Lamb dip CRDS of highly saturated transitions of water near 1.4 µm.

Doppler-free saturated-absorption Lamb dips were measured at sub-Pa pressures on rovibrational lines of H216O near 7180 cm-1, using optical feedback frequency stabilized cavity ring-down spectroscopy. The saturation of the considered lines is so high that at the early stage of the ring down, the cavity loss rate remains unaffected by the absorption. By referencing the laser source to an optical frequency comb, transition frequencies are determined down to 100 Hz precision and kHz accuracy. The developed setup allows resolving highly K-type blended doublets separated by about 10 MHz (to be compared to a HWHM Doppler width on the order of 300 MHz). A comparison with the most recent spectroscopic databases is discussed. The determined K-type splittings are found to be very well predicted by the most recent variational calculations.

SARS CoV subunit vaccine: antibody-mediated neutralisation and enhancement.

1. A SARS vaccine was produced based on recombinant native full-length Spike-protein trimers (triSpike) and efficient establishment of a vaccination procedure in rodents. 2. Antibody-mediated enhancement of SARS-CoV infection with anti-SARS-CoV Spike immune-serum was observed in vitro. 3. Antibody-mediated infection of SARS-CoV triggers entry into human haematopoietic cells via an FcγR-dependent and ACE2-, pH-, cysteine-protease-independent pathways. 4. The antibody-mediated enhancement phenomenon is not a mandatory component of the humoral immune response elicited by SARS vaccines, as pure neutralising antibody only could be obtained. 5. Occurrence of immune-mediated enhancement of SARS-CoV infection raises safety concerns regarding the use of SARS-CoV vaccine in humans and enables new ways to investigate SARS pathogenesis (tropism and immune response deregulation).

InterCarb: A Community Effort to Improve Interlaboratory Standardization of the Carbonate Clumped Isotope Thermometer Using Carbonate Standards.

Increased use and improved methodology of carbonate clumped isotope thermometry has greatly enhanced our ability to interrogate a suite of Earth-system processes. However, interlaboratory discrepancies in quantifying carbonate clumped isotope (Δ47) measurements persist, and their specific sources remain unclear. To address interlaboratory differences, we first provide consensus values from the clumped isotope community for four carbonate standards relative to heated and equilibrated gases with 1,819 individual analyses from 10 laboratories. Then we analyzed the four carbonate standards along with three additional standards, spanning a broad range of δ47 and Δ47 values, for a total of 5,329 analyses on 25 individual mass spectrometers from 22 different laboratories. Treating three of the materials as known standards and the other four as unknowns, we find that the use of carbonate reference materials is a robust method for standardization that yields interlaboratory discrepancies entirely consistent with intralaboratory analytical uncertainties. Carbonate reference materials, along with measurement and data processing practices described herein, provide the carbonate clumped isotope community with a robust approach to achieve interlaboratory agreement as we continue to use and improve this powerful geochemical tool. We propose that carbonate clumped isotope data normalized to the carbonate reference materials described in this publication should be reported as Δ47 (I-CDES) values for Intercarb-Carbon Dioxide Equilibrium Scale.

Most Earth-surface calcites precipitate out of isotopic equilibrium.

Oxygen-isotope thermometry played a critical role in the rise of modern geochemistry and remains extensively used in (bio-)geoscience. Its theoretical foundations rest on the assumption that 18O/16O partitioning among water and carbonate minerals primarily reflects thermodynamic equilibrium. However, after decades of research, there is no consensus on the true equilibrium 18O/16O fractionation between calcite and water (18αcc/w). Here, we constrain the equilibrium relations linking temperature, 18αcc/w, and clumped isotopes (Δ47) based on the composition of extremely slow-growing calcites from Devils Hole and Laghetto Basso (Corchia Cave). Equilibrium 18αcc/w values are systematically ~1.5‰ greater than those in biogenic and synthetic calcite traditionally considered to approach oxygen-isotope equilibrium. We further demonstrate that subtle disequilibria also affect Δ47 in biogenic calcite. These observations provide evidence that most Earth-surface calcites fail to achieve isotopic equilibrium, highlighting the need to improve our quantitative understanding of non-equilibrium isotope fractionation effects instead of relying on phenomenological calibrations.

