Catherine Gerard

Cloning, sequencing and expression of the human thyrotropin (TSH) receptor: evidence for binding of autoantibodies.

A human thyroid cDNA library was screened by hybridization with a dog thyrotropin receptor (TSHr) cDNA. Sequencing of the resulting clones identified a 2292 residue open reading frame encoding a 744 amino acid mature polypeptide presenting 90.3% similarity with the dog TSHr. Two major transcripts (4.6 and 4.4 kilobases) were identified in the human thyroid which suggests that alternative splicing could generate multiple forms of human TSHr. Transfection of the coding sequence in COS-7 cells conferred to a membrane preparation of these cells the ability to bind specifically TSH. TSH binding was completely displaced by immunoglobulin preparations from patients with idiopathic myxoedema.

Combining lapatinib (GW572016), a small molecule inhibitor of ErbB1 and ErbB2 tyrosine kinases, with therapeutic anti-ErbB2 antibodies enhances apoptosis of ErbB2-overexpressing breast cancer cells.

Antibodies and small molecule tyrosine kinase inhibitors targeting ErbB2 exhibit distinct, noncross resistant mechanisms of action. Here, apoptosis of ErbB2-overexpressing breast cancer cells was enhanced by combining lapatinib, an inhibitor of ErbB1 and ErbB2 tyrosine kinases, with anti-ErbB2 antibodies, including (i) trastuzumab, a humanized monoclonal antibody, and (ii) pAb, rabbit polyclonal antisera generated by vaccination with a human ErbB2 fusion protein. Treating ErbB2-overexpressing breast cancer cell lines with a relatively low concentration of lapatinib alone resulted in a minimal increase in tumor cell apoptosis with an associated decrease in steady-state protein levels of p-ErbB2, p-Akt, p-Erk1/2, and notably survivin, compared to baseline. Exposure to pAb alone reduced total ErbB2 protein, disrupting ErbB3 transactivation, leading to a marked inhibition of p-Akt; however, survivin protein levels remained unchanged and apoptosis only increased slightly. Treatment with trastuzumab alone had relatively little effect on survivin and apoptosis was unaffected. Combining lapatinib with either pAb or trastuzumab markedly downregulated survivin protein and enhanced tumor cell apoptosis. The association between the inhibition of survivin and enhanced apoptosis following the combination of ErbB2-targeted therapies provides a biological effect in order to identify therapeutic strategies that promote tumor cell apoptosis and might improve clinical response.

Stable expression of the human TSH receptor in CHO cells and characterization of differentially expressing clones

The human thyrotropin receptor cDNA was transfected in CHO cells and individual clones were isolated. They were tested for their response to thyrotropin, forskolin and antibodies from a patient with high levels of thyroid stimulating antibodies. Several clones were characterized extensively with respect to membrane binding of labeled thyrotropin, cAMP accumulation in response to thyrotropin and kinetics of cAMP production. Data for three representative clones are presented. Receptor number as assessed by membrane binding of labeled thyrotropin, and cAMP production, measured in a thyrotropin response bioassay, are correlated. The Kd value for the human thyrotropin receptor expressed in CHO was estimated to be 50 pM.

Modulation of the release of cytokines and reduction of the shock syndrome induced by anti-CD3 monoclonal antibody in mice by interleukin-10.

Since IL-10 was recently shown to inhibit several T cell functions in vitro, we investigated the effects of IL-10 on the cytokine release syndrome induced in mice by the 145–2C11 anti-CD3 mAb. As OKT3 in man, this mAb induces a massive polyclonal T cell activation before to induce immunosuppression. First, we found that administration of 1000 U of recombinant mouse IL-10 (mIL-10) 30 min before injection of 10 μg of the 145–2C11 antimouse CD3 mAb markedly reduced the systemic release of IFN-γ and TNF. In contrast, IL-10 pretreatment did not significantly modify the release of IL-6. To determine the effect of IL-10 pretreatment on the endogenous secretion of IL-10 induced by the 145–2C11 mAb, mice were injected with human IL-10 (hIL-10) which does not cross-react in the ELISA for mIL-10 determination. While hIL-10 was as efficient as mIL-10 in reducing TNF and IFN-γ release, it did not modify peak serum levels of IL-10. The modulation of cytokine production by mIL-10 was associated with a significant reduction of the toxicity of the 145–2C11 mAb, as assessed by the attenuation of hypothermia and by the reduced lethality in D-galac-tosamine-sensitized mice. We conclude that IL-10 differentially regulates the in vivo production of cytokines and decreases the systemic toxicity induced by the 145–2C11 mAb. These observations suggest potential therapeutic applications of IL-10 in organ transplantation, especially in association with anti-CD3 mAb.

