Low end-tidal CO2 as a real-time severity marker of intra-anaesthetic acute hypersensitivity reactions.

BACKGROUND: Prompt diagnosis of intra-anaesthetic acute hypersensitivity reactions (AHR) is challenging because of the possible absence and/or difficulty in detecting the usual clinical signs and because of the higher prevalence of alternative diagnoses. Delayed epinephrine administration during AHR, because of incorrect/delayed diagnosis, can be associated with poor prognosis. Low end-tidal CO2 (etCO2) is known to be linked to low cardiac output. Yet, its clinical utility during suspected intra-anaesthetic AHR is not well documented. METHODS: Clinical data from the 86 patients of the Neutrophil Activation in Systemic Anaphylaxis (NASA) multicentre study were analysed. Consenting patients with clinical signs consistent with intra-anaesthetic AHR to a neuromuscular blocking agent were included. Severe AHR was defined as a Grade 3-4 of the Ring and Messmer classification. Causes of AHR were explored following recommended guidelines. RESULTS: Among the 86 patients, 50% had severe AHR and 69% had a confirmed/suspected IgE-mediated event. Occurrence and minimum values of arterial hypotension, hypocapnia and hypoxaemia increased significantly with the severity of AHR. Low etCO2 was the only factor able to distinguish mild [median 3.5 (3.2;3.9) kPa] from severe AHR [median 2.4 (1.6;3.0) kPa], without overlap in inter-quartile range values, with an area under the receiver operator characteristic curve of 0.92 [95% confidence interval: 0.79-1.00]. Among the 41% of patients who received epinephrine, only half received it as first-line therapy despite international guidelines. CONCLUSIONS: An etCO2 value below 2.6 kPa (20 mm Hg) could be useful for prompt diagnosis of severe intra-anaesthetic AHR, and could facilitate early treatment with titrated doses of epinephrine. CLINICAL TRIAL REGISTRATION: NCT01637220.

Molecular Rods Based on Oligo-spiro-thioketals.

We report on an extension of the previously established concept of oligospiroketal (OSK) rods by replacing a part or all ketal moieties by thioketals leading to oligospirothioketal (OSTK) rods. In this way, some crucial problems arising from the reversible formation of ketals are circumvented. Furthermore, the stability of the rods toward hydrolysis is considerably improved. To successfully implement this concept, we first developed a number of new oligothiol building blocks and improved the synthetic accessibility of known oligothiols, respectively. Another advantage of thioacetals is that terephthalaldehyde (TAA) sleeves, which are too flexible in the case of acetals can be used in OSTK rods. The viability of the OSTK approach was demonstrated by the successful preparation of some OSTK rods with a length of some nanometers.

Contribution of humoral immune responses to the antitumor effects mediated by anthracyclines.

Immunogenic cell death induced by cytotoxic compounds contributes to the success of selected chemotherapies by eliciting a protective anticancer immune response, which is mediated by CD4(+) and CD8(+) T cells producing interferon-γ. In many instances, cancer progression is associated with high titers of tumor-specific antibodies, which become detectable in the serum, but whose functional relevance is elusive. Here, we explored the role of humoral immune responses in the anticancer efficacy of anthracyclines. Chemotherapy reduced the number of tumor-infiltrating B cells, and failed to promote humoral responses against immunodominant tumor antigens. Although anthracycline-based anticancer chemotherapies failed in T cell-deficient mice, they successfully reduced the growth of cancers developing in mice lacking B lymphocytes (due to the injection of a B-cell-depleting anti-CD20 antibody), immunoglobulins (Igs) or Ig receptors (Fc receptor) due to genetic manipulations. These results suggest that the humoral arm of antitumor immunity is dispensable for the immune-dependent therapeutic effect of anthracyclines against mouse sarcoma. In addition, we show here that the titers of IgA and IgG antibodies directed against an autoantigen appearing at the cell surface of tumor cells post chemotherapy (calreticulin, CRT) did not significantly increase in patients treated with anthracyclines, and that anti-CRT antibodies had no prognostic or predictive significance. Collectively, our data indicate that humoral anticancer immune responses differ from cellular responses in, thus far, that they do not contribute to the success of anthracycline-mediated anticancer therapies in human breast cancers and mouse sarcomas.

