Comparison of the predictive value of thromboelastography and Sonoclot analysis for postoperative bleeding in children undergoing corrective surgery for cyanotic congenital heart disease.

Background: The aim of the study was to compare the predictive value of Sonoclot analysis and thromboelastography (TEG) for postoperative bleeding in children younger than 12 years coming for cardiac surgery for congenital cyanotic heart disease. Methods: This is a prospective, observational study carried out in a single tertiary care military hospital. Ninety patients of the paediatric age group undergoing bypass cardiac surgery for correction of congenital cyanotic heart defect were included in the study. Laboratory-derived values to assess coagulation status (prothrombin time, international normalisation ratio, activated partial thromboplastin time) and point-of-care Sonoclot- and TEG-derived parameters were noted at the start of surgery and postoperatively in all patients. Bleeders were predefined on the basis of chest tube drainage. Results: The incidence of bleeders was 42.2% (38/90 patients), whereas 57.8% (52/90 patients) were non-bleeders. The postoperative R value and preoperative gbPF test were predictive for postoperative bleeders on multivariate analysis. Postoperative gbPF had the highest area under the curve (0.72), with a cut-off value of 1.75, and gbPF had 82% sensitivity and 71% specificity in predicting significant postoperative bleeding in paediatric cyanotic congenital heart surgeries. Transfusion requirements and mechanical ventilation duration were higher in bleeders; however; length of intensive care unit stay, incidence of sepsis and mortality were similar in both the groups. Conclusion: Bleeding in patients undergoing corrective surgery for cyanotic congenital heart disease could be predicted by the preoperative gbPF and postoperative R value. Among these, preoperative gbPF has the maximum predictive value.

Inadequacy of near-infrared spectroscopy cerebral oximetry monitoring for detecting neurological complication.

Near-infrared spectroscopy (NIRS) cerebral oximetry is an established and standard monitoring modality for surgery under extracorporeal circulation with circulatory arrest. It helps to reduce the neurological complication, but in many instances, it becomes not only technically challenging but also is difficult to interpret and take corrective action based on the NIRS values. In this case study, we aimed to present the inadequacy of cerebral oximetry for detecting neurological complication.

4-1BB ligand induces cell division, sustains survival, and enhances effector function of CD4 and CD8 T cells with similar efficacy.

A costimulatory member of the TNFR family, 4-1BB, is expressed on activated T cells. Although some reports have suggested that 4-1BB is primarily involved in CD8 T cell activation, in this report we demonstrate that both CD4 and CD8 T cells respond to 4-1BB ligand (4-1BBL) with similar efficacy. CD4 and CD8 TCR transgenic T cells up-regulate 4-1BB, OX40, and CD27 and respond to 4-1BBL-mediated costimulation during a primary response to peptide Ag. 4-1BBL enhanced proliferation, cytokine production, and CTL effector function of TCR transgenic T cells. To compare CD4 vs CD8 responses to 4-1BBL under similar conditions of antigenic stimulation, we performed MLRs with purified CD4 or CD8 responders from CD28(+/+) and CD28(-/-) mice. We found that CD8 T cells produced IL-2 and IFN-gamma in a 4-1BBL-dependent manner, whereas under the same conditions the CD4 T cells produced IL-2 and IL-4. 4-1BBL promoted survival of CD4 and CD8 T cells, particularly at late stages of the MLR. CD4 and CD8 T cells both responded to anti-CD3 plus s4-1BBL with a similar cytokine profile as observed in the MLR. CD4 and CD8 T cells exhibited enhanced proliferation and earlier cell division when stimulated with anti-CD3 plus anti-CD28 compared with anti-CD3 plus 4-1BBL, and both subsets responded comparably to anti-CD3 plus 4-1BBL. These data support the idea that CD28 plays a primary role in initial T cell expansion, whereas 4-1BB/4-1BBL sustains both CD4 and CD8 T cell responses, as well as enhances cell division and T cell effector function.

Chemical chaperones enhance superantigen and conventional antigen presentation by HLA-DM-deficient as well as HLA-DM-sufficient antigen-presenting cells and enhance IgG2a production in vivo.

