Targeted mutagenesis of duplicated genes in soybean with zinc-finger nucleases

We performed targeted mutagenesis of a transgene and nine endogenous soybean (Glycine max) genes using zinc-finger nucleases (ZFNs). A suite of ZFNs were engineered by the recently described context-dependent assembly platform--a rapid, open-source method for generating zinc-finger arrays. Specific ZFNs targeting dicer-like (DCL) genes and other genes involved in RNA silencing were cloned into a vector under an estrogen-inducible promoter. A hairy-root transformation system was employed to investigate the efficiency of ZFN mutagenesis at each target locus. Transgenic roots exhibited somatic mutations localized at the ZFN target sites for seven out of nine targeted genes. We next introduced a ZFN into soybean via whole-plant transformation and generated independent mutations in the paralogous genes DCL4a and DCL4b. The dcl4b mutation showed efficient heritable transmission of the ZFN-induced mutation in the subsequent generation. These findings indicate that ZFN-based mutagenesis provides an efficient method for making mutations in duplicate genes that are otherwise difficult to study due to redundancy. We also developed a publicly accessible Web-based tool to identify sites suitable for engineering context-dependent assembly ZFNs in the soybean genome.     

Therapeutic targeting of innate immunity with Toll-like receptor agonists and antagonists

The identification of the antigen recognition receptors for innate immunity, most notably the Toll-like receptors, has sparked great interest in therapeutic manipulation of the innate immune system. Toll-like receptor agonists are being developed for the treatment of cancer, allergies and viral infections, and as adjuvants for potent new vaccines to prevent or treat cancer and infectious diseases. As recognition grows of the role of inappropriate Toll-like receptor stimulation in inflammation and autoimmunity, significant efforts have begun to develop antagonists to Toll-like receptors as well.     

Protein turnover in adipose tissue from fasted or diabetic rats

Protein synthesis and degradation in vitro were compared in epididymal fat pads from animals deprived of food for 48 h or treated 6 or 12 days prior with streptozotocin to induce diabetes. Although both fasting and diabetes led to depressed (-24% to -57%) protein synthesis, the diminution in protein degradation (-63% to -72%) was even greater, so that net in vitro protein balance improved dramatically. Insulin failed to inhibit protein degradation in fat pads of these rats as it does for fed animals. Although insulin stimulated protein synthesis in fat pads of fasted and 12 day diabetic rats, the absolute change was much smaller than that seen in the fed state. The inhibition of protein degradation by leucine also seems to be less in fasted animals, probably because leucine catabolism is slower in fasting. These results show that fasting and diabetes may improve protein balance in adipose tissue but diminish the regulatory effects of insulin.     

Activity of a partially purified human BCGF on murine assays for B-cell stimulatory factors. I. BCGF II-like activity of human BCGF

To further characterize a human B-cell growth factor (BCGF) produced by phytohemagglutinin (PHA) P-stimulated peripheral blood T cells, a partially purified preparation of this material was tested in a number of murine assays for B-cell stimulatory factors (BSF). Human BCGF lacked murine BSF-1 activity as assessed via the induction of polyclonal proliferation of anti-IgM-stimulated murine B cells; however, this material consistently augmented the proliferative response of murine B cells to anti-IgM and a saturating dose of murine BSF-1. Human BCGF also induced proliferation in unstimulated murine B cells, and augmented the proliferative response of dextran sulfate activated murine B cells. Human BCGF is therefore capable of causing proliferation of unstimulated and activated murine B cells, and by these criteria closely resembles murine BCGF II. In contrast to murine BCGF II, however, human BCGF failed to stimulate proliferation or immunoglobulin (Ig) secretion by murine BCL1 B lymphoma cells. A murine analog of this human BCGF showing the same pattern of biological responses was found in concanavalin A-stimulated supernatants of the murine MB2.1 T-cell line and D9-Cl T-cell hybridoma. The active component of the human BCGF preparation was not due to contaminating PHA, interleukin 1, interleukin 2; interferon-gamma, or endotoxin. Comparison between the above human BCGF and a commonly used source of murine BCGF II, i.e., supernatant from antigen-stimulated D10.G4.1 T cells, provided information suggestive of BCGF II heterogeneity. Both human BCGF and D10.G4.1 supernatant caused proliferation of unstimulated and dextran sulfate-stimulated murine B cells; however, only the human BCGF preparation augmented the proliferative response of murine B cells to anti-IgM and a saturating dose of murine BSF-1, and only the D10.G4.1 supernatant stimulated BCL1 cell proliferation and immunoglobulin secretion. The data therefore indicate that the different assays for BCGF II used in this study respond to different factors, and suggest the existence of two BCGF II-like activities.     

Inhibition of basal and tumor necrosis factor-enhanced binding of murine tumor cells to murine endothelium by transforming growth factor-beta 1.