Apoptosis of mouse mast cells is reciprocally regulated by the IgG receptors FcγRIIB and FcγRIIIA.

BACKGROUND: Mast cells are important effector cells in allergy. They usually have a long life span and resist cell death induction. Fcγ receptor- and IgG immune complex-mediated apoptosis has been demonstrated in B-lineage cells, but not in mast cells. The aim of the current study was to investigate whether mast cells could respond to apoptosis induction by IgG immune complex aggregation of Fcγ receptors. It is known that mouse mast cells express the low-affinity Fcγ receptors FcγRIIB and FcγRIIIA, which bind IgG especially in the form of antigen-IgG immune complexes. METHODS: Mouse bone marrow-derived cultured mast cells were examined for surface expression of FcγRIIB and FcγRIIIA. Apoptosis of such cells from wild-type, FcγRIIB(-/-) or FcγRIIIA(-/-) mice was measured following receptor aggregation by IgG immune complexes. RESULTS: Our data demonstrate that aggregation of either FcγRIIB or FcγRIIIA by IgG immune complexes induced apoptosis of mouse bone marrow-derived cultured mast cells. However, mast cells expressing both FcγRIIB and FcγRIIIA were able to resist cell death induction by IgG immune complexes. CONCLUSION: Our findings reveal a fine-tuning system for regulating mast cell apoptosis through aggregating Fcγ receptors by IgG immune complexes. Such apoptosis regulation may have a substantial impact on mast cell homeostasis during allergic inflammation.

Is the center of mass (COM) a reliable parameter for the localization of brain function in fMRI?

The center of mass (COM) in functional MRI studies is defined as the center of a cerebral activation cluster. Although the COM is a well-accepted parameter for exactly localizing brain function, the reliability of COMs has not received much attention until now. Our goal was to investigate COM reliability as a function of the thresholding technique, the threshold level, and the type of COM calculation. Therefore 15 subjects were examined repeatedly using simple hand and tongue movement paradigms. Postprocessing was performed with uncorrected, corrected, and proportional thresholding as well as different threshold levels. Geometric and T-weighted COMs of left-hemispheric primary hand and tongue motor clusters were calculated. The COM variation was evaluated within and between repeated sessions depending on the different postprocessing setups. Mean COM variations over three repeated sessions varied between 1.6 mm and 9.8 mm for the hand paradigm and between 7.0 mm and 14.4 mm for the tongue task. Stringent thresholding techniques and high threshold levels were required to assess reliable results, whereas the kind of COM calculation was of lesser relevance. Thus, COM reliability cannot be presupposed; it depends strongly on the individual postprocessing techniques. This should be considered when using COMs for localizing brain function.

Reproducibility of activations in Broca area with two language tasks: a functional MR imaging study.

BACKGROUND AND PURPOSE: Functional MR imaging (fMRI) is rapidly evolving and claims to complement or even substitute intraoperative mapping (IOM) of language functions. However, little is known about the reproducibility of imaging data in the language domain. The aim of our study was to assess the reproducibility of activations for 2 widely used paradigms: naming and word generation. Individual analysis was focused on the Broca area and the left insula. MATERIALS AND METHODS: We examined 13 healthy right-handed subjects in 3 sessions with fMRI. Two conditions were assessed: overt naming and overt naming plus noun generation. The same stimuli were used in all of the sessions. A random-effects analysis was performed to analyze whole-brain activation on a group level. For the regions of interest, the number of voxels classified as active were counted for each subject, and individual reproducibility coefficients were calculated over sessions. RESULTS: For the naming condition, the random-effects analysis did not reveal significant activations in the specified regions; small individual activations were not reproducible. For the combined task, all of the subjects showed activations in the Broca area that were more extensive and reproducible than in the naming task. Activations in the insula were only poorly reproducible. CONCLUSION: Naming is an approved task in IOM but does not identify the Broca area with fMRI in a reproducible way. Priming may have affected our results, but the use of a combined task, in which naming is paired with noun generation, improves the reproducibility of activations and is also suitable for IOM.

Src homology 2 domain-containing inositol 5-phosphatase 1 mediates cell cycle arrest by FcgammaRIIB.