Chemical chaperones, first defined in studies of mutant cystic fibrosis transmembrane conductance regulator proteins, are small molecules that act as stabilizers of proteins in their native state and have the ability in some cases to rescue protein-folding mutants within cells. HLA-DM is an MHC II-specific molecular chaperone that facilitates peptide loading onto MHC II proteins and also stabilizes empty MHC II molecules prior to their acquisition of antigenic peptides. APC that lack HLA-DM exhibit quantitative defects in protein Ag as well as superantigen presentation. Here we show that both the superantigen and protein presentation defect in MHC II-transfected, HLA-DM-deficient T2 cells can be partially overcome by treating the APC with the chemical chaperones glycerol, DMSO, or trimethylamine oxide. These chemical chaperones also enhance superantigen and conventional Ag presentation by wild-type APC. In vivo, glycerol was found to act as an adjuvant and resulted in enhanced IgG2a production to trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH). In vitro, the enhancement of Ag presentation by chemical chaperones was found to take place at the level of the APC and took several hours to develop. Subcellular fractionation experiments show that HLA-DM enhances presentation of peptides by dense endosome fractions whereas chemical chaperones enhance presentation by light membrane fractions (early endosome or plasma membrane). The mechanism by which these chemical chaperones augment Ag presentation is not defined, but flow cytometric analysis suggests that the enhancement may be due to a subtle effect on the stability of several different proteins at the cell surface.

Quantitative defect in staphylococcal enterotoxin A binding and presentation by HLA-DM-deficient T2.Ak cells corrected by transfection of HLA-DM genes.

HLA-DM facilitates peptide acquisition by MHC class II proteins within the endosomes of APC by facilitating release of invariant chain peptide intermediates (CLIP) from the class II molecules. T2 cells have a deletion in the MHC II region which deletes HLA-DM and MHC II genes. T2 cells transfected with MHC class II proteins are defective in protein presentation, a defect that is corrected by HLA-DM transfection. Here we show that T2 cells transfected with Ak are also impaired in binding and presentation of the superantistaphylococcal enterotoxin A and that HLA-DM transfection corrects this defect. The poor ability of SEA to bind to Ak on DM-deficient cells is somewhat surprising since Ak has a low affinity for CLIP and is not predominantly occupied with CLIP on T2 cells compared to wide-type APC. These data suggest an influence of HLA-DM on the structure or composition of the Ak/peptide complex beyond its role in the release of invariant chain peptides.

Effect of HLA-DM transfection on hen egg lysozyme presentation by T2.Ak cells.

T2 cells have a large homozygous deletion in the MHC II region. Transfection of MHC class II genes into T2 cells allows presentation of peptide but not native protein Ags. This defect in protein presentation has been attributed to the lack of HLA-DM, an MHC class II-related protein that facilitates the release of an invariant chain peptide (CLIP) intermediate from nascent MHC class II proteins within the endocytic compartment of APC. Here, we show that Ak molecules within isolated late endosome fractions of T1.Ak (wild-type) vs T2.Ak (HLA-DM-deficient) bind biotin-HEL46-61 at comparable levels, consistent with previous observations that Ak molecules on T2 cells are not predominantly occupied with CLIP. However, Ak molecules in the late endosomes of T2.Ak fail to present peptide to a T hybrid, whereas the late endosomes from T1.Ak have no such defect. Transfection of HLA-DM A and B into T2.Ak partially restores protein Ag presentation by T2.Ak cells. These data suggest that HLA-DM can play a role in Ag presentation in addition to its role in CLIP release. However, even after DM transfection there remains a 10-fold difference in the dose-response curve for hen egg lysozyme presentation by T1.Ak vs T2.Ak/DM cells. In addition, HLA-DM transfection fails to restore presentation by late endosome fractions. The failure to fully restore Ag presentation in T2.Ak cells by DM transfection suggests that another gene product, required for efficient Ag presentation, may be absent from the late endosomes of T2.

Effects of MHC II conformation and pH on the recognition of peptide by T cells.

In this study we analysed the binding of the peptide HEL46-61 to purified Ak molecules which have been altered by site-directed mutagenesis at polymorphic positions to include amino acids from the Ad alpha-chain. We find that changes in the floor of the peptide binding groove, at positions 11, 14 and 28, abolish T cell recognition without changing peptide binding affinity. We further show that amino acid changes at these positions in the Ad molecule result in a conformationally altered molecule as evidenced by loss of binding of the Ad alpha specific monoclonal antibody K24. Thus the T cell receptor is highly sensitive to subtle changes in MHC II structure induced at sites that are unlikely to be involved in direct T cell contact. This has important implications with respect to allorecognition. The binding studies reported here were performed both at pH 7, to reflect binding of peptides at the cell surface, and at pH 5.5, to mimic binding in an intracellular acidic compartment. Binding to wild-type Ak was increased 2-3-fold at pH 5.5, whereas binding to some MHC II mutants was increased by greater than 20-fold at pH 5.5 relative to pH 7. These results show that the apparent peptide binding specificity for the mutants differs at pH 7 and 5.5, and suggest that caution should be used in defining the MHC-restriction of peptide epitopes at neutral pH.