Gerd Birkenmeier

Mast cell-derived exosomes induce phenotypic and functional maturation of dendritic cells and elicit specific immune responses in vivo

Mast cells (MCs) are considered major players in IgE-mediated allergic responses, but have also recently been recognized as active participants in innate as well as specific immune responses. Recent work provided evidence that MCs are able to activate B and T lymphocytes through the release of vesicles called exosomes. Here we demonstrate that exosomes, which are located in the endocytic pathway, harbor exogenous Ags that associate with other molecules endowed with immunomodulatory functions, including 60- and 70-kDa heat shock proteins. Administration to naive mice of Ag-containing exosomes in the absence of conventional adjuvants elicits specific Ab responses across the MHC II haplotype barrier. We demonstrate that MC-exosomes induce immature dendritic cells (DCs) to up-regulate MHC class II, CD80, CD86, and CD40 molecules and to acquire potent Ag-presenting capacity to T cells. Uptake and processing of Ag-associated exosomes by endogenous DCs were also demonstrated. Finally, exosome-associated heat shock proteins are critical for the acquisition by DCs of the Ag-presenting function. This work demonstrates a heretofore unrecognized collaborative interaction between MCs and DCs leading to the elicitation of specific immune responses.

Curcumin inhibits glyoxalase 1: a possible link to its anti-inflammatory and anti-tumor activity.

Background Glyoxalases (Glo1 and Glo2) are involved in the glycolytic pathway by detoxifying the reactive methylglyoxal (MGO) into D-lactate in a two-step reaction using glutathione (GSH) as cofactor. Inhibitors of glyoxalases are considered as anti-inflammatory and anti-carcinogenic agents. The recent finding that various polyphenols modulate Glo1 activity has prompted us to assess curcumins potency as an Glo1 inhibitor. Methodology/Principal Findings Cultures of whole blood cells and tumor cell lines (PC-3, JIM-1, MDA-MD 231 and 1321N1) were set up to investigate the effect of selected polyphenols, including curcumin, on the LPS-induced cytokine production (cytometric bead-based array), cell proliferation (WST-1 assay), cytosolic Glo1 and Glo2 enzymatic activity, apoptosis/necrosis (annexin V-FITC/propidium iodide staining; flow cytometric analysis) as well as GSH and ATP content. Results of enzyme kinetics revealed that curcumin, compared to the polyphenols quercetin, myricetin, kaempferol, luteolin and rutin, elicited a stronger competitive inhibitory effect on Glo1 (Ki = 5.1±1.4 µM). Applying a whole blood assay, IC50 values of pro-inflammatory cytokine release (TNF-α, IL-6, IL-8, IL-1β) were found to be positively correlated with the Ki-values of the aforementioned polyphenols. Moreover, whereas curcumin was found to hamper the growth of breast cancer (JIMT-1, MDA-MB-231), prostate cancer PC-3 and brain astrocytoma 1321N1 cells, no effect on growth or vitality of human primary hepatocytes was elucidated. Curcumin decreased D-lactate release by tumor cells, another clue for inhibition of intracellular Glo1. Conclusions/Significance The results described herein provide new insights into curcumins biological activities as they indicate that inhibition of Glo1 by curcumin may result in non-tolerable levels of MGO and GSH, which, in turn, modulate various metabolic cellular pathways including depletion of cellular ATP and GSH content. This may account for curcumins potency as an anti-inflammatory and anti-tumor agent. The findings support the use of curcumin as a potential therapeutic agent.

Interleukin-17-producing T helper cells in autoimmunity

With all the incredible progress in scientific research over the past two decades, the trigger of the majority of autoimmune disorders remains largely elusive. Research on the biology of T helper type 17 (T(H)17) cells over the last decade not only clarified previous observations of immune regulations and disease manifestations, but also provided considerable information on the signaling pathways mediating the effects of this lineage and its seemingly dual role in fighting the invading pathogens on one hand, and in frightening the host by inducing chronic inflammation and autoimmunity on the other hand. In this context, recent reports have implicated T(H)17 cells in mediating host defense as well as a growing list of autoimmune diseases in genetically-susceptible individuals. Herein, we summarize the current knowledge on T(H)17 in autoimmunity with emphasis on its differentiation factors and some mechanisms involved in initiating pathological events of autoimmunity.

Immobilized metal ion affinity partitioning, a method combining metal-protein interaction and partitioning of proteins in aqueous two-phase systems.