The adherence of cells to microvascular endothelium is important in a number of processes, including inflammatory responses and metastasis. It has been demonstrated that in human models, cytokines such as TNF, IL-1, IFN-gamma increase the adhesiveness of endothelium for cells of the immune and inflammatory system by stimulating the expression of cell adhesion molecules on endothelial cell surfaces. We and others have shown similar cytokine-induced endothelial adhesiveness for tumor cells in murine and human models. In contrast to the effect of those modulators, transforming growth factor-beta (TGF-beta) has been shown to inhibit the binding of human neutrophils and T lymphocytes to human endothelium, although the mechanism of TGF-beta action remains unknown. Little is known about the effect of TGF-beta on tumor cell-endothelial interaction. In the present study, we demonstrate that TGF-beta inhibits basal and TNF-enhanced binding of murine P815 mastocytoma cells to murine microvascular endothelium (MME). The alterations in MME mediated by TGF-beta, also lead to the inhibition of adherence of murine splenocytes, thymocytes, and human lymphoblastoid cells but do not inhibit adherence of murine B16 melanoma cells. The effect of TGF-beta is transient and inhibition of the endothelial adhesive phenotype is strongest 12 to 24 h after addition of the factor to MME. The TGF-beta-mediated inhibition of P815 basal binding to endothelium is dependent on protein synthesis because cycloheximide reverses the TGF-beta effect. TGF-beta does not appear to activate classical signal transduction pathways. Inhibitors of G proteins do not abolish TGF-beta action, protein kinase C and protein kinase A activators elicit an effect opposite to that of the factor, TGF-beta does not increase intracellular cAMP levels, and finally calcium-mobilizing agents do not mimic, but rather inhibit the effect of TGF-beta. However, TGF-beta-mediated inhibition of both basal binding and TNF-enhanced P815 binding to MME is completely abolished in the presence of the protein phosphatase inhibitor okadaic acid which suggests that TGF-beta may elicit its effect by stimulating protein phosphatase activity.     

Analysis of Th1 and Th2 cells in murine gut-associated tissues. Frequencies of CD4+ and CD8+ T cells that secrete IFN-gamma and IL-5

After Ag and/or mitogen stimulation, cloned mouse Th1 and Th2 cells produce different cytokines that contribute to induction of particular B cell isotype responses. In this regard, IL-5 produced by Th2 cells has been shown to enhance IgA synthesis in LPS-triggered splenic (SP) B cell or in unstimulated Peyer's patch (PP) B cell cultures. This raises the possibility that Th2 cells may occur in higher frequency in gut-associated tissues, because B cells in these areas are committed to IgA synthesis. We have used an ELISPOT assay to detect individual T cells producing IFN-gamma or IL-5. For the IL-5 assay, the mAb TRFK-5 and biotinylated TRFK-4 were used in coating and detection, respectively, whereas the mAb R4-6A2 and biotinylated XMG 1.2 were similarly used for enumeration of IFN-gamma-specific spot forming cells (SFC). Specificity of each assay was tested by using Con A-activated, cloned Th1 (H66-61) or Th2 (CDC-25) cells, where the Th1 cells only produced IFN-gamma SFC and the Th2 cells only gave IL-5-specific spots. Further, preincubation of biotinylated TRFK-4 or XMG 1.2 with rIL-5 or IFN-gamma, respectively, abrogated the formation of specific spots when tested with Con A-activated SP CD4+ T cells. Both IFN-gamma and IL-5 were produced de novo, because treatment of T cells with cycloheximide inhibited both IFN-gamma and IL-5 SFC. We have assessed the numbers of T cells spontaneously secreting these cytokines in PP and in lamina propria and intraepithelial lymphocyte (LPL and IEL) populations. Moderate levels of IL-5 SFC occurred in the IEL subset, whereas higher levels existed in the LPL population. Although significant numbers of IFN-gamma SFC (Th1-type) were also seen in LPLs, the frequency of IL-5 SFC was always higher (Th1:Th2 in LPL = 1:3). In IELs, equal numbers of IFN-gamma and IL-5 SFC were seen. Interestingly, CD8+ IEL T cells produced these two cytokines. In contrast, T cells freshly isolated from PP, an IgA inductive site, contained smaller numbers of IL-5- or IFN-gamma-secreting cells and SP T cells had essentially no SFC. When PP or SP T cells were stimulated with Con A, significant and approximately equal numbers of IFN-gamma- and IL-5-producing cells appeared.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ablation of eosinophil and IgE responses with anti-IL-5 or anti-IL-4 antibodies fails to affect immunity against Schistosoma mansoni in the mouse