Regulation of high-affinity IgE receptor-mediated mast cell activation by murine low-affinity IgG receptors.

Allergic symptoms result from the release of granular and lipidic mediators and of cytokines by inflammatory cells. The whole process is initiated by the aggregation of mast cell and basophil high-affinity IgE receptors (Fc epsilon RI) by IgE and antigen. We report here that IgE-induced release of mediator and cytokine can be inhibited by cross-linking Fc epsilon RI to low-affinity IgG receptors (Fc gamma RII) which are constitutively expressed on mast cells and basophils. Using a model of stable transfectants in RBL-2H3 cells expressing endogeneous rat Fc epsilon RI and recombinant murine Fc gamma RII, we showed that inhibition requires that Fc epsilon RI be crosslinked to Fc gamma RII by the same multivalent ligand. Inhibition of cross-linked receptors left non-cross-linked Fc epsilon RI capable of triggering mediator release and was reversible upon disengagement. Both isoforms of wild-type Fc gamma RII were equally capable of inhibiting Fc epsilon RI-mediated mast cell activation provided they had an intact intracytoplasmic domain. Our results demonstrate that mast cell secretory responses triggered by high-affinity receptors for IgE may be controlled by low-affinity receptors for IgG. This regulation of Fc epsilon RI-mediated mast cell activation is of potential interest in mast cell physiology and in allergic pathology.

Sequence and length heterogeneity of alpha Fc gamma R transcripts in AKR mice.

The murine low-affinity receptors for IgG, Fc gamma RIII and Fc gamma RII, are encoded by the alpha and the beta Fc gamma R genes, respectively. By contrast to the sequence and the molecular polymorphism of human Fc gamma RIII, no heterogeneity of the murine Fc gamma RIII has been reported and a single alpha Fc gamma R transcript was observed. We describe here a double heterogeneity of alpha Fc gamma R transcripts. First, by S1 mapping of alpha transcripts and by cloning of cDNA coding for Fc gamma RIII, we found a strain-related sequence heterogeneity: four amino acids in the coding region and two stretches of nucleotides in the 3' untranslated sequences differ between alpha transcripts of AKR and BALB/c mice. Second, in AKR mice, we found a cell-dependent length heterogeneity: a short 0.9 kb alpha transcript was present in peritoneal thioglycolate-elicited cells (PEC) from AKR mice. This transcript was present neither in mast cells and NK cells from AKR and BALB/c mice nor in PEC from BALB/c mice. A short cDNA, with a deletion of all the 3' untranslated sequences, has been cloned from AKR PEC, and corresponds to the short alpha transcript. All the differences found in the 3' untranslated sequences of AKR alpha transcripts are located within the fifth exon of the mouse alpha Fc gamma R gene.

The carbohydrate moieties of suppressor IgG-binding factor released by murine T cells.

The carbohydrate moieties of murine IgG-binding factor (IgG-BF) were studied using lectins binding N-glycosylated sequences such as Concanavalin A (Con A), Lens culinaris agglutinin (LcA), and wheat germ agglutinin (WGA), and lectins binding O-glycosylated sequences such as peanut agglutinin (PNA) and Helix pomatia Agglutinin (HpA). Sources of IgG-BF were: (1) supernatants from T2D4, a T cell hybridoma constitutively producing IgG-BF, and (2) factor purified by affinity chromatography on rabbit IgG-Sepharose, using T2D4 supernatants or supernatants of alloantigen-activated T cells (ATC) as starting material. The presence of IgG-BF was assessed by its ability to inhibit secondary anti-sheep red blood cell (SRBC) IgG antibody responses in vitro and to inhibit rosette formation between Fc gamma receptor (Fc gamma R)-positive spleen cells and erythrocytes sensitized with rabbit anti-Forssman IgG antibodies. Fractionation on immobilized lectins showed that IgG-BF: (1) is completely adsorbed by WGA and PNA and partially by Con A, LcA and HpA, and (2) can be eluted from the five different lectins using the competitor sugars. When produced in the presence of tunicamycin, an inhibitor of N-glycosylation, IgG-BF still binds to HpA which has affinity for O-glycosylated carbohydrate chains. These results indicate that IgG-BF is a glycoprotein with N- and O-glycosylated carbohydrate moieties.

