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L'étude s'est déroulée sur une portion de 25 Km de rivière de première catégorie du Pas-de-Calais, le long de laquelle se trouvent 4 salmonicultures contaminées par les virus SHV et/ou NHI. Des pêches électriques ont été effectuées en juin, septembre et octobre 1991 au niveau de 4 sites choisis en amont et en aval des salmonicultures. Elles ont permis de prélever un échantillonnage représentatif de poissons vivants autres que salmonidés pour une recherche virologique (85 analyses par lot de 3 ou 5 sujets) et de réaliser 96 sérologies SHV et NHI sur des prises de sang de truites capturées en rivière. Une soixantaine de sérologies ont également été faites dans chacune des piscicultures en décembre 1991. Au niveau de la rivière, toutes les virologies et les sérologies sont négatives. Au vu de ces résultats, le niveau de contamination et de portage de rhabdovirus dans le milieu naturel en relation avec des salmonicultures infectées semble relativement bas, ce qui n'exclut pas la possibilité de transmission par le biais des poissons sauvages.

We have reported previously that CD33 hi myeloid-derived suppressor cells (MDSCs) play a direct role in the pathogenesis of myelodysplastic syndromes (MDSs) and that their sustained activation contributes to hematopoietic and immune impairment, including modulation of PD1/PDL1. MDSCs can also limit the clinical activity of immune checkpoint inhibition in solid malignancies. We hypothesized that depletion of MDSCs may ameliorate resistance to checkpoint inhibitors and, hence, targeted them with AMV564 combined with anti-PD1 in MDS bone marrow (BM) mononuclear cells (MNCs) enhanced activation of cytotoxic T cells. AMV564 was active in vivo in a leukemia xenograft model when co-administered with healthy donor peripheral blood MNCs (PBMCs). Our findings provide a strong rationale for clinical investigation of AMV564 as a single agent or in combination with an anti-PD1 antibody and in particular for treatment of cancers resistant to checkpoint inhibitors., Competing Interests: Declaration of interests S.E. and V.S. are employees of Amphivena Therapeutics, Inc. S.W. and A.L. are scientific/medical advisors for Amphivena Therapeutics, Inc. The S.W. and A.F.L. laboratories received unrelated support from Celgene and Genentech. A.F.L. has provided consulting services and has received research funding support from multiple other pharmaceutical and biotechnology companies that pursue cancer research, including Celgene, Precision BioSciences, Prelude Therapeutics, and CTI Biopharma, and serves on the scientific advisory boards of Aileron and CBMG. A.F.L. also has potential royalty income from IP he developed related to cancer treatment and is a member of the MDS Foundation board of directors. S.W. has received unrelated support from Blackbird BioFinance, LLC. J.A.F. is a consultant for and owns Amphivena stocks and is currently employed by Sunesis Pharmaceuticals., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: Randomized studies with gemtuzumab ozogamicin have validated CD33 as a target for antigen-specific immunotherapy of acute myelogenous leukemia (AML). Here, we investigated the potential of CD33/CD3-directed tandem diabodies (TandAbs) as novel treatment approach for AML. These tetravalent bispecific antibodies provide two binding sites for each antigen to maintain the avidity of a bivalent antibody and have a molecular weight exceeding the renal clearance threshold, thus offering a longer half-life compared to smaller antibody constructs., Experimental Design: We constructed a series of TandAbs composed of anti-CD33 and anti-CD3 variable domains of diverse binding affinities and profiled their functional properties in CD33 + human leukemia cell lines, xenograft models, and AML patient samples., Results: Our studies demonstrated that several CD33/CD3 TandAbs could induce potent, dose-dependent cytolysis of CD33 + AML cell lines. This effect was modulated by the effector-to-target cell ratio and strictly required the presence of T cells. Activation and proliferation of T cells and maximal AML cell cytolysis correlated with high avidity to both CD33 and CD3. High-avidity TandAbs were broadly active in primary specimens from patients with newly diagnosed or relapsed/refractory AML in vitro, with cytotoxic properties independent of CD33 receptor density and cytogenetic risk. Tumor growth delay and inhibition were observed in both prophylactic and established HL-60 xenograft models in immunodeficient mice., Conclusions: Our data show high efficacy of CD33/CD3 TandAbs in various preclinical models of human AML. Together, these findings support further study of CD33/CD3 TandAbs as novel immunotherapeutics for patients with AML. Clin Cancer Res; 22(23); 5829-38. ©2016 AACR., (©2016 American Association for Cancer Research.)

