Dual-specific T cells combine proliferation and antitumor activity

An effective immune response against cancer requires the activation and expansion of specific T cells. Tumor antigens, however, are generally poor immunogens. To achieve expansion of tumor-reactive T cells in vivo, we used a strategy of generating dual-specific T cells that could respond to a powerful immunogen while also possessing tumor reactivity. We generated dual-specific T cells by genetic modification of alloreactive T cells with a chimeric receptor recognizing folate-binding protein, an ovarian cancer-associated antigen. Mouse dual-specific T cells responded in vitro to both allogeneic antigen and tumor cells expressing folate-binding protein, and expanded in number in vivo in response to immunization with allogeneic cells. Most importantly, the combination of dual-specific T cells and immunization had an antitumor effect in vivo. We also generated human dual-specific T cells and characterized the dual-specific nature of individual clones. Assigning the tasks of expansion and tumor reactivity to different receptors within the same lymphocyte may help to overcome the problem of poor immunogenicity of tumor antigens.     

Dose-effect of interleukin-10 and its immunoregulatory role in Crohn's disease

BACKGROUND: Interleukin-10 (IL-10) is currently being extensively studied in clinical trials for the treatment of Crohn's disease (CD). Only marginal effects have, however, been reported, and the dose-response curve was bell-shaped contrasting with the reported data from in vitro experiments. AIM: To use another in vitro model to analyze the effect of rhIL-10 and rhIL-4 on the spontaneous mucosal TNF-alpha secretion in patients with CD, and to characterize the phenotype of the cells targeted by rhIL-10. METHODS: Non-inflamed colon biopsies from CD patients were cultured for 16 hours in presence of different concentrations of rhIL-10 or rhIL-4. The numbers of TNF-alpha-secreting cells among isolated lamina propria mononuclear cells (LPMNC) were estimated by Elispot. RESULTS: Both rhIL-10 and rhIL-4 down-regulate TNF-alpha secretion by LPMNC from CD patients, with a more pronounced effect with rhIL-10. These effects were closely linked to the cytokine concentrations used, with a bell-shaped dose-response curve. Residual TNF-alpha secretion, in the presence of optimal rhIL-10 concentration was mainly attributable to CD3+ T cells. In contrast, at higher rhIL-10 concentrations, CD3- cells contributed significantly to the TNF-alpha secretion. CONCLUSIONS: The in vitro model we used, demonstrates that IL-4, but mostly IL-10, efficiently suppresses TNF-alpha secretion in LPMNC from CD patients, with a dose-response curve similar to results obtained in vivo. Resistance at high rhIL-10 concentrations was associated with a change in the phenotype of TNF-alpha-secreting cells.     

Nonparametric methods for identifying differentially expressed genes in microarray data

MOTIVATION: Gene expression experiments provide a fast and systematic way to identify disease markers relevant to clinical care. In this study, we address the problem of robust identification of differentially expressed genes from microarray data. Differentially expressed genes, or discriminator genes, are genes with significantly different expression in two user-defined groups of microarray experiments. We compare three model-free approaches: (1). nonparametric t-test, (2). Wilcoxon (or Mann-Whitney) rank sum test, and (3). a heuristic method based on high Pearson correlation to a perfectly differentiating gene ('ideal discriminator method'). We systematically assess the performance of each method based on simulated and biological data under varying noise levels and p-value cutoffs. RESULTS: All methods exhibit very low false positive rates and identify a large fraction of the differentially expressed genes in simulated data sets with noise level similar to that of actual data. Overall, the rank sum test appears most conservative, which may be advantageous when the computationally identified genes need to be tested biologically. However, if a more inclusive list of markers is desired, a higher p-value cutoff or the nonparametric t-test may be appropriate. When applied to data from lung tumor and lymphoma data sets, the methods identify biologically relevant differentially expressed genes that allow clear separation of groups in question. Thus the methods described and evaluated here provide a convenient and robust way to identify differentially expressed genes for further biological and clinical analysis.     