Identification of the Fc gamma RII-related component of murine IgG-BF.

Suppressor murine IgG-BF produced by the T cell hybrid (T2D4) expressing low affinity Fc gamma R (Fc gamma RII) contain four biologically active polypeptides of pI 5.2, 6.3, 7.7 and 8.3, respectively. They were fractionated by affinity chromatography on immunoadsorbents coupled with F(ab')2 fragments of the monoclonal anti-Fc gamma RII antibody 2.4G2 and by hydrophobic interaction chromatography. Both methods led to the identification of biologically active IgG-BF which react with 2.4G2 and of IgG-BF which do not react with 2.4G2. Molecules bearing the epitope recognized by 2.4G2 had an apparent pI of 5.3 while the pI of those which did not express this epitope were 6.3, 7.8 and 8.5, respectively. Therefore, one IgG-BF polypeptide of pI 5.3 is probably related to Fc gamma RII.

21.1.1, a novel activation marker of T and B cells.

A new rat mAb designated mAb 21.1.1 was raised against a T cell hybridoma of mouse origin, T2D4. This antibody, an IgG2b, immunoprecipitates from the membrane extracts of iodinated T2D4 cells a 56-kDa glycoprotein of apparent pI 4.6 which gives a 34-kDa polypeptide after treatment with endoglycosidase F. MAb 21.1.1 reacts with an antigen expressed on murine mitogen-activated thymocytes and T cells, and on B cells stimulated by anti-IgM antibodies. Cells isolated from the spleen, lymph nodes and bone marrow are negative, as are purified resting B cells or T cells. This antigen is strongly expressed on most day-16 fetal thymocytes whereas adult thymocytes are almost negative. mAb 21.1.1 may be useful for the study of activation and differentiation of T and B cells.

Regulation of the expression of murine alpha- and beta-Fc gamma R genes.

Murine low-affinity receptors for the Fc portion of IgG are of two types: Fc gamma RII and Fc gamma RIII. Murine Fc gamma RII and III have 95% homologous extracellular (EC) domains and bind the same ligands, but different transmembrane (TM) and intracytoplasmic (IC) domains. They, however, have unrelated TM and IC domains. Murine Fc gamma RII are single-chain receptors, encoded by the beta-Fc gamma R gene. Murine Fc gamma RIII are composed of two subunits: the ligand-binding alpha-subunit, encoded by the alpha-Fc gamma R gene and the gamma-subunit, encoded by another gene which belongs to a family of genes encoding dimeric subunits of multichain receptors. The expression of murine Fc gamma RII and Fc gamma RIII depends on a number of mechanisms which do the following: (1) determine the tissue-specific expression of the alpha- and beta-Fc gamma R genes by selectively unmethylating DNA in specific 5' sequences in different cell types; (2) regulate the initiation of the transcription of the alpha- and beta-Fc gamma R genes via several transcription factors; (3) up- and downregulate the amount of alpha- and beta-Fc gamma R transcripts in response to cytokines; (4) decide the alternative splicing of IC exons of the beta-Fc gamma R gene and generate the different Fc gamma RII isoforms; (5) possibly regulate the translation of alpha- and beta-Fc gamma R transcripts in different cells; (6) control the assembly of the Fc gamma RIII subunits and their membrane insertion, and (7) determine the turnover of Fc gamma RII and III in the presence and absence of ligands by affecting the internalization, shedding and proteolytic cleavage of the receptors. These mechanisms altogether contribute to make a variety of cells capable of responding differently to antigen-antibody complexes, depending on environmental stimuli.

Building up the family of ITIM-bearing negative coreceptors.