To investigate the role of anaphylactic immune responses in protective immunity against schistosomiasis, mice vaccinated with irradiated cercariae of Schistosoma mansoni were treated with neutralizing mAb antibodies against either IL-5 or IL-4 before and during challenge infection. Anti-IL-5-treated vaccinated mice showed a complete ablation of circulating as well as tissue eosinophils present in inflammatory reactions to migrating schistosomula in the skin and lungs but nevertheless eliminated challenge infections as effectively as vaccinated animals treated with a control mAb. Similarly, treatment of vaccinated mice with an anti-IL-4 mAb markedly reduced serum IgE although failing to diminish immunity. The effect of anti-IL-5 mediated eosinophil depletion was also assessed in a second model in which resistance is induced by concomitant chronic infection. Again, normal, unaltered protection was observed in the absence of circulating and tissue eosinophils. In contrast to the above findings, treatment with anti-IFN-gamma was found to cause a partial depletion of immunity in vaccinated mice whereas, paradoxically, increasing the numbers of inflammatory reactions against invading schistosomula in the lungs. These observations argue against a requirement for either eosinophils or IgE in the anti-schistosome immunity induced by vaccination with irradiated cercariae or for eosinophils in the resistance resulting from previous infection in mice and support previous data suggesting a role for an IFN-gamma dependent cell-mediated effector mechanism in vaccine-induced resistance.     

Thromboxane receptor blockade improves cyclosporine nephrotoxicity in rats

Cyclosporine A (CyA) nephrotoxicity is associated with impaired renal hemodynamic function and increased production of the vasoconstrictor eicosanoid thromboxane A2 (TxA2). In CyA toxic rats, renal dysfunction can be partially reversed by inhibitors of thromboxane synthase. However, interpretation of these results is complicated since inhibition of thromboxane synthase may cause accumulation of prostaglandin endoperoxides that can act as partial agonists at the TxA2 receptor and may blunt the efficacy of treatment. Furthermore, these endoperoxides may be used as substrate for production of vasodilator prostaglandins causing beneficial effects on hemodynamics which are independent of thromboxane inhibition. To more specifically examine the role of TxA2 in CyA toxicity, we investigated the effects of the thromboxane receptor antagonist GR32191 on renal hemodynamics in a rat model of CyA nephrotoxicity. In this model, administration of CyA resulted in a significant decrease in glomerular filtration rate (GFR) (2.85 +/- 0.26 [CyA] vs 6.82 +/- 0.96 ml/min/kg [vehicle]; p less than 0.0005) and renal blood flow (RBF) (21.65 +/- 2.31 [CyA] vs 31.87 +/- 3.60 ml/min/kg [vehicle]; p less than 0.025). Renal vascular resistance (RVR) was significantly higher in rats given CyA compared to animals treated with CyA vehicle (5.32 +/- 0.55 [CyA] vs. 3.54 +/- 0.24 mm Hg/min/ml/kg [vehicle]; p less than 0.05). These renal hemodynamic alterations were associated with a significant increase in urinary excretion of unmetabolized, "native" thromboxane B2 (TxB2) (103 +/- 18 [CyA] vs 60 +/- 16 pg/hour [vehicle]; p less than 0.05). Only minimal histomorphologic changes were apparent by light microscopic examination of kidneys from both CyA and vehicle treated animals. However, with immunoperoxidase staining, a significantly greater number of cells expressing the rat common leukocyte antigen was found in the renal interstitium of rats given CyA. There was no detectable increase in monocytes/macrophages in the kidneys of CyA toxic animals. In rats treated with CyA, intraarterial infusion of GR32191 at maximally tolerated doses significantly increased GFR and RBF, and decreased RVR. Although both RBF and RVR were restored to levels not different from controls, GFR remained significantly reduced following administration of GR32191. These data suggest that the potent vasoconstrictor TxA2 plays an important role in mediating renal dysfunction in CyA nephrotoxicity. However, other factors may be important in producing nephrotoxicity associated with CyA.     

IL-4 induces a Th2 response in Leishmania major-infected mice.

The infection of mice with Leishmania major can cause either a fatal disseminated disease or a localized healing disease, depending on the genetic background of the mice. A strong correlation has been shown between disease outcome and the nature of the T cell response, with healer strains developing a Th1-like response and nonhealer strains a Th2-like response. The treatment of nonhealer BALB/c mice with a single dose of an anti-IL-4 antibody, given at the time of infection with L. major, allowed these mice to develop healing Th1-like responses, suggesting that IL-4 is required in BALB/c mice for the differentiation of Th cells into Th2 cells. Anti-IL-4 had to be present during the first 2 wk of infection to have this effect. Anti-IL-4 caused a marked shift from a Th2 to a Th1 pattern of cytokine expression within 4 days, in vivo, and injections of IL-4 had the opposite effect on the early response in healer C3H/HeN mice. These findings demonstrate that IL-4 can induce the development of Th2 response to L. major infection in vivo.