Wolfgang E. Kaminski

The human ABC transporter pseudogene family: Evidence for transcription and gene-pseudogene interference

BackgroundPseudogenes are an integral component of the human genome. Little attention, however, has so far been paid to the phenomenon that some pseudogenes are transcriptionally active. Recently, we demonstrated that the human ortholog of the rodent testis-specific ATP-binding cassette (ABC) transporter Abca17 is a ubiquitously transcribed pseudogene (ABCA17P). The aim of the present study was to establish a complete inventory of all ABC transporter pseudogenes in the human genome and to identify transcriptionally active ABC transporter pseudogenes. Moreover, we tested the hypothesis that a regulatory interdependency exists between ABC transporter pseudogenes and their parental protein coding equivalents.ResultsSystematic bioinformatic analysis revealed the existence of 22 ABC transporter pseudogenes within the human genome. We identified two clusters on chromosomes 15 and 16, respectively, which harbor almost half of all pseudogenes (n = 10). Available information from EST and mRNA databases and RT-PCR expression profiling indicate that a large portion of the ABC transporter pseudogenes (45%, n = 10) are transcriptionally active and some of them are expressed as alternative splice variants. We demonstrate that both pseudogenes of the pseudoxanthoma elasticum gene ABCC6, ABCC6P1 and ABCC6P2, are transcribed. ABCC6P1 and ABCC6 possess near-identical promoter sequences and their tissue-specific expression profiles are strikingly similar raising the possibility that they form a gene-pseudogene dual transcription unit. Intriguingly, targeted knockdown of the transcribed pseudogene ABCC6P1 resulted in a significant reduction of ABCC6 mRNA expression levels.ConclusionThe human genome contains a surprisingly small number of ABC transporter pseudogenes relative to other known gene families. They are unevenly distributed across the chromosomes. Importantly, a significant portion of the ABC transporter pseudogenes is transcriptionally active. The downregulation of ABCC6 mRNA levels by targeted suppression of the expression of its pseudogene ABCC6P1 provides evidence, for the first time, for a regulatory interdependence of a transcribed pseudogene and its protein coding counterpart in the human genome.

A TNF-Regulated Recombinatorial Macrophage Immune Receptor Implicated in Granuloma Formation in Tuberculosis

Macrophages play a central role in host defense against mycobacterial infection and anti- TNF therapy is associated with granuloma disorganization and reactivation of tuberculosis in humans. Here, we provide evidence for the presence of a T cell receptor (TCR) αβ based recombinatorial immune receptor in subpopulations of human and mouse monocytes and macrophages. In vitro, we find that the macrophage-TCRαβ induces the release of CCL2 and modulates phagocytosis. TNF blockade suppresses macrophage-TCRαβ expression. Infection of macrophages from healthy individuals with mycobacteria triggers formation of clusters that express restricted TCR Vβ repertoires. In vivo, TCRαβ bearing macrophages abundantly accumulate at the inner host-pathogen contact zone of caseous granulomas from patients with lung tuberculosis. In chimeric mouse models, deletion of the variable macrophage-TCRαβ or TNF is associated with structurally compromised granulomas of pulmonary tuberculosis even in the presence of intact T cells. These results uncover a TNF-regulated recombinatorial immune receptor in monocytes/macrophages and demonstrate its implication in granuloma formation in tuberculosis.

A-Subclass ATP-Binding Cassette Proteins in Brain Lipid Homeostasis and Neurodegeneration

The A-subclass of ATP-binding cassette (ABC) transporters comprises 12 structurally related members of the evolutionarily highly conserved superfamily of ABC transporters. ABCA transporters represent a subgroup of “full-size” multispan transporters of which several members have been shown to mediate the transport of a variety of physiologic lipid compounds across membrane barriers. The importance of ABCA transporters in human disease is documented by the observations that so far four members of this protein family (ABCA1, ABCA3, ABCA4, ABCA12) have been causatively linked to monogenetic disorders including familial high-density lipoprotein deficiency, neonatal surfactant deficiency, degenerative retinopathies, and congenital keratinization disorders. Recent research also point to a significant contribution of several A-subfamily ABC transporters to neurodegenerative diseases, in particular Alzheimer’s disease (AD). This review will give a summary of our current knowledge of the A-subclass of ABC transporters with a special focus on brain lipid homeostasis and their involvement in AD.

Benefit of combining quantitative cardiac CT parameters with troponin I for predicting right ventricular dysfunction and adverse clinical events in patients with acute pulmonary embolism.