The acronym (ITAM) for immunoreceptor tyrosine-based activation motif was first proposed in September 1994, during the 8th Meeting on Signals and Signal Processing in the Immune System held in Kecskemet, Hungary, to designate the di-tyrosine-based YxxL activation motifs that had been previously understood by Michael Reth to account for the cell-triggering properties of BCR, TCR and FcR. It was then agreed, by those who signed the collective letter John Cambier had been commissioned to submit to Immunology Today (Cambier, J.C. (1994) Immunol. Today 16, 110-110) that it was premature to propose ITIM (for immunoreceptor tyrosine-based inhibition motif) to designate the one inhibitory sequence containing a single Ys1L motif that had been identified in the intracytoplasmic domain of a low-affinity Fc receptor for IgG. Right away, ITAM became unanimously accepted and widely used in the literature. Remarkably, ITIM was soon adopted too and, in September 1996, a whole session of the 9th Signal Meeting, held in Tihany, Hungary, was devoted to ITIM. During the last 2 years, evidence accumulated that indeed accredited the ITIM concept.

The SH2 domain containing inositol 5-phosphatase SHIP2 associates to the immunoreceptor tyrosine-based inhibition motif of Fc gammaRIIB in B cells under negative signaling.

Fc gammaRIIB are single-chain low-affinity receptors for IgG that bear an immunoreceptor tyrosine-based inhibition motif (ITIM) in their intracytoplasmic domain and that negatively regulate immunoreceptor tyrosine-based activation motif (ITAM)-dependent cell activation. In B cells, coaggregation of the B cell receptor (BCR) and Fc gammaRIIB leads to an inhibition of B cell activation. Inhibitory properties of Fc gammaRIIB have been related to the recruitment of SHIP, an SH2 domain-containing inositol 5-phosphatase (referred to as SHIP1), via ITIM phosphorylated Fc gammaRIIB. Here, we demonstrate that the second SH2 domain-containing inositol 5-phosphatase SHIP2 could also bind to the Fc gammaRIIB ITIM. As a model, a Fc gammaRIIB deficient B cell line (IIA1.6), transfected with a cDNA encoding either w.t. Fc gammaRIIB1' or Fc gammaRIIB1' whose ITIM tyrosine was mutated has been used. SHIP2 tyrosine phosphorylation and association to the adaptator protein Shc were only found in transfectants expressing w.t. Fc gammaRIIB1'. SHIP2 was also found to bind to a phosphopeptide corresponding to the ITIM sequence of Fc gammaRIIB. There was no binding to the nonphosphorylated peptide. Finally, both SHIP2 and SHIP1 were coprecipitated with Fc gammaRIIB1' upon coaggregation with BCR in IIA1.6 transfectants.

Regulation of tyrosine-containing activation motif-dependent cell signalling by Fc gamma RII.

Crosslinking of the B-cell receptor (BCR) for antigen to low-affinity receptors for IgG (Fc gamma RII) inhibits B-cell activation induced by BCR aggregation. The cell-triggering properties of the BCR depend on tyrosine-containing activation motifs (TAM), in the intracytoplasmic domain of its Ig alpha and Ig beta subunits. TAMs also account for the cell-triggering capabilities of the T-cell receptor (TCR) for antigen, in T lymphocytes, and of the high-affinity receptor for IgE (Fc epsilon RI), in mast cells. Using as a model, rat basophilic leukemia cells (RBL-2H3) stably transfected with cDNA encoding wild-type or mutated murine or human Fc gamma RIIB and chimeric molecules having the intracytoplasmic domain of the FcR gamma subunit or of TCR-CD3 zeta subunit, we found that the inhibitory properties of Fc gamma RII are neither restricted to B cells nor to BCR-dependent cell activation, but can be extended to other cells and, as a general rule, to TAM-dependent cell activation.

Selective in vivo recruitment of the phosphatidylinositol phosphatase SHIP by phosphorylated Fc gammaRIIB during negative regulation of IgE-dependent mouse mast cell activation.

