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The parametrization and validation of the OPLS3 force field for small molecules and proteins are reported. Enhancements with respect to the previous version (OPLS2.1) include the addition of off-atom charge sites to represent halogen bonding and aryl nitrogen lone pairs as well as a complete refit of peptide dihedral parameters to better model the native structure of proteins. To adequately cover medicinal chemical space, OPLS3 employs over an order of magnitude more reference data and associated parameter types relative to other commonly used small molecule force fields (e.g., MMFF and OPLS_2005). As a consequence, OPLS3 achieves a high level of accuracy across performance benchmarks that assess small molecule conformational propensities and solvation. The newly fitted peptide dihedrals lead to significant improvements in the representation of secondary structure elements in simulated peptides and native structure stability over a number of proteins. Together, the improvements made to both the small molecule and protein force field lead to a high level of accuracy in predicting protein-ligand binding measured over a wide range of targets and ligands (less than 1 kcal/mol RMS error) representing a 30% improvement over earlier variants of the OPLS force field.

Using an acute promyelocytic leukemia (APL) preclinical model, we show that oncogene-specific PCR (Polymerase Chain Reaction)-based assays allow to evaluate the efficacy of immunotherapy combining all-trans retinoic acid (ATRA) and a DNA-based vaccine targeting the promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα) oncogene. Kaplan-Meier survival analysis according to the peripheral blood PML-RARα normalized copy number (NCN) clearly shows that ATRA + DNA-treated mice with an NCN lower than 10 (43%) formed the group with a highly significant (p < 0.0001) survival advantage. Furthermore, a PCR assay was used to assess various tissues and organs for the presence of PML-RARα-positive cells in long-term survivors (n = 15). As expected, the majority of mice (n = 10) had no measurable tissue level of PML-RARα. However, five mice showed a weak positive signal in both the brain and spleen (n = 2), in the brain only (n = 2) and in the spleen only (n = 1). Thus tracking the oncogene-positive cells in long-term survivors reveals for the first time that extramedullary PML-RARα-positive cell reservoirs such as the brain may persist and be involved in relapses., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Connexins are transmembrane proteins involved in gap junction intercellular communication. They present cell- and tissue-specific expression, with own electric and metabolic coupling specificities. These proteins are involved in numerous physiological processes in the brain and among them neuronal synchronization and trafficking of glucose. Such proteins are also described as being misregulated in various pathologies in the central nervous system. Thus, connexin blockers have been proposed as pharmacological tools to dissect these implications. However, such approaches lack accurate characterization of known inhibitors toward gap junction isoform specificity. In addition, those compounds are limited to few chemical classes and exhibit other activities, for example, an anti-inflammatory effect. The aims of this study were to evaluate the selectivity of described inhibitors and to enrich this pharmacopeia by new chemical classes. In this study, we present the specificity of published inhibitors toward several connexin isoforms expressed in the brain. Furthermore, after a screening of compounds using cellular models, we identified seven new inhibitors, with high functional reversibility and different relative selectivity toward isoforms. They constitute new chemical classes of connexin modulators completing those previously described. These new inhibitors might also provide new insights in understanding numerous pathophysiological processes involving gap junctions.

DNA vaccination and all-trans retinoic acid (ATRA) result in a survival advantage in a mouse model of acute promyelocytic leukemia (APL). Depletion of CD4(+) or CD8(+) cells abolished this effect. CD4(+) depletions of long-term survivors resulted in relapse and death within 3 months, thus demonstrating the need of both CD4(+) and CD8(+) subsets for the generation of DNA-driven antileukemic immune responses and underscoring a crucial role of CD4(+) cells in the maintenance of durable remissions. Degranulation and cytotoxic carboxyfluorescein diacetate succinimidyl ester-based assays showed major histocompatibility complex-restricted APL-specific T cell-mediated immune responses. Sorted APL-specific CD8(+)CD107a(+) T cells showed an increase of antileukemic activity. Effectors from ATRA + DNA-treated mice were shown to secrete interferon-gamma when stimulated with either APL cells or peptides from the promyelocytic leukemia-RARalpha vaccine-derived sequences as detected by ELISpot assays. Our results demonstrate that DNA vaccination with ATRA confers the effective boosting of interferon-gamma-producing and cytotoxic T cells in the leukemic mice.

