Temporally regulated and tissue-specific gene manipulations in the adult and embryonic heart using a tamoxifen-inducible Cre protein.

The advent of conditional and tissue-specific recombination systems in gene-targeted or transgenic mice has permitted an assessment of single gene function in a temporally regulated and cell-specific manner. Here we generated transgenic mice expressing a tamoxifen-inducible Cre recombinase protein fused to two mutant estrogen-receptor ligand-binding domains (MerCreMer) under the control of the alpha-myosin heavy chain promoter. These transgenic mice were crossed with the ROSA26 lacZ-flox-targeted mice to examine Cre recombinase activity and the fidelity of the system. The data demonstrate essentially no Cre-mediated recombination in the embryonic, neonatal, or adult heart in the absence of inducing agent but >80% recombination after only four tamoxifen injections. Expression of the MerCreMer fusion protein within the adult heart did not affect cardiac performance, cellular architecture, or expression of hypertrophic marker genes, demonstrating that the transgene-encoded protein is relatively innocuous. In summary, MerCreMer transgenic mice represent a tool for temporally regulated inactivation of any loxP-targeted gene within the developing and adult heart or for specifically directing recombination and expression of a loxP-inactivated cardiac transgene in the heart.

[Treatment of anterior segment fibrinous reactions and hemorrhage with intracameral low dose rt-PA: clinical study and review of the literature]. / Intérêt des faibles doses de rt-PA dans les syndromes inflammatoires et hémorragiques du segment antérieur: étude clinique et revue de la littérature.

PURPOSE: to evaluate the efficacy and the safety of low dose intraocular tissue plasminogen activator (rt-PA) in the treatment of traumatic hyphema and postoperative fibrinous membrane. METHODS: Six microg to 10 microg of rt-PA was injected into the anterior chamber to treat severe fibrinous postoperative membranes and total traumatic hyphemae. RESULTS: Thirteen eyes of 13 patients were treated. Four cases of traumatic hyphema and 9 cases of fibrinous membranes were included. Complete fibrinolysis within 24 hours was observed in 4 cases (30.8%). A partial success was noted in 7 eyes (53.8%). No beneficial effect was observed in two cases of traumatic hyphema associated with intravitreal hemorrhage after penetrating trauma. No side effect attributable to rt-PA occurred. CONCLUSION: Low dose intraocular rt-PA appears to be safe and effective in the treatment of postoperative fibrinous membrane and endocular hemorrhage limited to the anterior chamber.

[Early limbal autograft after alkali burn of the ocular surface]. / Autogreffe de limbe précoce après brûlure alcaline de la surface oculaire.

Limbal autograft transplantation is recommended for treatment of limbal stem cell destruction and its complications. It is appropriate to restore corneal reepithelialization and arrest corneal vascularization and scarring. In unilateral alkali injury, limbal transplantation was first recommended for late management of corneal conjunctivalization. Our patient had severe ocular unilateral alkali injury. Early autologous limbal transplant was effective in restoring an excellent corneal surface and a good visual function.

Defective liver formation and liver cell apoptosis in mice lacking the stress signaling kinase SEK1/MKK4.

The stress signaling kinase SEK1/MKK4 is a direct activator of stress-activated protein kinases (SAPKs; also called Jun-N-terminal kinases, JNKs) in response to a variety of cellular stresses, such as changes in osmolarity, metabolic poisons, DNA damage, heat shock or inflammatory cytokines. We have disrupted the sek1 gene in mice using homologous recombination. Sek1(-/- )embryos display severe anemia and die between embryonic day 10.5 (E10.5) and E12.5. Haematopoiesis from yolk sac precursors and vasculogenesis are normal in sek1(-/- )embryos. However, hepatogenesis and liver formation were severely impaired in the mutant embryos and E11.5 and E12.5 sek1(-/- )embryos had greatly reduced numbers of parenchymal hepatocytes. Whereas formation of the primordial liver from the visceral endoderm appeared normal, sek1(-/-) liver cells underwent massive apoptosis. These results provide the first genetic link between stress-responsive kinases and organogenesis in mammals and indicate that SEK1 provides a crucial and specific survival signal for hepatocytes.

