Adoptive transfer of genetically modified macrophages elucidated TGF-beta-mediated 'self-defence' of the glomerulus against local action of macrophages.
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TGF-beta has several anti-inflammatory properties which may be relevant to prevention of or recovery from acute glomerular inflammation. Using genetically modified mesangial cells and a technique for in vivo macrophage transfer, this article provides evidence for TGF-beta-mediated 'self-defence' of the glomerulus against macrophages. Rat mesangial cells stably transfected with TGF-beta1 showed a blunted response to the macrophage-derived, proinflammatory cytokine IL-1beta. In contrast, mesangial cells expressing the dominant-interfering TGF-beta receptor showed an enhanced response to IL-1. Similarly, externally added TGF-beta1 inhibited the cytokine response of normal glomeruli, and isolated nephritic glomeruli producing active TGF-beta1 showed a depressed response to IL-1beta, compared to normal glomeruli. Consistent with these in vitro results, in vivo transfer of activated macrophages revealed that the TGF-beta-producing glomeruli are insensitive to the effector action of macrophages. These results indicate that TGF-beta1 functions as an endogenous 'defender' that counteracts local action of activated macrophages in the glomerulus. (+info)
Effects of the Chinese traditional medicine mao-bushi-saishin-to on therapeutic efficacy of a new benzoxazinorifamycin, KRM-1648, against Mycobacterium avium infection in mice.
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The Chinese traditional medicine mao-bushi-saishin-to (MBST), which has anti-inflammatory effects and has been used to treat the common cold and nasal allergy in Japan, was examined for its effects on the therapeutic activity of a new benzoxazinorifamycin, KRM-1648 (KRM), against Mycobacterium avium complex (MAC) infection in mice. In addition, we examined the effects of MBST on the anti-MAC activity of murine peritoneal macrophages (M phi s). First, MBST significantly increased the anti-MAC therapeutic activity of KRM when given to mice in combination with KRM, although MBST alone did not exhibit such effects. Second, MBST treatment of M phi s significantly enhanced the KRM-mediated killing of MAC bacteria residing in M phi s, although MBST alone did not potentiate the M phi anti-MAC activity. MBST-treated M phi s showed decreased levels of reactive nitrogen intermediate (RNI) release, suggesting that RNIs are not decisive in the expression of the anti-MAC activity of such M phi populations. MBST partially blocked the interleukin-10 (IL-10) production of MAC-infected M phi s without affecting their transforming growth factor beta (TGF-beta)-producing activity. Reverse transcription-PCR analysis of the lung tissues of MAC-infected mice at weeks 4 and 8 after infection revealed a marked increase in the levels of tumor necrosis factor alpha, gamma interferon (IFN-gamma), IL-10, and TGF-beta mRNAs. KRM treatment of infected mice tended to decrease the levels of the test cytokine mRNAs, except that it increased TGF-beta mRNA expression at week 4. MBST treatment did not affect the levels of any cytokine mRNAs at week 8, while it down-regulated cytokine mRNA expression at week 4. At week 8, treatment of mice with a combination of KRM and MBST caused a marked decrease in the levels of the test cytokines mRNAs, especially IL-10 and IFN-gamma mRNAs, although such effects were obscure at week 4. These findings suggest that down-regulation of the expression of IL-10 and TGF-beta is related to the combined therapeutic effects of KRM and MBST against MAC infection. (+info)
Lack of regulation in the heart forming region of avian embryos.