At present, there is no effective therapy for any of the neurodegenerative amyloidoses, despite renewed efforts to identify compounds active against the various implicated pathogenetic molecules. We have screened a library of 2960 natural and synthetic compounds in two cell lines chronically infected with mouse prions, and have identified eight new inhibitors of prion replication in vitro. They belong to two distinct chemical families that have not previously been recognised as effective in the field of transmissible spongiform encephalopathies: seven are 3-aminosteroids and one is a derivative of erythromycin A with an oxime functionality. Our results suggest that these aminosteroids inhibit prion replication by triggering a common target, possibly implicated in the regulatory pathways of cellular prion protein metabolism. Furthermore, using a quantitative approach for the study of protein stability, it was shown that the erythromycin A derivative altered prion protein stability by direct interaction. Such direct targeting of this amyloid precursor might provide new clues for the understanding of prion diseases and, more importantly, help to define new molecules that are active against prion diseases.

Myelodysplastic syndromes (MDS) are clonal stem cell hematologic disorders that evolve to acute myeloid leukemia (AML) and thus model multistep leukemogenesis. Activating RAS mutations and overexpression of BCL-2 are prognostic features of MDS/AML transformation. Using NRASD12 and BCL-2, we created two distinct models of MDS and AML, where human (h)BCL-2 is conditionally or constitutively expressed. Our novel transplantable in vivo models show that expression of hBCL-2 in a primitive compartment by mouse mammary tumor virus-long terminal repeat results in a disease resembling human MDS, whereas the myeloid MRP8 promoter induces a disease with characteristics of human AML. Expanded leukemic stem cell (Lin(-)/Sca-1(+)/c-Kit(+)) populations and hBCL-2 in the increased RAS-GTP complex within the expanded Sca-1(+) compartment are described in both MDS/AML-like diseases. Furthermore, the oncogenic compartmentalizations provide the proapoptotic versus antiapoptotic mechanisms, by activating extracellular signal-regulated kinase and AKT signaling, in determination of the neoplastic phenotype. When hBCL-2 is switched off with doxycycline in the MDS mice, partial reversal of the phenotype was observed with persistence of bone marrow blasts and tissue infiltration as RAS recruits endogenous mouse (m)BCL-2 to remain active, thus demonstrating the role of the complex in the disease. This represents the first in vivo progression model of MDS/AML dependent on the formation of a BCL-2:RAS-GTP complex. The colocalization of BCL-2 and RAS in the bone marrow of MDS/AML patients offers targeting either oncogene as a therapeutic strategy.

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of disorders characterized by ineffective hematopoiesis, with an increased propensity to develop acute myelogenous leukemia (AML). The molecular basis for MDS progression is unknown, but a key element in MDS disease progression is loss of chromosomal material (genomic instability). Using our two-step mouse model for myeloid leukemic disease progression involving overexpression of human mutant NRAS and BCL2 genes, we show that there is a stepwise increase in the frequency of DNA damage leading to an increased frequency of error-prone repair of double-strand breaks (DSB) by nonhomologous end-joining. There is a concomitant increase in reactive oxygen species (ROS) in these transgenic mice with disease progression. Importantly, RAC1, an essential component of the ROS-producing NADPH oxidase, is downstream of RAS, and we show that ROS production in NRAS/BCL2 mice is in part dependent on RAC1 activity. DNA damage and error-prone repair can be decreased or reversed in vivo by N-acetyl cysteine antioxidant treatment. Our data link gene abnormalities to constitutive DNA damage and increased DSB repair errors in vivo and provide a mechanism for an increase in the error rate of DNA repair with MDS disease progression. These data suggest treatment strategies that target RAS/RAC pathways and ROS production in human MDS/AML.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. In recent years, the outcome has been globally improved by current therapies, but it remains poor in patients with high, persistent residual disease following the first course of chemotherapy, prompting evaluation of the possible beneficial effects of immunotherapy protocols. In this study, we hypothesized that the disruption of two immunoregulatory pathways controlling the auto-reactive T cell response might synergize with dendritic cell-based immunotherapy of the disease, which is considered to be poorly immunogenic. In this study, we used TAL1xLMO1 leukemia cells adoptively transferred in mice, to generate murine leukemia with poorly immunogenic cells as a model for human T-ALL. Subsequently, these animals were treated with several different immunotherapeutic protocols. We compared the efficiency of a classical, dendritic cell-based immunotherapy (injection of dendritic cells loaded with tumor-derived antigenic products), to a combined treatment associating injection of antigen-loaded dendritic cells and disruption of the two immunoregulatory pathways: CD25+ suppressive T cells and cytotoxic T lymphocyte-associated antigens (CTLA-4). We show that this combined treatment resulted in cure, concomitantly with in vivo generation of immune memory, and TNF-alpha secretion. This study demonstrates that the disruption of these two immunoregulatory pathways synergized with immunostimulation by dendritic cells loaded with tumor-derived antigens, and paves the way for the testing of this combination in clinical trials.

