Tumor-promoted constraints negatively affect cytotoxic T lymphocyte (CTL) trafficking to the tumor core and, as a result, inhibit tumor killing. The production of reactive nitrogen species (RNS) within the tumor microenvironment has been reported in mouse and human cancers. We describe a novel RNS-dependent posttranslational modification of chemokines that has a profound impact on leukocyte recruitment to mouse and human tumors. Intratumoral RNS production induces CCL2 chemokine nitration and hinders T cell infiltration, resulting in the trapping of tumor-specific T cells in the stroma that surrounds cancer cells. Preconditioning of the tumor microenvironment with novel drugs that inhibit CCL2 modification facilitates CTL invasion of the tumor, suggesting that these drugs may be effective in cancer immunotherapy. Our results unveil an unexpected mechanism of tumor evasion and introduce new avenues for cancer immunotherapy.

Background: The national programs for the harmonization of hemoglobin (Hb)A_1c measurements in the US [National Glycohemoglobin Standardization Program (NGSP)], Japan [Japanese Diabetes Society (JDS)/Japanese Society of Clinical Chemistry (JSCC)], and Sweden are based on different designated comparison methods (DCMs). The future basis for international standardization will be the reference system developed by the IFCC Working Group on HbA_1c Standardization. The aim of the present study was to determine the relationships between the IFCC Reference Method (RM) and the DCMs.

HbA1C is the stable glucose adduct to the N-terminal group of the beta-chain of HbA0. The measurement of HbA1c in human blood is most important for the long-term control of the glycaemic state in diabetic patients. Because there was no internationally agreed reference method the IFCC Working Group on HbA1c Standardization developed a reference method which is here described. In a first step haemoglobin is cleaved into peptides by the enzyme endoproteinase Glu-C, and in a second step the glycated and non-glycated N-terminal hexapeptides of the beta-chain obtained are separated and quantified by HPLC and electrospray ionisation mass spectrometry or in a two-dimensional approach using HPLC and capillary electrophoresis with UV-detection. Both principles give identical results. HbA1c is measured as ratio between the glycated and non-glycated hexapeptides. Calibrators consisting of mixtures of highly purified HbA1c and HbA0 are used. The analytical performance of the reference method has been evaluated by an international network of reference laboratories comprising laboratories from Europe, Japan and the USA. The intercomparison studies of the network showed excellent results with intra-laboratory CVs of 0.5 to 2% and inter-laboratory CVs of 1.4 to 2.3%. Possible interferences have been carefully investigated. Due to the higher specificity of the reference method the results are lower than those generated with most of the present commercial methods which currently are calibrated with unspecific designated comparison methods. The new reference method has been approved by the member societies of the International Federation of Clinical Chemistry and Laboratory Medicine and will be the basis for the future uniform standardization of HbA1c routine assays worldwide.

Subsets of rodent neurons are reported to express major histocompatibility complex class I (MHC-I), but such expression has not been reported in normal adult human neurons. Here we provide evidence from immunolabel, RNA expression and mass spectrometry analysis of postmortem samples that human catecholaminergic substantia nigra and locus coeruleus neurons express MHC-I, and that this molecule is inducible in human stem cell-derived dopamine (DA) neurons. Catecholamine murine cultured neurons are more responsive to induction of MHC-I by gamma-interferon than other neuronal populations. Neuronal MHC-I is also induced by factors released from microglia activated by neuromelanin or alpha-synuclein, or high cytosolic DA and/or oxidative stress. DA neurons internalize foreign ovalbumin and display antigen derived from this protein by MHC-I, which triggers DA neuronal death in the presence of appropriate cytotoxic T cells. Thus, neuronal MHC-I can trigger antigenic response, and catecholamine neurons may be particularly susceptible to T-cell-mediated cytotoxic attack.

Human CD4(+)CD25(hi)Foxp3(+)CD127(-) Treg and CD4(+)CD25(-)Foxp3(-) Tconv cell functions are governed by their metabolic requirements. Here we report a comprehensive comparative analysis between ex vivo human Treg and Tconv cells that comprises analyses of the proteomic networks in subcellular compartments. We identified a dominant proteomic signature at the metabolic level that primarily impacted the highly-tuned balance between glucose and fatty-acid oxidation in the two cell types. Ex vivo Treg cells were highly glycolytic while Tconv cells used predominantly fatty-acid oxidation (FAO). When cultured in vitro, Treg cells engaged both glycolysis and FAO to proliferate, while Tconv cell proliferation mainly relied on glucose metabolism. Our unbiased proteomic analysis provides a molecular picture of the impact of metabolism on ex vivo human Treg versus Tconv cell functions that might be relevant for therapeutic manipulations of these cells.

Cell therapy trials using cardiac-resident progenitor cells (CPCs) and bone marrow-derived mesenchymal stem/progenitor cells (BMCs) in patients after myocardial infarction have provided encouraging results. Exosomes, nanosized extracellular vesicles of endosomal origin, figure prominently in the bioactivities of these cells. However, a head-to-head comparison of exosomes from the two cell types has not been performed yet. CPCs and BMCs were derived from cardiac atrial appendage specimens and sternal bone marrow, respectively, from patients (n = 20; age, 69.9 ± 10.9) undergoing heart surgery for aortic valve disease and/or coronary artery disease. Vesicles were purified from cell conditioned media by centrifugation/filtration and ultracentrifugation. Vesicle preparations were predominantly composed of exosomes based on particle size and marker expression (CD9, CD63, CD81, Alix, and TSG-101). CPC-secreted exosomes prevented staurosporine-induced cardiomyocyte apoptosis more effectively than BMC-secreted exosomes. In vivo, CPC-secreted exosomes reduced scar size and improved ventricular function after permanent coronary occlusion in rats more efficiently than BMC-secreted exosomes. Both types of exosomes stimulated blood vessel formation. CPC-secreted exosomes, but not BMC-derived exosomes, enhanced ventricular function after ischaemia/reperfusion. Proteomics profiling identified pregnancy-associated plasma protein-A (PAPP-A) as one of the most highly enriched proteins in CPC vs. BMC exosomes. The active form of PAPP-A was detected on CPC exosome surfaces. These vesicles released insulin-like growth factor-1 (IGF-1) via proteolytic cleavage of IGF-binding protein-4 (IGFBP-4), resulting in IGF-1 receptor activation, intracellular Akt and ERK1/2 phosphorylation, decreased caspase activation, and reduced cardiomyocyte apoptosis. PAPP-A knockdown prevented CPC exosome-mediated cardioprotection both in vitro and in vivo. These results suggest that CPC-secreted exosomes may be more cardioprotective than BMC-secreted exosomes, and that PAPP-A-mediated IGF-1 release may explain the benefit. They illustrate a general mechanism whereby exosomes may function via an active protease on their surface, which releases a ligand in proximity to the transmembrane receptor bound by the ligand.