Characterisation of monoclonal antibodies against human interleukin-10 and their use in an ELISA for the measurement of this cytokine

Biological and biochemical characteristics of monoclonal antibodies (MABs) raised against human interleukin-10 (IL-10) are described as well as their use in the design of a specific ELISA for the measurement of the cytokine. 21 murine anti-human interleukin-10 (IL-10) MABs were obtained by fusion of splenocytes from mice immunized against human recombinant IL-10 with SP2/0 myelomatous cells. These antibodies define three major antigenic areas on the IL-10 molecule, one of which comprises epitopes involved in receptor binding and induction of biological activity. They recognize recombinant human IL-10 with affinities ranging from 1.3 x 10(-7) to 3 x 10(-11), as well as natural IL-10. Most of them also recognize viral IL-10 (vIL-10) encoded by the Epstein-Barr virus (EBV). A specific human-IL-10 ELISA has been developed using two MABs (18 and 19) as capture antibody and one MAB (17) as detector. The sensitivity (3 pg/ml), precision (intra-assays < 4%), reproducibility (interassay < 3%), and accuracy (recoveries, ranging between 84 and 107%, in several fluids) of the assay, plus its excellent performance in dilution tests, and the lack of interference when in the presence of possible cross-reactive substances, permits accurate cytokine measurement in biological fluids such as serum, plasma, bronchoalveolar lavage, urine and culture supernatants. Using the assay, IL-10 was measurable in the plasma of patients with septic shock (range 11-2740 pg/ml) whereas IL-10 plasma levels were < 7.8 pg/ml in healthy volunteers.

Spontaneous and cycloheximide-induced interleukin-10 mRNA expression in human mononuclear cells

Interleukin-10 (IL-10) is known to be spontaneously produced by human peripheral blood mononuclear cells. In order to define the cell type in which IL-10 gene is spontaneously expressed we used the reverse polymerase chain reaction for IL-10 mRNA expression, which was also used to study the effects of cycloheximide (CHX). First, we found that IL-10 mRNA is spontaneously expressed in monocytes and B cells but not T cells from healthy donors, and second, we demonstrated that CHX superinduces IL-10 mRNA in monocytes and B cells. Experiments including nuclear run-on analyses established that the effects of CHX on IL-10 gene expression involve both gene transcription and mRNA stabilization.

Collaboration and Performance: Perspectives From Public Managers and NGO Leaders

ABSTRACT: This article reports on exploratory, mixed-method research using three different datasets to provide a qualitative comparison describing how U.S. local public managers, U.S. federal public managers, and U.S.-based transnational NGO leaders understand the links between collaboration and performance. We augment a growing literature within the field of public administration, which has rarely undertaken comparative cross-sectoral examinations of collaboration and performance, and has largely neglected the perspectives of transnational NGO leaders. Insights are compiled from multiple waves of surveys and interviews with leaders from the U.S. federal Senior Executive Service (n = 305), the International City/County Management Association (n = 1,417), and U.S.-based transnational NGOs (n = 152). Mixed-method analysis reveals that for all three groups of respondents, the perceived positive link between collaboration and performance is the main catalyst for engaging in collaboration as a management strategy. The results also identify specific concerns about the cost of collaboration in terms of power, time, conflict, stress, process, suboptimal outcomes, and resources.

Differential regulation of thyrotropin receptor and thyroglobulin mRNA accumulation at the cellular level: An in situ hybridization study☆