Vav regulates peptide-specific apoptosis in thymocytes.

The protooncogene Vav functions as a GDP/GTP exchange factor (GEF) for Rho-like small GTPases involved in cytoskeletal reorganization and cytokine production in T cells. Gene-targeted mice lacking Vav have a severe defect in positive and negative selection of T cell antigen receptor transgenic thymocytes in vivo, and vav-/- thymocytes are completely resistant to peptide-specific and anti-CD3/anti-CD28-mediated apoptosis. Vav acts upstream of mitochondrial pore opening and caspase activation. Biochemically, Vav regulates peptide-specific Ca2+ mobilization and actin polymerization. Peptide-specific cell death was blocked both by cytochalasin D inhibition of actin polymerization and by inhibition of protein kinase C (PKC). Activation of PKC with phorbol ester restored peptide-specific apoptosis in vav-/- thymocytes. Vav was found to bind constitutively to PKC-theta in thymocytes. Our results indicate that peptide-triggered thymocyte apoptosis is mediated via Vav activation, changes in the actin cytoskeleton, and subsequent activation of a PKC isoform.

Cooperation between CD44 and LFA-1/CD11a adhesion receptors in lymphokine-activated killer cell cytotoxicity.

IL-2-activated NK cells exhibit cytotoxic activity against a wide variety of tumor cells in a non-MHC-restricted fashion and in the absence of prior sensitization. The molecular mechanisms that regulate the cytotoxicity and attachment of activated killer cells to tumor target cells are not known. We provide genetic evidence in CD44(-/-) and LFA-1(-/-) mice that the cell adhesion receptors LFA-1 and CD44 regulate the cytotoxic activity of IL-2-activated NK cells against a variety of different tumor cells. This defect in cytotoxicity was significantly enhanced in mice that carried a double mutation of both CD44 and LFA-1. In vitro differentiation, TNF-alpha and IFN-gamma production, and expression of the cytolytic effector molecules perforin and Fas-L were comparable among IL-2-activated NK cells from LFA-1(-/-), CD44(-/-), CD44(-/-)LFA-1(-/-), and control mice. However, CD44(-/-), LFA-1(-/-), and CD44(-/-)LFA-1(-/-) IL-2-activated NK cells showed impaired binding and conjugate formation with target cells. We also show that hyaluronic acid is the principal ligand on tumor cells for CD44-mediated cytotoxicity of IL-2-activated NK cells. These results provide the first genetic evidence of the role of adhesion receptors in IL-2-activated NK killing. These data also indicate that distinct adhesion receptors cooperate to mediate binding between effector and target cells required for the initiation of "natural" cytotoxicity.

Vav is a regulator of cytoskeletal reorganization mediated by the T-cell receptor.

BACKGROUND: Vav is a guanine-nucleotide exchange factor for the Rho-like small GTPases RhoA, Rac1 and Cdc42, which regulate cytoskeletal reorganization and activation of stress-activated protein kinases (SAPK/JNKs). Vav is expressed in hematopoietic cells and is phosphorylated in T and B cells following activation of various growth factor or antigen receptors. Vav interacts with several signaling molecules in T cells, but the functional relevance of these interactions is established only for Slp76: they cooperate to induce activity of the transcription factor NF-AT and interleukin-2 expression. We have investigated the role of Vav in T cells by generating vav-/- mice. RESULTS: Mice deficient for vav were viable and healthy, but had impaired T-cell development. In vav-/- T cells, in response to activation of the T-cell receptor (TCR), cell cycle progression, induction of NF-ATc1 activity, downregulation of the cell-cycle inhibitor p27Kip1, interleukin-2 production, actin polymerization and the clustering of TCRs into patches and caps--a cytoskeletal reorganization process--were defective. TCR-mediated activation of mitogen-activated protein kinase and SAPK/JNK was unaffected. Ca2+ mobilization was impaired in vav-/- thymocytes and T cells. In wild-type cells, Vav constitutively associated with the cytoskeletal membrane anchors talin and vinculin. In the absence of Vav, phosphorylation of Slp76, Slp76-talin interactions, and recruitment of the actin cytoskeleton to the CD3 zeta chain of the TCR co-receptor were impaired. CONCLUSIONS: Vav is a crucial regulator of TCR-mediated Ca2+ flux, cytoskeletal reorganization and TCR clustering, and these are required for T-cell maturation, interleukin-2 production and cell cycle progression.