Nonclassical major histocompatibility complex (MHC) class I human leukocyte antigen E (HLA-E) and HLA-G molecules differ from classical ones by specific patterns of transcription, protein expression, and immunotolerant functions. The HLA-G molecule can be expressed as four membrane-bound (HLA-G1 to -G4) and three soluble (HLA-G5 to -G7) proteins upon alternative splicing of its primary transcript. In this study, we describe a new set of monoclonal antibodies (mAbs) called MEM-G/01, -G/04, -G/09, -G/13, MEM-E/02, and -E/06 recognizing HLA-G or HLA-E. The pattern of reactivity of these mAbs were analyzed on transfected cells by flow cytometry, Western blotting, and immunochemistry. MEM-G/09 and -G/13 mAbs react exclusively with native HLA-G1 molecules, as the 87G mAb. MEM-G/01 recognizes (similar to the 4H84 mAb) the denatured HLA-G heavy chain of all isoforms, whereas MEM-G/04 recognizes selectively denatured HLA-G1, -G2, and -G5 isoforms. MEM-E/02 and -E/06 mAbs bind the denatured and cell surface HLA-E molecules, respectively. These mAbs were then used to analyze the expression of HLA-G and HLA-E on freshly isolated cytotrophoblast cells, on the JEG-3 placental tumor cell line, and on cryopreserved and paraffin-embedded serial sections of trophoblast tissue. These new mAbs represent valuable tools to study the expression of HLA-G and HLA-E molecules in cells and tissues under normal and pathologic conditions.

Human cytomegalovirus has evolved multiple strategies to interfere with immune recognition by the host. A variety of mechanisms affect antigen presentation by major histocompatibility complex class I molecules resulting in a reduced class I cell-surface expression. This downregulation is expected to trigger natural killer (NK) cytotoxicity, requiring counteraction by the virus to establish long-term infection. Here we describe that the human cytomegalovirus gpUS6 protein, which has been demonstrated to downregulate the expression of human leukocyte antigen (HLA) class I and the presentation of cytotoxic T lymphocyte epitopes by blocking transporter associated with antigen presentation (TAP function), does not affect the ability of HLA-E to inhibit NK cell mediated lysis of K-562 cells by interaction with CD94/NKG2A expressed on NK cells. Cell surface expression and function of HLA-E is not altered although gpUS6 inhibits TAP-dependent peptide transport by 95%. Moreover, HLA-E molecules presenting HLA class I signal sequence-derived peptides are functionally detectable on transfected TAP-deficient RMA-S cells.

Objective: To describe a fast, simple method to acquire pressure-volume curves of the respiratory system and to compare this with a classic method in terms of reliability of the data and speed., Design: Acquisition of pressure-volume curves by low flow inflation technique (P-Vlf) versus the occlusion technique (P-Vst) using the standard equipment of a Cesar ventilator., Setting: General ICU - Aix en Provence Hospital., Patients: Ten sedated, curarized patients undergoing mechanical ventilation., Interventions: P-Vlf curves were acquired by setting the ventilator parameters at f = 5 c./min, duty time Ti/Ttot = 80 %, VT = 1100 ml, pause time = 0. The pressure and volume data were collected directly on the ventilator screen. P-Vst curves were acquired using an airway occlusion technique. The pressures obtained for the same inflation volumes and times necessary for performance of the two techniques were compared., Results: The time needed to acquire a P-Vlf curve was 3 min versus 38 min for P-Vst curve. Concordance analysis between the two methods showed a 95 % confidence interval of (-0.5 cm H2O, + 1.8 cm H2O) for pressure., Conclusions: P-Vlf curves are close to P-Vst curves, are much less time-consuming, easy to acquire with Cesar ventilator equipment, and may be used in clinical routine to assess the elastic properties of the respiratory system.