We demonstrated previously that the low-affinity IgG receptors Fc gammaRIIB, which are coexpressed with the high-affinity IgE receptors Fc epsilonRI in mouse mast cells, can inhibit IgE-induced release of inflammatory mediators and cytokines by these cells. Inhibition was found to require the coaggregation of the two receptors and to depend on the presence of a tyrosine-based inhibition motif (ITIM) in the intracytoplasmic domain of Fc gammaRIIB. We report here that the coaggregation with Fc gammaRIIB does not prevent Fc epsilonRI from triggering activation signals in BMMC and induces the tyrosine phosphorylation of Fc gammaRIIB. Phosphorylated ITIM peptides bound in vitro to three SH2 domain-containing phosphatases present in BMMC lysates: the phosphotyrosine phosphatases SHP-1 and SHP-2. and the inositolphosphate phosphatase SHIP. Using BMMC generated from the SHP-1-deficient motheaten mice, SHP-1 was found to be dispensable for inhibition of mast cell activation. When analyzed for in vivo association, SHIP coprecipitated with phosphorylated Fc gammaRIIB, whereas SHP-1 or SHP-2 did not. These observations altogether indicate that Fc epsilonRI actively participates in its own regulation and that the mechanisms by which Fc gammaRIIB inhibit cell activation might be different in mast cells and in B-cells.

Structural bases of Fc gamma R functions.

This review describes structures which determine the biological activities triggered by Fc gamma R and account for the cell-mediated functions of IgG antibodies in physiology and pathology. The binding specificity and affinity of Fc gamma R depend primarily on IgG-binding structures, in their immunoglobulin-like extracellular domains. Binding is however also influenced by subunits that associate to multichain Fc gamma R. Effector and regulatory intracytoplasmic sequences that are unique to molecules of the Fc gamma RIIB family determine the internalization properties of these receptors. Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) are intracytoplasmic effector sequences shared by Fc gamma R and other receptors involved in the recognition of antigen, which trigger cell activation and internalization. Immunoreceptor Tyrosine-based Inhibition Motifs (ITIMs) are intracytoplasmic sequences, shared by Fc gamma RIIB and a growing number of negative coreceptors which negatively regulate cell activation via ITAM-bearing receptors. Altogether, these structures enable IgG antibodies to exert a variety of finely tuned biological effects during the immune response.

FCgammaRII (CD32)-dependent induction of interferon-alpha by serum from patients with lupus erythematosus.

Interferon-alpha (IFN-alpha) is detected in the serum of 70-80% of patients with systemic lupus erythematosus (SLE). Furthermore, soluble factors in SLE serum can induce peripheral blood mononuclear cells (PBMC) to produce IFN-alpha. The purpose of this work was to investigate the mechanism of this IFN-alpha induction. In eleven of fifteen SLE serum samples, an IFN-alpha inducing activity was detected, whereas serum from healthy controls, patients with other autoimmune disease and patients with viral infections were ineffective under the same conditions. After gel filtration of the serum, the inducing activity was found in the same fraction as IgG. The IFN-alpha inducing activity was inhibited by native monoclonal antibodies to the receptors for the Fc portion of IgG: FcgammaRIIA/C and FcgammaRIIB subclasses (CD32) and by their F(ab)'2 fragments. Purified Fc fragments of human IgG were also effective in abolishing the IFN-alpha-inducing activity. Since no anti-CD32 autoantibodies were found in SLE serum, this IFN-alpha-inducing activity may be due to immune complex antibodies. Such results may allow better understand the origin of endogenous IFN-alpha, which has a deleterious effect on the course of this autoimmune disease. The inhibition of this function by the CD32 antibody could lead to new therapeutic approach in SLE.

Chimeric immunoglobulin E reactive with tumor-associated antigen activates human Fc epsilon RI bearing cells.

