficoll and that cyclosporin A (CsA) prevents the expression of these B memory cells when DNP-Ficoll and not TNP-LPS is the challenging antigen. The possibility that the mitogenic signal delivered by TNP-LPS circumvents the inhibition exerted by CsA was investigated. It is demonstrated here that TNP-LPS, in its non-mitogenic (polymyxin-B-treated) as well as in its mitogenic form, is capable of driving TNP-specific B memory cells into the antibody-secreting stage, regardless of the presence or absence of CsA. It is suggested that both forms of TNP-LPS activate the same CsA-resistant subpopulation of B memory cells. The lack of DNP-Ficoll-induced memory expression in the presence of CsA is related to an intrinsic sensitivity to CsA inhibition of a subpopulation of B memory cells.

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PubMedDate not supplied

Anamnestic response to a thymus-independent antigen, TNP-LPS in C57BL/6 and DBA/2 mice: dominance of the IgG secondary response of the C57BL/6 mice in the F1 hybrids.

The primary and secondary immune responses to a thymus-independent antigen, TNP-LPS, were investigated in C57BL/6, DBA/2 and (C57BL/6 x DBA/2)F1 mice. While there is no evidence that TNP-LPS induces immunological memory in DBA/2 mice, a definite priming effect has been observed in C57BL/6 mice. Memory is revealed by the appearrance of antibody-forming cells of the IgG isotype. The differentiation of B micron precursors into B gamma memory cells is a dominant phenotype, since it is also found in the F1 C57BL/6 x DBA/2 hybrids.

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PubMedDate not supplied

Differentiation signal defect of splenic cells from LPS-treated mice.

Spleen cells from mice sensitized with 10 microgram of LPS given intravenously are unable, when stimulated in vitro 48 h after this treatment, to respond to sheep erythrocytes (SRBC). Addition of T-cell replacing factors (TRF) to these cells restores their capacity to mount an anti-SRBC immune response. Killing of the cells proliferating under antigen stimulation by highly radioactive thymidine leads to the suppression of the anti-SRBC response observed in the presence of TRF. These experiments suggests that the proliferative events leading to the expansion of B cell precursors under antigen stimulation is not impaired by the treatment by LPS. These preliminary data show that the defect is linked to the lack of signals leading to the differentiation of B cells into antibody-secreting cells.

Adjuvants, Immunologic↗