Lymphoid organs exhibit complex structures tightly related to their function. Surprisingly, although the thymic medulla constitutes a specialized microenvironment dedicated to the induction of T cell tolerance, its three-dimensional topology remains largely elusive because it has been studied mainly in two dimensions using thymic sections. To overcome this limitation, we have developed an automated method for full organ reconstruction in three dimensions, allowing visualization of intact mouse lymphoid organs from a collection of immunolabeled slices. We validated full organ reconstruction in three dimensions by reconstructing the well-characterized structure of skin-draining lymph nodes, before revisiting the complex and poorly described corticomedullary organization of the thymus. Wild-type thymi contain ~200 small medullae that are connected to or separated from a major medullary compartment. In contrast, thymi of immunodeficient Rag2(-/-) mice exhibit only ~20 small, unconnected medullary islets. Upon total body irradiation, medullary complexity was partially reduced and then recovered upon bone marrow transplantation. This intricate topology presents fractal properties, resulting in a considerable corticomedullary area. This feature ensures short distances between cortex and medulla, hence efficient thymocyte migration, as assessed by mathematical models. Remarkably, this junction is enriched, particularly in neonates, in medullary thymic epithelial cells expressing the autoimmune regulator. The emergence of a major medullary compartment is induced by CD4(+) thymocytes via CD80/86 and lymphotoxin-α signals. This comprehensive three-dimensional view of the medulla emphasizes a complex topology favoring efficient interactions between developing T cells and autoimmune regulator-positive medullary thymic epithelial cells, a key process for central tolerance induction.

The thymic medulla is dedicated for purging the T-cell receptor (TCR) repertoire of self-reactive specificities. Medullary thymic epithelial cells (mTECs) play a pivotal role in this process because they express numerous peripheral tissue-restricted self-antigens. Although it is well known that medulla formation depends on the development of single-positive (SP) thymocytes, the mechanisms underlying this requirement are incompletely understood. We demonstrate here that conventional SP CD4⁺ thymocytes bearing autoreactive TCRs drive a homeostatic process that fine-tunes medullary plasticity in adult mice by governing the expansion and patterning of the medulla. This process exhibits strict dependence on TCR-reactivity with self-antigens expressed by mTECs, as well as engagement of the CD28-CD80/CD86 costimulatory axis. These interactions induce the expression of lymphotoxin α in autoreactive CD4⁺ thymocytes and RANK in mTECs. Lymphotoxin in turn drives mTEC development in synergy with RANKL and CD40L. Our results show that Ag-dependent interactions between autoreactive CD4⁺ thymocytes and mTECs fine-tune homeostasis of the medulla by completing the signaling axes implicated in mTEC expansion and medullary organization.

Molecular features of ligand binding to MHC class II HLA-DR molecules have been elucidated through a combination of peptide structure-activity studies and structure-based drug design, resulting in analogues with nanomolar affinity in binding assays. Stabilization of lead compounds against cathepsin B cleavage by N-methylation of noncritical backbone NH groups or by dipeptide mimetic substitutions has generated analogues that compete effectively against protein antigens in cellular assays, resulting in inhibition of T-cell proliferation. Crystal structures of four ternary complexes of different peptide mimetics with the rheumatoid arthritis-linked MHC DRB10401 and the bacterial superantigen SEB have been obtained. Peptide-sugar hybrids have also been identified using a structure-based design approach in which the sugar residue replaces a dipeptide. These studies illustrate the complementary roles played by phage display library methods, peptide analogue SAR, peptide mimetics substitutions, and structure-based drug design in the discovery of inhibitors of antigen presentation by MHC class II HLA-DR molecules.

We have identified a heptapeptide with high affinity to rheumatoid arthritis-associated class II major histocompatibility (MHC) molecules. Using a model of its interaction with the class II binding site, a variety of mimetic substitutions were introduced into the peptide. Several unnatural amino acids and dipeptide mimetics were found to be appropriate substituents and could be combined into compounds with binding affinities comparable to that of the original peptide. Compounds were designed that were several hundred-fold to more than a thousand-fold more potent than the original peptide in inhibiting T-cell responses to processed protein antigens presented by the target MHC molecules. Peptidomimetic compounds of this type could find therapeutic use as MHC-selective antagonists of antigen presentation in the treatment of autoimmune diseases.

Mutant mice generated by disrupting the H2-Aab major histocompatibility complex (Mhc) gene are demonstrated here to express Abetab chains in the absence of alpha chains. These mice possess a CD4(+) helper T cell (Th) repertoire that uses predominantly the Vbeta7 T-cell antigen receptor (Tcr) segment for recognition of any protein antigen presented by the alpha-free Abeta molecule. As an alloantigen, the Aalpha-free Abeta molecule is recognized very poorly by T cells from a series of class II disparate mouse strains, indicating that it is grossly different from normal alpha/beta heterodimers. Indeed, molecular modeling suggests a beta/beta homodimer arrangement with an altered geometry of the Tcr contact area. Interestingly, the mutant mice exhibit normal alloreactivity, without a restricted Vbeta usage, toward a series of foreign alpha/beta class II heterodimers, although their T cells developed in the absence of such heterodimers. Thus, the complementarity of Tcr to normal alpha/beta heterodimers, and thereby also alloreactivity, appears to be an ontogeny independent (i. e., germline-encoded) feature.