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The ability to regenerate a heart after ablation of cardiogenic mesoderm has been demonstrated in early stage fish and amphibian embryos but this type of regulation of the heart field has not been seen in avians or mammals. The regulative potential of the cardiogenic mesoderm was examined in avian embryos and related to the spatial expression of genes implicated in early cardiogenesis. With the identification of early cardiac regulators such as bmp-2 and nkx-2.5, it is now possible to reconcile classical embryological studies with molecular mechanisms of cardiac lineage determination in vivo. The most anterior lateral embryonic cells were identified as the region that becomes the heart and removal of all or any subset of these cells resulted in the loss of corresponding cardiac structures. In addition, removal of the lateral heart forming mesoderm while leaving the lateral endoderm intact also results in loss of cardiac structures. Thus the medial anterior mesoderm cannot be recruited into the heart lineage in vivo even in the presence of potentially cardiac inducing endoderm. In situ analysis demonstrated that genes involved in early events of cardiogenesis such as bone morphogenetic protein 2 (bmp-2) and nkx-2.5 are expressed coincidentally with the mapped far lateral heart forming region. The activin type IIa receptor (actR-IIa) is a potential mediator of BMP signaling since it is expressed throughout the anterior mesoderm with the highest level of expression occurring in the lateral prospective heart cells. The posterior boundary of actR-IIa is consistent with the posterior boundary of nkx-2.5 expression, supporting a model whereby ActR-IIa is involved in restricting the heart forming region to an anterior subset of lateral cells exposed to BMP-2. Analysis of the cardiogenic potential of the lateral plate mesoderm posterior to nkx-2.5 and actR-IIa expression demonstrated that these cells are not cardiogenic in vitro and that removal of these cells from the embryo does not result in loss of heart tissue in vivo. Thus, the region of the avian embryo that will become the heart is defined medially, laterally, and posteriorly by nkx-2.5 gene expression. Removal of all or part of the nkx-2.5 expressing region results in the loss of corresponding heart structures, demonstrating the inability of the chick embryo to regenerate cardiac tissue in vivo at stages after nkx-2.5 expression is initiated. (+info)
BMP7 acts in murine lens placode development.
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Targeted inactivation of the Bmp7 gene in mouse leads to eye defects with late onset and variable penetrance (A. T. Dudley et al., 1995, Genes Dev. 9, 2795-2807; G. Luo et al., 1995, Genes Dev. 9, 2808-2820). Here we report that the expressivity of the Bmp7 mutant phenotype markedly increases in a C3H/He genetic background and that the phenotype implicates Bmp7 in the early stages of lens development. Immunolocalization experiments show that BMP7 protein is present in the head ectoderm at the time of lens placode induction. Using an in vitro culture system, we demonstrate that addition of BMP7 antagonists during the period of lens placode induction inhibits lens formation, indicating a role for BMP7 in lens placode development. Next, to integrate Bmp7 into a developmental pathway controlling formation of the lens placode, we examined the expression of several early lens placode-specific markers in Bmp7 mutant embryos. In these embryos, Pax6 head ectoderm expression is lost just prior to the time when the lens placode should appear, while in Pax6-deficient (Sey/Sey) embryos, Bmp7 expression is maintained. These results could suggest a simple linear pathway in placode induction in which Bmp7 functions upstream of Pax6 and regulates lens placode induction. At odds with this interpretation, however, is the finding that expression of secreted Frizzled Related Protein-2 (sFRP-2), a component of the Wnt signaling pathway which is expressed in prospective lens placode, is absent in Sey/Sey embryos but initially present in Bmp7 mutants. This suggests a different model in which Bmp7 function is required to maintain Pax6 expression after induction, during a preplacodal stage of lens development. We conclude that Bmp7 is a critical component of the genetic mechanism(s) controlling lens placode formation. (+info)
Blockade of type beta transforming growth factor signaling prevents liver fibrosis and dysfunction in the rat.
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We eliminated type beta transforming growth factor (TGF-beta) signaling by adenovirus-mediated local expression of a dominant-negative type II TGF-beta receptor (AdCATbeta-TR) in the liver of rats treated with dimethylnitrosamine, a model of persistent liver fibrosis. In rats that received a single application of AdCATbeta-TR via the portal vein, liver fibrosis as assessed by histology and hydroxyproline content was markedly attenuated. All AdCATbeta-TR-treated rats remained alive, and their serum levels of hyaluronic acid and transaminases remained at low levels, whereas all the AdCATbeta-TR-untreated rats died of liver dysfunction. The results demonstrate that TGF-beta does play a central role in liver fibrogenesis and indicate clearly in a persistent fibrosis model that prevention of fibrosis by anti-TGF-beta intervention could be therapeutically useful. (+info)
A binding site for homeodomain and Pax proteins is necessary for L1 cell adhesion molecule gene expression by Pax-6 and bone morphogenetic proteins.