Immobilized metal ions were used for the affinity extraction of proteins in aqueous two-phase systems composed of polyethylene glycol (PEG) and dextran or PEG and salt. Soluble chelating polymers were prepared by covalent attachment of metal-chelating groups to PEG. The effect on the partitioning of proteins of such chelating PEG derivatives coordinated with different metal ions is demonstrated. The proteins studied were alpha 2-macroglobulin, tissue plasminogen activator, superoxide dismutase and monoclonal antibodies. The results indicate that immobilized metal ion affinity partitioning provides excellent potential for the extraction of proteins.

High-resolution mapping of the 8p23.1 beta-defensin cluster reveals strictly concordant copy number variation of all genes.

One unexpected feature of the human genome is the high structural variability across individuals. Frequently, large regions of the genome show structural polymorphisms and many vary in their abundance. However, accurate methods for the characterization and typing of such copy number variations (CNV) are needed. The defensin cluster at the human region 8p23.1 is one of the best studied CNV regions due to its potential clinical relevance for innate immunity, inflammation, and cancer. The region can be divided into two subclusters, which harbor predominantly either alpha‐ or beta‐defensin genes. Previous studies assessing individual copy numbers gave different results regarding whether the complete beta‐defensin cluster varies or only particular genes therein. We applied multiplex ligation‐dependent probe amplification (MLPA) to measure defensin locus copy numbers in 42 samples. The data show strict copy number concordance of all 10 loci typed within the beta‐defensin cluster in each individual, while seven loci within the alpha‐defensin cluster are consistently found as single copies per chromosome. The exception is DEFA3, which is located within the alpha‐defensin cluster and was found to also differ in copy number interindividually. Absolute copy numbers ranged from two to nine for the beta‐defensin cluster and zero to four for DEFA3. The CNV‐typed individuals, including HapMap samples, are publicly available and may serve as a universal reference for absolute copy number determination. On this basis, MLPA represents a reliable technique for medium‐ to high‐throughput typing of 8p23.1 defensin CNV in association studies for diverse clinical phenotypes. Hum Mutat 0,1–8, 2008.

Posttranslational Modification of Human Glyoxalase 1 Indicates Redox-Dependent Regulation

Background Glyoxalase 1 (Glo1) and glyoxalase 2 (Glo2) are ubiquitously expressed cytosolic enzymes that catalyze the conversion of toxic α-oxo-aldehydes into the corresponding α-hydroxy acids using L-glutathione (GSH) as a cofactor. Human Glo1 exists in various isoforms; however, the nature of its modifications and their distinct functional assignment is mostly unknown. Methodology/Principal Findings We characterized native Glo1 purified from human erythrocytes by mass spectrometry. The enzyme was found to undergo four so far unidentified posttranslational modifications: (i) removal of the N-terminal methionine 1, (ii) N-terminal acetylation at alanine 2, (iii) a vicinal disulfide bridge between cysteine residues 19 and 20, and (iv) a mixed disulfide with glutathione on cysteine 139. Glutathionylation of Glo1 was confirmed by immunological methods. Both, N-acetylation and the oxidation state of Cys19/20, did not impact enzyme activity. In contrast, glutathionylation strongly inhibited Glo1 activity in vitro. The discussed mechanism for enzyme inhibition by glutathionylation was validated by molecular dynamics simulation. Conclusion/Significance It is shown for the first time that Glo1 activity directly can be regulated by an oxidative posttranslational modification that was found in the native enzyme, i.e., glutathionylation. Inhibition of Glo1 by chemical reaction with its co-factor and the role of its intramolecular disulfides are expected to be important factors within the context of redox-dependent regulation of glucose metabolism in cells.

Cerebral small vessel disease-induced Apolipoprotein E leakage is associated with Alzheimer disease and the accumulation of amyloid [beta]-protein in perivascular astrocytes

Apolipoprotein E (apoE) plays a role in the pathogenesis of Alzheimer disease (AD). It is involved in the receptor-mediated cellular clearance of the amyloid &bgr;-protein (A&bgr;) and in the perivascular drainage of the extracellular fluid. Microvascular changes are also associated with AD and have been discussed as a possible reason for altered perivascular drainage. To further clarify the role of apoE in the perivascular and vascular pathology in AD patients, we studied its occurrence and distribution in the perivascular space, the perivascular neuropil, and in the vessel wall of AD and control cases with and without small vessel disease (SVD). Apolipoprotein E was found in the perivascular space and in the neuropil around arteries of the basal ganglia from control and AD cases disclosing no major differences. Western blot analysis of basal ganglia tissue also revealed no significant differences pertaining to the amount of full-length and C-terminal truncated apoE in AD cases compared with controls. In contrast, A&bgr; occurred in apoE-positive perivascular astrocytes in AD cases but not in controls. In blood vessels, apoE and immunoglobulin G were detected within the SVD-altered vessel wall. The severity of SVD was associated with the occurrence of apoE in the vessel wall and with that of A&bgr; in perivascular astrocytes. These results point to an important role of apoE in the perivascular clearance of A&bgr; in the human brain. The occurrence of apoE and immunoglobulin G in SVD lesions and in the perivascular space suggests that the presence of SVD results in plasma-protein leakage into the brain. It is therefore tempting to speculate that apoE represents a pathogenetic link between SVD and AD.