We previously found that low affinity receptors for the Fc portion of IgG, FcgammaRIIB, which are widely expressed by hematopoietic cells, can negatively regulate receptor tyrosine kinase-dependent cell proliferation. We investigated here the mechanisms of this inhibition. We used as experimental models wild-type mast cells, which constitutively express the stem cell factor receptor Kit and FcgammaRIIB, FcgammaRIIB-deficient mast cells reconstituted with wild-type or mutated FcgammaRIIB, and Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1)-deficient mast cells. We found that, upon coaggregation with Kit, FcgammaRIIB are tyrosyl-phosphorylated, recruit SHIP1, but not SHIP2, SH2 domain-containing protein tyrosine phosphatase-1 or -2, abrogate Akt phosphorylation, shorten the duration of the activation of mitogen-activated protein kinases of the Ras and Rac pathways, abrogate cyclin induction, prevent cells from entering the cell cycle, and block thymidine incorporation. FcgammaRIIB-mediated inhibition of Kit-dependent cell proliferation was reduced in SHIP1-deficient mast cells, whereas inhibition of IgE-induced responses was abrogated. Cell proliferation was, however, inhibited by coaggregating Kit with FcgammaRIIB whose intracytoplasmic domain was replaced with the catalytic domain of SHIP1. These results demonstrate that FcgammaRIIB use SHIP1 to inhibit pathways shared by receptor tyrosine kinases and immunoreceptors to trigger cell proliferation and cell activation, respectively, but that, in the absence of SHIP1, FcgammaRIIB can use other effectors that specifically inhibit cell proliferation.

Insufficient phosphorylation prevents fc gamma RIIB from recruiting the SH2 domain-containing protein-tyrosine phosphatase SHP-1.

Fc gamma RIIB are IgG receptors that inhibit immunoreceptor tyrosine-based activation motif (ITAM)-dependent cell activation. Inhibition depends on an immunoreceptor tyrosine-based inhibition motif (ITIM) that is phosphorylated upon Fc gamma RIIB coaggregation with ITAM-bearing receptors and recruits SH2 domain-containing phosphatases. Agarose bead-coated phosphorylated ITIM peptides (pITIMs) bind in vitro the single-SH2 inositol 5-phosphatases (SHIP1 and SHIP2) and the two-SH2 protein tyrosine phosphatases (SHP-1 and SHP-2). Phosphorylated Fc gamma RIIB, however, recruit selectively SHIP1/2 in vivo. We aimed here at explaining this discordance. We found that beads coated with low amounts of pITIM bound in vitro SHIP1, but not SHP-1, i.e. behaved as phosphorylated Fc gamma RIIB in vivo. The reason is that SHP-1 requires its two SH2 domains to bind on adjacent pITIMs. Consequently, the binding of SHP-1, but not of SHIP1, increased with pITIM density on beads. When trying to increase Fc gamma RIIB phosphorylation in B cells and mast cells, we found that concentrations of ligands optimal for Fc gamma RIIB phosphorylation failed to induce SHP-1 recruitment. SHP-1 was, however, recruited by Fc gamma RIIB when hyperphosphorylated following cell treatment with pervanadate. Our data suggest that Fc gamma RIIB phosphorylation may not be sufficient in vivo to enable the recruitment of SHP-1 but that (pathological?) conditions that would hyperphosphorylate Fc gamma RIIB might enable SHP-1 recruitment.

Molecular basis of the recruitment of the SH2 domain-containing inositol 5-phosphatases SHIP1 and SHIP2 by fcgamma RIIB.