To evaluate the role of single residues of a MHC class I molecule in the induction of a primary allogeneic response, we have tested the ability of various point mutants (of the alpha-helices or beta-sheet of the alpha1 and alpha2 domains) of the Kd molecule to induce a primary cytotoxic T cell response in mice carrying the wild-type molecule. For that, we have used an in vivo model in which cells expressing mutant molecules were injected into the hind footpads of mice carrying wild-type Kd, and the recipient graft-draining popliteal lymph nodes were tested for the presence of alloreactive CTL. Under these experimental conditions, only 7 of the 25 mutant Kd molecules induced a primary allogeneic response. All of these mutations (positions 62, 65, 69, 72, 155, 163, 166) concern residues of the alpha-helices, demonstrating that very small variances from self in a class I molecule, located outside the peptide-binding groove, can be antigenic. To determine the peptide requirements for the generation of a primary allogeneic response, we have analyzed the repertoire of peptides selected by individual mutant molecules shown to be able or unable to induce a CTL response. No correlation was observed between the peptidic make-up presented by a given mutant and its capacity to induce a primary allogeneic response. On the whole, our data point to the alloantigenicity of potentially TCR-contacting surface residues of the MHC class I molecules.

As a part of our continuing effort to study the antigenic structure of class I molecules, we have undertaken two types of studies. First, we have studied the capacity of five different Kd-reactive mAbs to recognize a panel of 25 site-directed mutants of the H-2Kd molecule. Both the gain and the decrease in Ab binding resulted from a single amino acid substitution at different positions. All mutations that increase the binding of the tested mAbs are located on the alpha-helices, indicating that the replacement of an Ig-contacting surface residue with a charged or polar side chain by a short one generally favors Ab binding. Mutation of two alpha-helix-situated residues, 58 and 166, completely abolished the binding of one mAb (Tu191.7.1), indicating that these two residues contribute to the antigenic determinant defined by this mAb. The overwhelming majority of mutations that diminished Ab binding concerns residues buried within the Ag binding groove, suggesting the possibility of peptide contribution to serologic epitopes defined by alloreactive Abs. We have addressed this issue by comparison of the repertoire of peptides eluted from Kd molecules precipitated by different Kd-reactive mAbs. The results reveal that the two-dimensional profile obtained with one (F35.119.18) of the alloreactive mAbs is clearly different. The use of 21 single amino acid variants of a Kd-restricted 10-mer peptide allowed us to identify the residue of the bound peptide contributing to the epitope recognized by this mAb. Thus, we have shown that at least in some instances, changes induced in the MHC molecules by the binding of distinct peptides can be recognized as alterations in serologic determinants expressed on the class I molecules.

A set of mouse HLA-B27-reactive cytotoxic T lymphocyte clones were found to recognize the HLA-B27 molecule in an H-2-unrestricted manner, i.e. independently of any mouse major histocompatibility complex (MHC) molecule. The reactivity patterns of these clones on HLA-B27 variants (positive only on HLA-B*2702 and HLA-B*2701) allowed the identification of residues N77 and A81 of the HLA-B27 molecule as important for their reactivity. The location of these residues in the peptide-binding groove (specificity pocket F) suggested that the reactivity of the clones is dependent on HLA-B27-bound peptide(s). However, several other class I molecules sharing these residues (N77 and A81) were not recognized, indicating that other residues might also be involved. One of the clones was found to display an interesting cross-reactivity with allogeneic H-2Kk molecules, sharing N77 and A81 with HLA-B*2702. Sequence comparison suggested the involvement of residue H9, located in specificity pocket B of the peptide-binding groove, and revealed some similarity of pockets B in HLA-B27 and H-2Kk. The structural basis of such T cell-mediated MHC cross-reactions across species barriers is discussed.