α2-Macroglobulin-Mediated Degradation of Amyloid β1–42: A Mechanism to Enhance Amyloid β Catabolism

Abstract Peptides derived from proteolytic degradation of the amyloid precursor protein, e.g., amyloid β (Aβ), are considered to be central to the pathology of Alzheimers disease (AD). Soluble Aβ is present in measurable concentrations in cerebrospinal fluid and blood. There are indications that soluble Aβ present in circulation can cross the blood–brain barrier via transcytosis mediated by brain capillary endothelial cells. It implies that Aβ originating from circulation may contribute to vascular and parenchymal Aβ deposition in AD. Enhancing of Aβ catabolism mediated by proteolytic degradation or receptor-mediated endocytosis could be a key mechanism to maintain low concentrations of soluble Aβ. To launch Aβ clearance we have exploited the Aβ-degrading activity of diverse α2-macroglobulin (α2-M)–proteinase complexes. Complexes with trypsin, α-chymotrypsin, and bromelain strongly degrade 125I-Aβ1–42 whereas complexes with endogenous proteinases, e.g., plasmin and prostate-specific antigen, were not effective. Aβ degradation by the complexes was not inhibited by α1-antichymotrypsin and soybean trypsin inhibitor which normally would inactivate the free serine proteinases. A prerequisite for Aβ degradation is its binding to specific binding sites in α2-M that may direct Aβ to the active site of the caged proteinase. Ex vivo, enhanced degradation of 125I-Aβ1–42 in blood could be achieved upon oral administration of high doses of proteinases to volunteers. These results suggest that up-regulation of Aβ catabolism could probably reduce the risk of developing AD by preventing Aβ accumulation in brain and vasculature.

α2-Macroglobulin Inhibits the Malignant Properties of Astrocytoma Cells by Impeding β-Catenin Signaling

Targets that could improve the treatment of brain tumors remain important to define. This study of a transformation-associated isoform of alpha2-macroglobulin (A2M*) and its interaction with the low-density lipoprotein receptor-related protein-1 (LRP1) suggests a new mechanism for abrogating the malignant potential of astrocytoma cells. LRP1 bound A2M* found to be associated with an inhibition of tumor cell proliferation, migration, invasion, spheroid formation, and anchorage-independent growth. Transcriptional studies implicated effects on the Wnt/beta-catenin signaling pathway. Notably, LRP1 antibodies could phenocopy the effects of A2M*. Our findings suggest a pathway of tumor suppression in astrocytoma that might be tractable to therapeutic exploitation.

Production of conformation-specific monoclonal antibodies against α2 macroglobulin and their use for quantitation of total and transformed α2 macroglobulin in human blood

Abstract Monoclonal antibodies against the human proteinase inhibitor, α 2 macroglobulin, have been produced by immunizing BALB/c mice with α 2 macroglobulin reacted with methylamine. Two antibodies have been characterized in detail with respect to their binding to native α 2 macroglobulin and to different derivatives of the inhibitor. The antibody α-1 was found to recognize only those forms of the inhibitor which were transformed by reaction with different proteinaseso with methylamine. Binding of α-1 was mapped to a specific epitope localized within a distance of 138 amino acid residues from the C terminal end of α 2 macroglobulin. The C terminal end is assumed to be exposed during the transformation of the inhibitor and harbours the receptor recognition site. The monoclonal antibody α-11 was found to bind to all forms of the inhibitor indicating that its epitope is located in a region not involved in major conformational changes of the inhibitor. On the basis of the different reactivity patterns of α-1 and α-11 two enzyme-linked immunosorption assays were established for quantitation of total and transformed α 2 macroglobulin in human blood. The concentration of the two forms have been determined in a population of 114 healthy individuals giving values of 254 ± 6.6 mg/dl (mean ± SEM) of total α 2 macroglobulin and 1.07 ± 0.05 mg/dl (means ± SEM) of the transformed inhibitor.