FcgammaRIIB are single-chain low affinity receptors for IgG that negatively regulate immunoreceptor tyrosine-based activation motif-dependent cell activation. They bear one immunoreceptor tyrosine-based inhibition motif (ITIM) that becomes tyrosyl-phosphorylated upon coaggregation of FcgammaRIIB with immunoreceptor tyrosine-based activation motif-bearing receptors and that recruits SH2 domain-containing inositol 5-phosphatases (SHIPs) in vivo. Synthetic FcgammaRIIB ITIM phosphopeptides, however, also bind SH2 domain-containing protein-tyrosine phosphatases (SHPs) in vitro. To identify SHIP-binding sites, we exchanged residues between the FcgammaRIIB ITIM and the N-terminal ITIM of a killer cell Ig-like receptor that does not bind SHIPs. Loss of function and gain of function substitutions identified the Y+2 leucine, in the FcgammaRIIB ITIM, as determining the binding of both SHIP1 and SHIP2, but not the binding of SHP-1 or SHP-2. Conversely, the Y-2 isoleucine that determines the in vitro binding of SHP-1 and SHP-2 affected neither the binding nor the recruitment of SHIP1 or SHIP2. One hydrophobic residue, in the ITIM of FcgammaRIIB therefore determines the affinity for SHIPs. This residue is symmetrical to the hydrophobic residue that determines the affinity of all ITIMs for SHPs. It defines a SHIP-binding site, distinct from a SHP-binding site, that enables FcgammaRIIB to recruit SHIP1 and SHIP2 and that is preferentially used in vivo.

Mutational analysis reveals multiple distinct sites within Fc gamma receptor IIB that function in inhibitory signaling.

The low-affinity receptor for IgG, FcgammaRIIB, functions broadly in the immune system, blocking mast cell degranulation, dampening the humoral immune response, and reducing the risk of autoimmunity. Previous studies concluded that inhibitory signal transduction by FcgammaRIIB is mediated solely by its immunoreceptor tyrosine-based inhibition motif (ITIM) that, when phosphorylated, recruits the SH2-containing inositol 5'- phosphatase SHIP and the SH2-containing tyrosine phosphatases SHP-1 and SHP-2. The mutational analysis reported here reveals that the receptor's C-terminal 16 residues are also required for detectable FcgammaRIIB association with SHIP in vivo and for FcgammaRIIB-mediated phosphatidylinositol 3-kinase hydrolysis by SHIP. Although the ITIM appears to contain all the structural information required for receptor-mediated tyrosine phosphorylation of SHIP, phosphorylation is enhanced when the C-terminal sequence is present. Additionally, FcgammaRIIB-mediated dephosphorylation of CD19 is independent of the cytoplasmic tail distal from residue 237, including the ITIM. Finally, the findings indicate that tyrosines 290, 309, and 326 are all sites of significant FcgammaRIIB1 phosphorylation following coaggregation with B cell Ag receptor. Thus, we conclude that multiple sites in FcgammaRIIB contribute uniquely to transduction of FcgammaRIIB-mediated inhibitory signals.

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.

The pseudo-immunoreceptor tyrosine-based activation motif of CD5 mediates its inhibitory action on B-cell receptor signaling.

Genetic studies revealed that CD5 could be a negative regulator of the B-cell antigen receptor (BCR). We explore here the effect of human CD5 on BCR-triggered responses. B cells were obtained expressing a chimera composed of extracellular and transmembrane domains of Fcgamma type IIB receptor fused to CD5 cytoplasmic domain (CD5cyt). Coligation of the chimera with the BCR induces CD5cyt tyrosine phosphorylation. A rapid inhibition of BCR-induced calcium response is observed, as well as a partial but delayed inhibition of phospholipase Cgamma-1 phosphorylation. Activation of extracellular regulated kinase-2 is also severely impaired. Moreover, at the functional level, interleukin-2 production is abolished. Src homology 2 domain-bearing tyrosine phosphatase SHP-1 and Src homology 2 domain-bearing inositol 5'-phosphatase SHIP usually participate in negative regulation of the BCR. We show that they do not associate with the phosphorylated CD5 chimera. We finally demonstrate that the pseudo-immunoreceptor tyrosine based activation motif present in CD5cyt is involved because its deletion eliminates the inhibitory effect of the chimera, both at biochemical and functional levels. These results demonstrate the inhibitory role of CD5 pseudo-immunoreceptor tyrosine based activation motif tyrosine phosphorylation on BCR signaling. They further support the idea that CD5 uses mechanisms different from those already described to negatively regulate the BCR pathway.

The RasGAP-binding protein p62dok is a mediator of inhibitory FcgammaRIIB signals in B cells.