Genetic diversity and molecular detection of three toxic dinoflagellate genera (Alexandrium,Dinophysis, and Karenia) from French coasts

The objectives of this study were 1) to study the genetic diversity of the Alexandrium, Dinophysis and Karenia genera along the French coasts in order to design probes targeting specific DNA regions, and 2) to apply PCR-based detection to detect these three toxic dinoflagellate genera in natural samples. Genetic diversity of these toxic taxa was first studied from either cultures or cells isolated from Lugol-fixed field samples. By this way, partial sequences of the large ribosomal subunit (LSU rDNA) including the variable domains D1 and D2 of A. minutum, Alexandrium species inside the tamarensis complex, the D. acuminata complex and K. mikimotoi were obtained. Next, specific primers were designed for a selection of toxic algae and used during semi-nested PCR detection. This method was tested over a 3-month period on water samples from the Bay of Concarneau (Brittany, France) and on sediment from the Antifer harbor (The English Channel, France). Specificity and sensitivity of this molecular detection were evaluated using the occurrence of target taxa reported by the IFREMER (Institut Fran?ais de Recherche pour l'Exploitation de la Mer) monitoring network based on conventional microscopic examination. This work presents the first results obtained on the biogeographical distribution of genotypes of these three toxic genera along the French coasts.     

Synthesis, and functional properties of a modified human insulin A-chain: implication in a 'mini-insulin' structure determination

The design and total synthesis of a novel insulin A-chain mutant, analogue 3, is reported. In this compound, the cysteines implied in the two insulin inter-chain disulfide bridges are replaced by two serines (residues Ser(A7) and Ser(A20)) and the intra-A-chain disulfide bridge (residues Cys(A6) and Cys(A11)) is conserved. This A-chain analogue (3) has been tested in three in vitro cell culture assays, using insulin as a reference. The data clearly showed that analogue 3 mimics insulin effects on DNA synthesis, glucose uptake and glycogen synthesis without loss of potency as compared to insulin. To our knowledge, these are the first results showing that an isolated insulin chain displays functional properties similar to those of insulin. The implication of these new findings in insulin structure-function relationships and in a 'mini-insulin' structure determination is discussed.     

Association of MPF, MAPK, and nuclear progression dynamics during activation of young and aged bovine oocytes

We have previously shown that bovine oocytes parthenogenetically activated after 40 hours (hr) of in vitro maturation proceed through the cell cycle faster than those after 20 hr of maturation. In the present study, we used this model of different speed of nuclear progression to investigate the correlation of two hallmarks of nuclear events, exit of metaphase arrest and pronuclear formation, with dynamics of MPF and MAPK. Bovine oocytes were matured in vitro for 20 hr (young) or 40 hr (aged) and activated in 7% ethanol followed by incubation in cycloheximide for 0, 0.5, 1, 3, 5, or 7 hr. Activity of MPF and MAPK was lower in aged than young oocytes. The responses to oocyte activation by both the two kinases and nuclear progression were faster in aged than in young oocytes. The activity of MPF declined to undetectable levels (P < 0.05) as early as 0.5 hr after activation in aged oocytes, while this did not happen in young oocytes until 3 hr after activation. The inactivation of MAPK occurred approximately 2 hr earlier in aged oocytes (5 hr post-activation) than in young oocytes (7 hr post-activation). Furthermore, the decline in MPF activity preceded that of MAPK in both young and aged oocytes by about 2 hr. The decrease in activity of MPF and MAPK corresponded with the exit from meiosis and pronuclei formation regardless of the speed of nuclear progression. Despite dramatic changes in activity of MPF and MAPK, the levels of Cdc2 and Erk2 proteins were unchanged (P > 0.05) during the first 7 hr of activation. These observations suggest that inactivation of MPF and MAPK are pre-requisite for the release from metaphase arrest and formation of pronuclei in bovine oocytes.     

Noninvasive imaging of peripherally injected Alzheimer's disease type synthetic A beta amyloid in vivo

The pathological hallmark of Alzheimer's disease (AD) is accumulation in the brain of amyloid composed of the 40-mer peptide A beta. Many fundamental questions about the biology of (AD) remain unanswered because there is currently no method of quantifying A beta amyloid in vivo. A noninvasive method of detecting and quantifying A beta amyloid in vivo would have wide application for the premortem diagnosis of AD and the efficient evaluation of candidate therapeutics aimed at inhibiting the formation and growth of A beta amyloid. Taking advantage of the extraordinarily high affinity of A beta for itself, we have synthesized an N'-terminal diethylenetriaminepentaacetic acid (DTPA) derivative of A beta possessing the kinetic activity and specificity for A beta amyloid desired of a probe to be used for noninvasive imaging. DTPA-A beta(3-40) is readily labeled with (111)InOAc(3) to yield a stable probe with exquisite specificity for naturally occurring and synthetic A beta amyloid in vitro. Moreover, (111)In-DTPA-A beta(3-40), administered intravascularly can specifically deposit onto and label previously injected synthetic A beta amyloid and be imaged in vivo with a gamma camera. The present results demonstrate the design, synthesis, and use of an A beta amyloid-specific probe and methods for its use as a noninvasive imaging agent. In vivo imaging of A beta amyloid represents an important step toward the development of biochemically based objective tools for the assessment of progression of AD and efficacy of potential therapeutics.     