Regulation of TSH receptor (TSHr) mRNA accumulation has been investigated in canine thyrocytes in primary culture by in situ hybridization experiments; the effects of the mitogenic thyrotropin (TSH), epidermal growth factor (EGF), and phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) have been compared. Apart from their mitogenic action, TSH enhances, while EGF and phorbol ester inhibit, the expression of differentiation. The TSHr gene was transcribed in almost all the cells cultured in control conditions (serum free medium supplemented with insulin). Addition of TSH slightly upregulated (twofold) the expression (mRNA) of the TSHr gene. This positive effect was maintained for 20 and 44 h of treatment. EGF and TPA reduced transiently the TSHr mRNA accumulation but did not suppress it. In these different conditions, the TSHr mRNA was homogeneously distributed within the cell population. This contrasted strongly with the effects of TSH, EGF, and TPA on the expression of the thyroglobulin gene, a prominent marker of thyroid cell differentiation: in this case, the regulation was much tighter (high range of stimulation by TSH, strong inhibition by EGF, and suppression of Tg gene expression by TPA) and displayed a great variability of the level of individual cellular response. The fact that the TSHr gene was little modulated and remained expressed regardless of the treatment may reflect the physiological role of the receptor which is the main connection of the thyrocyte to the regulation network.

The contributions of leadership to the movement from violence to incorporation

1. Introduction Bruce Dayton and Louis Kriesberg 2. Protagonist Strategies that Help End Violence Louis Kriesberg and Gearoid Millar 3. The Contributions of Leadership to the Movement from Violence to Incorporation Margaret Hermann and Catherine Gerard 4. Challenges to Conflict Transformation from the Streets Elham Atashi 5. Useful but Insufficient: Third Party Intermediaries in Peacebuilding Bruce W. Dayton 6. Rhetorical Arts of Praise and Blame in Political Transformation Bradford Vivian 7. From Violence to Political Engagement: Ending Violence, Expanding Political incorporation Terrence Lyons 8. Insecurity and Opportunity in Conflict Settings Gavan Duffy 9. Globalization and the Transformation of Conflict Galia Golan and Adir Gal 10. Mozambique - Renamo Andrea Bartoli, Aldo Civico and Leone Gianturco 11. Revolution deferred: from armed struggle to liberal democracy: The African National Congress in South Africa Tom Lodge 12. The Nepali Maoists: Successful transformation or compliance with a strategic plan? Thania Paffenholz 13. Opportunity Lost: The Guatemalan National Revolutionary Unit (URNG) Michael Allison 14. Mainstreaming the Revolutionaries: National Liberating Action and the Shift from Resistance to Democracy in Brazil, 1964-Present Kenneth P. Serbin 15. Factors Influencing the Level of Violence in the Basque Country Juan Gutierrez 16. The Palestine Liberation Organization and the Oslo Process: Incorporation without accommodation Nigel Parsons 17. Domesticating Tigers: The LTTE and Peacemaking in Sri Lanka Camilla Orjuela

A Comprehensive Preclinical Model Evaluating the Recombinant PRAME Antigen Combined With the AS15 Immunostimulant to Fight Against PRAME-expressing Tumors

The PRAME tumor antigen is a potential target for immunotherapy. We assessed the immunogenicity, the antitumor activity, and the safety and the tolerability of a recombinant PRAME protein (recPRAME) combined with the AS15 immunostimulant (recPRAME+AS15) in preclinical studies in mice and Cynomolgus monkeys. Four groups of 12 CB6F1 mice received 4 injections of phosphate-buffered saline (PBS), recPRAME, AS15, or recPRAME+AS15. Immunized mice were injected with tumor cells expressing PRAME (CT26-PRAME) 2 weeks or 2 months after the last injection. The mean tumor surface was measured twice a week. Two groups of 10 monkeys received 7 injections of saline or recPRAME+AS15. T-cell responses were measured by flow cytometry using intracellular cytokine staining (ICS). In CB6F1 mice, repeated injections of recPRAME+AS15 induced high PRAME-specific antibody titers and mostly CD4+ T cells producing cytokines. This immune response was long-lasting in these animals and was associated with protection against a challenge with PRAME-expressing tumor cells (CT26-PRAME) applied either 2 weeks or 2 months after the last injection; these data indicate the induction of an immune memory. In HLA-A02.01/HLA-DR1 transgenic mice, recPRAME+AS15 induced both CD4+ and CD8+ T-cell responses, indicating that this antigen can be processed by the human leukocyte antigen and is potentially immunogenic in humans. In addition, a repeated-dose toxicity study in monkeys showed that 7 biweekly injections of recPRAME+AS15 were well tolerated, and induced PRAME-specific antibodies and T cells. In conclusion, these preclinical data indicate that repeated injections of the PRAME cancer immunotherapeutic are immunogenic and have an acceptable safety profile.