The low affinity receptor for IgG, FcgammaRIIB, functions to dampen the antibody response and reduce the risk of autoimmunity. This function is reportedly mediated in part by inhibition of B cell antigen receptor (BCR)-mediated p21ras activation, though the basis of this inhibition is unknown. We show here that FcgammaRIIB-BCR coaggregation leads to increased tyrosine phosphorylation of the RasGAP-binding protein p62dok, with a concomitant increase in its binding to RasGAP. These effects require the recruitment and tyrosine phosphorylation of the phosphatidylinositol 5-phosphatase SHIP, which further recruits p62dok via the latter's phosphotyrosine-binding domain. Using chimeric FcgammaRIIB containing the RasGAP-binding domain of p62dok, we demonstrate that p62dok contains all structural information required to mediate the inhibitory effect of FcgammaRIIB on Erk activation.

Signal regulatory proteins negatively regulate immunoreceptor-dependent cell activation.

Signal regulatory proteins of the alpha subtype (SIRPalpha) are ubiquitous molecules of the immunoglobulin superfamily that negatively regulate protein tyrosine kinase receptor-dependent cell proliferation. Their intracytoplasmic domain contains four motifs that resemble immunoreceptor tyrosine-based inhibition motifs (ITIMs) and that, when tyrosyl-phosphorylated, recruit cytoplasmic SH2 domain-bearing protein tyrosine phosphatases (SHPs). ITIMs are borne by molecules that negatively regulate cell activation induced by receptors bearing immunoreceptor tyrosine-based activation motifs (ITAMs). Because SIRPalpha are coexpressed with ITAM-bearing receptors in hematopoietic cells, we investigated whether SIRPalpha could negatively regulate ITAM-dependent cell activation. We found SIRPalpha transcripts in human mast cells, and we show that a chimeric molecule having the transmembrane and intracytoplasmic domains of SIRPalpha could inhibit IgE-induced mediator secretion and cytokine synthesis by mast cells. Inhibition required that the SIRPalpha chimera was coaggregated with ITAM-bearing high affinity IgE receptors (FcepsilonRI). It was correlated with the tyrosyl phosphorylation of the SIRPalpha chimera and the recruitment of SHP-1 and SHP-2. The phosphorylation of FcepsilonRI ITAMs was decreased; the mobilization of intracellular Ca(2+) and the influx of extracellular Ca(2+) were reduced, and the activation of the mitogen-activated protein kinases Erk1 and Erk2 was abolished. SIRPalpha can therefore negatively regulate not only receptor tyrosine kinase-dependent cell proliferation but also ITAM-dependent cell activation.

Differential roles of N- and C-terminal immunoreceptor tyrosine-based inhibition motifs during inhibition of cell activation by killer cell inhibitory receptors.

Killer cell inhibitory receptors (KIRs) inhibit NK and T cell cytotoxicity when recognizing MHC class I molecules on target cells. They possess two tandem intracytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs) that, when phosphorylated, each bind to the two Src homology 2 domain-bearing protein tyrosine phosphatases SHP-1 and SHP-2 in vitro. Using chimeric receptors having an intact intracytoplasmic KIR domain bearing both ITIMs (N + C-KIR), a deleted domain containing the N-terminal ITIM only (N-KIR), or a deleted domain containing the C-terminal ITIM only (C-KIR), we examined the respective contributions of the two ITIMs in the inhibition of cell activation in two experimental models (a rat mast cell and a mouse B cell line) that have been widely used to analyze KIR functions. We found that the two KIR ITIMs play distinct roles. When coaggregated with immunoreceptor tyrosine-based activation motif-bearing receptors such as high-affinity IgE receptors or B cell receptors, the N + C-KIR and the N-KIR chimeras, but not the C-KIR chimera, inhibited mast cell and B cell activation, became tyrosyl-phosphorylated, and recruited phosphatases in vivo. The N + C-KIR chimera recruited SHP-1 as expected, but also SHP-2. Surprisingly, the N-KIR chimera failed to recruit SHP-1; however, it did recruit SHP-2. Consequently, the N-terminal ITIM is sufficient to recruit SHP-2 and to inhibit cell activation, whereas the N-terminal and the C-terminal ITIMs are both necessary to recruit SHP-1. The two KIR ITIMs, therefore, are neither mandatory for inhibition nor redundant. Rather than simply amplifying inhibitory signals, they differentially contribute to the recruitment of distinct phosphatases that may cooperate to inhibit cell activation.