As a basis for the characterization of mouse T cells involved in the recognition of xenogeneic HLA molecules, a panel of HLA-B27-reactive cytotoxic T-cell clones was generated upon stimulation by cells from HLA-B27-transgenic mice. The HLA-B27-induced T-cell response was found to comprise two categories of clones: some recognizing HLA-B27 independent of H-2 molecules expressed by the target cells (unrestricted clones), others recognizing HLA-B27 in an H-2-restricted manner. The unrestricted clones exhibited diverse specificities, as judged from their various cross-reactivities with other xenogeneic (HLA) or allogeneic (H-2) molecules. In addition, although most of the unrestricted clones were able to react with both mouse and human HLA-B27-transgenic mice. The HLA-B27-induced T-cell which reacted only with HLA-B27-positive mouse, and not human cells. These findings illustrate that both H-2-restricted and unrestricted T cells with diverse specificities contribute to HLA-B27-xenorecognition.

This case of recurrent migratory superficial phlebitis is reported because of the highly unusual nature of the lesion observed. The picture was dominated by periphlebitis with inflammatory granuloma, giant cells and elastophagia. When the condition had been present for sixteen months, an aortic arch syndrome developed in an inflammatory context. In this light, various auto-immunological etiologies were considered, among them Takayashu's arteritis of which, in the author's opinion, this would be the first case to be combined with venopathy.

The HL-A groups of 138 Kel Diniq Twaregs were determined by the platelet complement fixation microtechnique. Their HL-A characteristics, compared to those of Caucasoid populations, are: decrease in HL-A2, 9, W17 and W15; increase in W28, W32, HL-A7, W5, W10 and second locus blank; absence of Da25 (W30 plus W31), HL-A13, W15, W18, W27. This genetic structure is in accordance with the isolated condition of this population. The most frequent haplotypes are W28,HL-A7, common to both the Kel Diniq and the Kel Kummer Twaregs previously studied; W32,W10 and HL-A3, HL-A5, Kel Diniq only. These two populations are both isolates, with a common origin the seventeenth century, but separated as from that date. Genealogical studies have enabled the haplotype W28,HL-A7 to be attributed to the two brothers who founded the populations in the seventeenth century. A comparison of these two populations constitutes a model for the study of genetic drift and the founder effect.

Xeroderma pigmentosum is an autosomal recessive disease. HLA-A and -B typing was performed on peripheral blood lymphocytes and platelets. Sixteen Tunisian families were typed with 37 patients and 108 relatives. Genetic transmission of the disease and of the HLA system seemed to be independent in this study. Comparison of HLA gene frequencies between (unrelated) parents of patients and a control population showed no difference, proving that there is no clear association in populations between deleterious XP genes and a particular HLA gene. However, an excess of identical HLA among pairs of diseased siblings would suggest that the disease is polymorphic and a form of the XP could be linked to HLA.

The frequencies of 30 HL-A antigens were studied in an Indonesian population of 95 individuals from the city of Jakarta. The antigens HL-A9, or more precisely W24, and HL-A11 (first series) and W15 (second series) occurred with high frequencies, whereas HL-A8, W14 and W22 were completely absent. These results are consistent with previous reports of HL-A typing in South East Asian populations.

Naturally occurring H-2-specific antibodies can be detected rather frequently in sera of non-alloimmunized mice by sufficiently sensitive techniques (Cerny-Provaznik et al., 1985a; Cerny-Provaznik & Ivanyi, 1985). In this report, we summarize our experiences with the preparation of monoclonal anit-H-2 antibodies obtained from hybridization experiments from non-alloimmunized mice. From a total of 30 spleen cell hybridization experiments, we could isolate only four anti-H-2 monoclonal antibodies (mAB). Two of the mAB are described in this report. Monoclonal antibody By-2 is anti-Kf and mAB By-3 is anti-Db, Ds. We investigated which conditions favour the isolation of monoclonal H-2-specific antibodies from non-alloimmunized mice. The presence of naturally occurring serum antibodies, the age of the spleen donor mouse or non-specific B cell stimulation were not critical for the isolation of natural anti-H-2 mAB. We hypothesise that the 'natural' H-2-specific antibodies represent compartments of the B cell repertoire which were triggered by modified or aberrant self-MHC expression.