mdr1a deficiency corrects sterility in Niemann-Pick C1 protein deficient female mice

Niemann-Pick type C disease is a progressive neurological disease with cholesterol storage in liver, and npc1-/- mice share these features and are sterile. We have searched for the cause of sterility and found normal folliculogenesis and progesterone levels but lack of implantation. Multiple drug resistance (MDR) P-glycoproteins are plasma membrane proteins implicated in the movement of drugs and lipids across membranes. Their functions are inhibited by progesterone, which has been shown to alter cellular cholesterol homeostasis and has implicated P-glycoproteins in the movement of cholesterol to the endoplasmic reticulum. We have introduced the mdr1a knockout into the npc1 mutant line. While the neurological disease continues at its usual rate, preventing the females from taking care of their litters, npc1-/-, mdr1a-/- females became fertile. Although the mdr1a P-glycoprotein co-localizes with caveolae, neither caveolin-1 nor npc1 levels were significantly altered in the livers of double homozygotes. The absence of mdr1a was confirmed by immunoblotting, but npc1 deficiency was not associated with consistent changes in cerebellar mdr1a in mdr1a+/+ mice. The results show that a mdr1a mutation is an in vivo suppressor of female sterility in npc1 deficient mice.     

The Arabidopsis SKU5 gene encodes an extracellular glycosyl phosphatidylinositol-anchored glycoprotein involved in directional root growth

To investigate how roots respond to directional cues, we characterized a T-DNA-tagged Arabidopsis mutant named sku5 in which the roots skewed and looped away from the normal downward direction of growth on inclined agar surfaces. sku5 roots and etiolated hypocotyls were slightly shorter than normal and exhibited a counterclockwise (left-handed) axial rotation bias. The surface-dependent skewing phenotype disappeared when the roots penetrated the agar surface, but the axial rotation defect persisted, revealing that these two directional growth processes are separable. The SKU5 gene belongs to a 19-member gene family designated SKS (SKU5 Similar) that is related structurally to the multiple-copper oxidases ascorbate oxidase and laccase. However, the SKS proteins lack several of the conserved copper binding motifs characteristic of copper oxidases, and no enzymatic function could be assigned to the SKU5 protein. Analysis of plants expressing SKU5 reporter constructs and protein gel blot analysis showed that SKU5 was expressed most strongly in expanding tissues. SKU5 was glycosylated and modified by glycosyl phosphatidylinositol and localized to both the plasma membrane and the cell wall. Our observations suggest that SKU5 affects two directional growth processes, possibly by participating in cell wall expansion.     

Downregulation of a pathogen-responsive tobacco UDP-Glc:phenylpropanoid glucosyltransferase reduces scopoletin glucoside accumulation,enhances oxidative stress,and weakens virus resistance

Plant UDP-Glc:phenylpropanoid glucosyltransferases (UGTs) catalyze the transfer of Glc from UDP-Glc to numerous substrates and regulate the activity of compounds that play important roles in plant defense against pathogens. We previously characterized two tobacco salicylic acid- and pathogen-inducible UGTs (TOGTs) that act very efficiently on the hydroxycoumarin scopoletin and on hydroxycinnamic acids. To identify the physiological roles of these UGTs in plant defense, we generated TOGT-depleted tobacco plants by antisense expression. After inoculation with Tobacco mosaic virus (TMV), TOGT-inhibited plants exhibited a significant decrease in the glucoside form of scopoletin (scopolin) and a decrease in scopoletin UGT activity. Unexpectedly, free scopoletin levels also were reduced in TOGT antisense lines. Scopolin and scopoletin reduction in TOGT-depleted lines resulted in a strong decrease of the blue fluorescence in cells surrounding TMV lesions and was associated with weakened resistance to infection with TMV. Consistent with the proposed role of scopoletin as a reactive oxygen intermediate (ROI) scavenger, TMV also triggered a more sustained ROI accumulation in TOGT-downregulated lines. Our results demonstrate the involvement of TOGT in scopoletin glucosylation in planta and provide evidence of the crucial role of a UGT in plant defense responses. We propose that TOGT-mediated glucosylation is required for scopoletin accumulation in cells surrounding TMV lesions, where this compound could both exert a direct antiviral effect and participate in ROI buffering.