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The cell adhesion molecule L1 regulates axonal guidance and fasciculation during development. We previously identified the regulatory region of the L1 gene and showed that it was sufficient for establishing the neural pattern of L1 expression in transgenic mice. In the present study, we characterize a DNA element within this region called the HPD that contains binding motifs for both homeodomain and Pax proteins and responds to signals from bone morphogenetic proteins (BMPs). An ATTA sequence within the core of the HPD was required for binding to the homeodomain protein Barx2 while a separate paired domain recognition motif was necessary for binding to Pax-6. In cellular transfection experiments, L1-luciferase reporter constructs containing the HPD were activated an average of 4-fold by Pax-6 in N2A cells and 5-fold by BMP-2 and BMP-4 in Ng108 cells. Both of these responses were eliminated on deletion of the HPD from L1 constructs. In transgenic mice, deletion of the HPD from an L1-lacZ reporter resulted in a loss of beta-galactosidase expression in the telencephalon and mesencephalon. Collectively, our experiments indicate that the HPD regulates L1 expression in neural tissues via homeodomain and Pax proteins and is likely to be a target of BMP signaling during development. (+info)
Characterization and mutation analysis of human LEFTY A and LEFTY B, homologues of murine genes implicated in left-right axis development.
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Members of the transforming growth factor (TGF)-beta family of cell-signaling molecules have been implicated recently in mammalian left-right (LR) axis development, the process by which vertebrates lateralize unpaired organs (e.g., heart, stomach, and spleen). Two family members, Lefty1 and Lefty2, are expressed exclusively on the left side of the mouse embryo by 8.0 days post coitum. This asymmetry is lost or reversed in two murine models of abnormal LR-axis specification, inversus viscerum (iv) and inversion of embryonic turning (inv). Furthermore, mice homozygous for a Lefty1 null allele manifest LR malformations and misexpress Lefty2. We hypothesized that Lefty mutations may be associated with human LR-axis malformations. We now report characterization of two Lefty homologues, LEFTY A and LEFTY B, separated by approximately 50 kb on chromosome 1q42. Each comprises four exons spliced at identical positions. LEFTY A is identical to ebaf, a cDNA previously identified in a search for genes expressed in human endometrium. The deduced amino acid sequences of LEFTY A and LEFTY B are more similar to each other than to Lefty1 or Lefty2. Analysis of 126 human cases of LR-axis malformations showed one nonsense and one missense mutation in LEFTY A. Both mutations lie in the cysteine-knot region of the protein LEFTY A, and the phenotype of affected individuals is very similar to that typically seen in Lefty1-/- mice with LR-axis malformations. (+info)
Ultramicroscopic structures of the leptomeninx of mice with communicating hydrocephalus induced by human recombinant transforming growth factor-beta 1.
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An experimental model of communicating hydrocephalus was developed based on intrathecal injection of human recombinant transforming growth factor-beta 1 (hrTGF-beta 1) in the mouse. To clarify the mechanism of this hydrocephalus model, the ultrastructure of the leptomeninx in the process of ventricular dilation was examined in C57/BL6 mice injected intrathecally with 60 ng of hrTGF-beta 1. The leptomeninx was examined at various periods after injection by light and electron microscopy. Immunostaining for fibroblasts and macrophages was also performed. Leptomeninx within a week after injection showed that the thin cytoplasmic processes of leptomeningeal cells formed a laminated structure with a meshwork, which was almost the same as the controls. In the second week, many cells with a round nucleus appeared in the leptomeninx. Immunohistochemically, these cells were positive for anti-fibroblast antibody and negative for anti-Mac-1 and anti-macrophage BM-8 antibodies. Three weeks later, the laminated structure was disrupted and abundant deposition of collagen fibers was found in the inter-cellular space of the leptomeninx. Such inter-meningeal fibrosis would disturb cerebrospinal fluid flow in the mouse leptomeninx and cause slowly progressive ventricular dilation. (+info)