The prevailing concept about a major influence of thymic positive selection on shaping the T cell repertoire during ontogeny is confronted with an old idea emphasizing a dominant role for genetic (evolutionary) factors in molding the recognition potential of mature T cells. Our recent results are not readily interpreted without introducing a new version of the old concept, according to which complementarity to the major histocompatibility complex peptide-binding site is a major evolutionary selective pressure on T cell antigen receptor variable genes, with alloreactivity being a reflection of this fact.

The electrostatic potential surfaces were characterized for trp repressor models that bind to DNA with sequence specificity, without specificity, and not at all. Comparisons among the surfaces were used to isolate protein surface features likely to be important in DNA binding. Models that differ in protein conformation and tryptophan-analogue binding consistently showed positive potential associated with the protein surfaces that interact with the DNA major groove. However, negative potential is associated with the trp repressor surface that contacts the DNA minor groove. This negative potential is significantly neutralized in the protein conformation that is bound to DNA. Positive potential is also associated with the tryptophan binding-site surface, a consequence of the tryptophan- or tryptophan analogue-induced allosteric change. This protein region is complementary to the strongest negative potential associated with the DNA phosphate backbone and is also present in the isolated protein structure from the protein-DNA complex. The effects of charge-change mutation, pH dependence, and salt dependence on the electrostatic potential surfaces were also examined with regard to their effects on protein-DNA binding constants. A consistent model is formed that defines a role for long-range electrostatics early in the protein-DNA association process and complements previous structural, molecular association, and mutagenesis studies.

Although aqueous simulations with periodic boundary conditions more accurately describe protein dynamics than in vacuo simulations, these are computationally intensive for most proteins. Trp repressor dynamic simulations with a small water shell surrounding the starting model yield protein trajectories that are markedly improved over gas phase, yet computationally efficient. Explicit water in molecular dynamics simulations maintains surface exposure of protein hydrophilic atoms and burial of hydrophobic atoms by opposing the otherwise asymmetric protein-protein forces. This properly orients protein surface side chains, reduces protein fluctuations, and lowers the overall root mean square deviation from the crystal structure. For simulations with crystallographic waters only, a linear or sigmoidal distance-dependent dielectric yields a much better trajectory than does a constant dielectric model. As more water is added to the starting model, the differences between using distance-dependent and constant dielectric models becomes smaller, although the linear distance-dependent dielectric yields an average structure closer to the crystal structure than does a constant dielectric model. Multiplicative constants greater than one, for the linear distance-dependent dielectric simulations, produced trajectories that are progressively worse in describing trp repressor dynamics. Simulations of bovine pancreatic trypsin were used to ensure that the trp repressor results were not protein dependent and to explore the effect of the nonbonded cutoff on the distance-dependent and constant dielectric simulation models. The nonbonded cutoff markedly affected the constant but not distance-dependent dielectric bovine pancreatic trypsin inhibitor simulations. As with trp repressor, the distance-dependent dielectric model with a shell of water surrounding the protein produced a trajectory in better agreement with the crystal structure than a constant dielectric model, and the physical properties of the trajectory average structure, both with and without a nonbonded cutoff, were comparable.

The structures of five basic pancreatic trypsin inhibitor (BPTI) molecules are compared to establish the extent and nature of the conformational variability resulting from crystal packing effects. BPTI is an ideal system to evaluate such factors because of the availability of high resolution X-ray models of five different BPTI structures, each in a different crystal packing environment. Differences observed among the structures are found to be distributed throughout the molecule, although the regions that display most variability are associated with the loop structures (residues 14-17 and 24-29). The regions of structure that show the largest rms deviations from the mean of the five packing motifs correlate well with the presence of intermolecular contacts in the crystal lattice. For most of the molecules there is also a correspondence between a larger number of intermolecular contacts and systematically higher B-factors, although it is not apparent whether this is induced by the crystal contact or results from the fact that the contacts are made predominantly through surface loops. The conformational differences seen among the X-ray models constitute more than local shifts at the lattice contact surfaces, and in fact involve in some cases the making and breaking of intramolecular H-bonds. The magnitudes of the differences among packing models are significantly larger than those usually associated with changes induced by mutagenesis; for instance; the structural differences at the site of mutation observed on removing an internal disulfide from the molecule are significantly less than those associated with lattice contact effects. The crystal packing conformations are compared to representative structures of BPTI generated during a 96-psec molecular dynamics (MD) simulation. This comparison shows a high level of correspondence between the protein flexibility indicated by the X-ray and MD analyses, and specifically between those regions that are most variable. This suggests that the regions that show most variability among the crystal packing models are not artifacts of crystallization, but rather represent true low-energy conformers that have been preferentially selected by crystallization factors.