Crosslinking of immunoglobulin E molecules that are bound to the Fc epsilon receptors expressed on mast cells or basophils triggers activation of these cells, resulting in the development of a type I hypersensitivity. Targeting this potent immune reaction towards tumors by using IgE that reacts with a tumor-associated antigen, may induce a local inflammation at the tumor site, and may therefore promote tumor regression. We have previously shown that murine IgE bound to tumor cells can activate murine mast cells to release TNF-alpha and histamine. To further investigate the therapeutic potential of IgE-mediated immunotherapy of carcinomas, we have developed human/murine chimeric versions, containing the murine variable regions and human constant regions, of both G250 and 323/A3 IgE. These chimeric IgEs are reactive respectively with the G250 renal cell carcinoma antigen and the Ep-CAM molecule, which is highly expressed by most carcinomas. Transfection of the respective chimeric heavy and light chain genes into recipient Sp2/0 myeloma cells yielded chimeric IgE-producing clones. Chimeric G250 and 323/A3 IgE reacted with tumor cells expressing the G250 antigen or Ep-CAM, respectively. To generate a cell line that expresses Fc receptors for human or chimeric IgE, the rat basophilic leukemia cell line RBL-7 was transfected with the human Fc epsilon RI alpha chain (RBL-7TZ) and subsequently tested for binding of chimeric IgE. Functional assays showed that both chimeric IgEs activated RBL-7TZ cells to release TNF-alpha when cultured with tumor cells that express the respective specific antigen. Furthermore, both chimeric IgEs were able to activate freshly isolated human basophils.

Intracytoplasmic sequences involved in the biological properties of low-affinity receptors for IgG expressed by murine macrophages.

Murine macrophages express several receptors for the Fc portion of IgG antibodies (Fc gamma R). These are high-affinity Fc gamma RI, which bind monomeric IgG2a, and low-affinity Fc gamma RII and Fc gamma RIII, which bind IgG1, IgG2a and IgG2b immune complexes. Fc gamma Ri and Fc gamma RIII are multichain receptors. They are composed of an IgG-binding alpha subunit, associated with the same FcR gamma subunit that also associates with mast cell high-affinity IgE receptors (Fc epsilon RI). Fc gamma RII are single-chain receptors. They exist as two isoforms, Fc gamma RIIb1 and Fc gamma RIIb2, differing by a 47-amino acid insertion in the intracytoplasmic domain of Fc gamma RIIb1. Using a model of stable transfectants, we analyzed the biological activities triggered by Fc gamma R and, by site-directed mutagenesis, we mapped functional sequences in the intracytoplasmic domains of recombinant Fc gamma R. A single tyrosine-based activation motif (ITAM), in the intracytoplasmic domain of the FcR gamma subunit of Fc gamma RIII and Fc gamma RI, triggers cell activation, endocytosis and phagocytosis. Two distinct motifs, in the intracytoplasmic domain of Fc gamma RIIb2, trigger endocytosis and phagocytosis, respectively. The Fc gamma RIIb1-specific intracytoplasmic insertion mediates capping and down-regulates Fc gamma RII-dependent internalization. A tyrosine-based inhibitory motif (ITIM), in intracytoplasmic sequences common to Fc gamma RIIb1 and Fc gamma RIIb2, was identified as down-regulating ITAM-dependent cell activation. The variety of biological properties of Fc gamma R implies that macrophage responses triggered by IgG antibodies depend on the complex interplay between different Fc gamma R having common ligands that are coexpressed by mouse macrophages.

[Regulation and dysregulations of immunoglobulin E synthesis]. / Régulation et dérégulations de la synthèse d'immunoglobines E.

Compared to other isotypes of immunoglobulins, IgE antibodies are present in exceedingly low concentrations in normal serum, because their synthesis is maintained under the strict control of several intricate regulatory mechanisms. These normally unapparent mechanisms become accessible to investigations in a number of pathological dysregulations. One may distinguish three orders of controls. An elementary isotypic circuit, constructed on the interactions between IgE Fc portions and IgE-binding molecules, directly regulates IgE synthesis by IgE-secreting B cells. IgE-binding molecules are themselves controlled by several lymphokine cascades, depending on external and genetic factors. The functioning of the IgE isotypic circuit is coordinated with other circuits, corresponding to other immunoglobulin isotypes, by means of an isotypic network, connected to other homeostatic systems of the organism. The dissection of these regulatory mechanisms and an understanding of their interactions are potentially useful for new therapeutic approaches.