The interferon regulatory transcription factor IRF-1 controls positive and negative selection of CD8+ thymocytes.

Little is known about the molecular mechanisms and transcriptional regulation that govern T cell selection processes and the differentiation of CD4+ and CD8+ T cells. Mice lacking the interferon regulatory transcription factor-1 (IRF-1) have reduced numbers of mature CD8+ cells within the thymus and peripheral lymphatic organs. Here we show that positive and negative T cell selection of two MHC class I-restricted TCR alphabeta transgenes, H-Y and P14, are impaired in IRF-1-/- mice. The absence of IRF-1 resulted in decreased expression of LMP2, TAP1, and MHC class I on thymic stromal cells. Despite decreased MHC class I expression on IRF-1-/- thymic stromal cells, the defect in CD8+ T cells development did not reside in the thymic environment, and IRF-1-/- stromal cells can fully support development of CD8+ thymocytes in in vivo bone marrow chimeras and in vitro reaggregation cultures. Moreover, IRF-1-/- thymocytes displayed impaired TCR-mediated signal transduction, and the induction of negative selection in TCR Tg thymocytes from IRF-1-/- mice required a 1000-fold increase in selecting peptide. We also provide evidence that IRF-1 is mainly expressed in mature, but not immature, thymocytes and that expression of IRF-1 in immature thymocytes is induced after peptide-specific TCR activation. These results indicate that IRF-1 regulates gene expression in developing thymocytes required for lineage commitment and selection of CD8+ thymocytes.

HIV infection decreases intracellular nicotinamide adenine dinucleotide [NAD].

We report that HIV-1 infection of human cells in vitro leads to significant decreases in the intracellular concentration of NAD. This decrease varies with viral load and HIV strain. In tissue culture, cells lacking CD4 receptors or cells incubated with heat inactivated virus do not demonstrate this decrease in NAD. Nicotinamide, the amide form of the vitamin niacin, increases intracellular NAD levels in uninfected cells as expected. Our data demonstrate that nicotinamide also maintains increased intracellular NAD concentrations in HIV infected cells. We conclude that HIV induces a state of intracellular pellagra which is reversed by the administration of nicotinamide.

Measuring swainsonine in serum of cancer patients: phase I clinical trial.

Swainsonine, an indolizidine alkaloid and competitive inhibitor of Golgi alpha-mannosidase II (EC 3.2.1.114), reduces tumor growth and stimulates immune function in mice. On the basis of these observations, a phase I clinical trial was initiated to determine whether swainsonine could be administered safely to cancer patients. We describe a method for extraction, acetylation, and quantification of swainsonine in human serum samples. Methyl alpha-D-mannopyranoside and methyl beta-D-galactopyranoside were added to serum samples as internal standards and, after sequential extraction of lipids and proteins with chloroform and acetonitrile, respectively, samples were acetylated with acetic anhydride and 4-dimethylaminopyridine and separated by gas-liquid chromatography. The identity of swainsonine and the internal standards after their extraction from serum and acetylation was confirmed by gas chromatography/mass spectrometry. Swainsonine was recovered at an efficiency of 90%, relative to internal standards, and calibration graphs were rectilinear from 3 to 18 mg/L with a detection limit of approximately 0.1 mg/L. The CV for multiple samples was < or = 6.7%. In patients receiving swainsonine (50-550 micrograms/kg per day) continuously for 5 days by intravenous infusion, serum concentrations of the drug reached 3-11.8 mg/L, 100 to 400 times greater than the 50% inhibitory concentration for Golgi alpha-mannosidase II and lysosomal alpha-mannosidases. Accurate measurements of swainsonine in biological fluids with this method should facilitate further clinical studies with the drug.