OBJECTIVE To prospectively evaluate the diagnostic accuracy of quantitative cardiac CT parameters alone and in combination with troponin I for the assessment of right ventricular dysfunction (RVD) and adverse clinical events in patients with acute pulmonary embolism (PE). MATERIALS AND RESULTS This prospective study had institutional review board approval and was HIPAA compliant. In total 83 patients with confirmed PE underwent echocardiography and troponin I serum level measurements within 24 h. Three established cardiac CT measurements for the assessment of RVD were obtained (RV/LVaxial, RV/LV4-CH, and RV/LVvolume). CT measurements and troponin I serum levels were correlated with RVD found on echocardiography and adverse clinical events according to Management Strategies and Prognosis in Pulmonary Embolism Trial-3 (MAPPET-3 criteria. 31 of 83 patients with PE had RVD on echocardiography and 39 of 83 patients had adverse clinical events. A RV/LVvolume ratio>1.43 showed the highest area under the curve (AUC) (0.65) for the prediction of adverse clinical events when compared to RV/LVaxial, RV/LV4Ch and troponin I. The AUC for the detection of RVD of RV/LVaxial, RV/LV4Ch, RV/LVvolume, and troponin I were 0.86, 0.86, 0.92, and 0.69, respectively. Combination of RV/LVaxial, RV/LV4Ch, RV/LVvolume with troponin I increased the AUC to 0.87, 0.87 and 0.93, respectively. CONCLUSION A combination of cardiac CT parameters and troponin I measurements improves the diagnostic accuracy for detecting RVD and predicting adverse clinical events if compared to either test alone.

The neutrophil recombinatorial TCR-like immune receptor is expressed across the entire human life span but repertoire diversity declines in old age.

Recent evidence has revealed the existence of T cell receptor (TCR) αβ-based recombinatorial immune receptors in phagocytes. Here, we performed a systematic survey of the variable β-chain repertoires of the neutrophil TCR-like αβ immunoreceptor (referred to as TCRL(n)αβ) in defined cohorts of young and old individuals. Peripheral blood CD15(+) neutrophils from young adults (age 30 ± 7 years, n=12) expressed an average number of 13 ± 6 distinct TCRL(n) Vβ-chains from the total pool of 25 human Vβ-chains. Neutrophils from aged subjects (age 76 ± 6 years, n=12) also consitutively express the TCRL(n), however, only a small number of Vβ-chains is used (4 ± 2). Consistent with this, the average number of expressed CDR3 Vβ length variants was fourfold higher in young individuals than in aged subjects (33 ± 24 vs. 8 ± 3). Young adults showed broad usage of all TCRL(n) Vβ-chains. In contrast, >70years individuals displayed a striking repertoire polarization towards the TCRL(n) Vβ1 and Vβ5b chains and a high degree of Vβ5b clonotype sharing. Our study reveals broad TCRL(n) repertoire diversity in young adults and demonstrates that the neutrophil variable immune receptor is expressed throughout the entire human life span. The marked decline in TCRL(n) repertoire diversity in old age identifies a novel mechanism of immunosenescence in neutrophils.

An autoimmune double attack

Institute for Clinical Chemistry (T Fuchs PhD, S Schneider MD, M Neumaier MD, Prof W E Kaminski MD), Department of Medicine (J Kruth MD), and Institute for Transfusion Medicine and Immunology (T J Schulze MD); University of Heidelberg Medical Faculty Mannheim, Mannheim, Germany; Department of Haematology, Haemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany (K Puellmann MD); and Department of Surgery, University of Gottingen, Gottingen, Germany (A W Beham MD)

A second combinatorial immune receptor in monocytes/macrophages is based on the TCRγδ

Recent evidence indicates that monocytes and macrophages express T cell receptor (TCR)αβ-like combinatorial immune receptors. Here, we demonstrate the presence of a second recombinatorial immunoreceptor, which is structurally based on the TCR γ- and δ-chains, in human and murine monocytes and differentially activated macrophages (referred to here as TCRL(m)γδ). In vitro, infection of macrophages with mycobacteria and gram positive or gram negative bacteria induced expression of donor-specific and differential TCRL(m)Vδ repertoires indicating that the novel immunoreceptor represents a dynamic flexible host defense system that responds to bacterial challenge. In vivo, we find that TCRL(m)γδ bearing macrophages, which express highly restricted repertoires of the antigen-binding Vδ chain, accumulate in the cerebrospinal fluid in acute bacterial meningitis and in advanced lesions of atherosclerosis. These results identify an as yet unrecognized monocyte/macrophage subpopulation that bears combinatorial TCRL(m)γδ immune receptors, and is associated with both acute and chronic inflammatory diseases. Moreover, they indicate that the monocytic lineage uses the same bipartite system of TCRαβ/TCRγδ-based combinatorial immune receptors that is present in T cells. Our findings suggest specific roles of monocytes/macrophages in various inflammatory conditions and lend further evidence that flexible immune recognition in higher vertebrates operates on a broader cellular basis than previously thought.

A combinatorial αβ T cell receptor expressed by macrophages in the tumor microenvironment.

Recent evidence indicates the presence of macrophage subpopulations that express the TCRαβ in two major inflammatory diseases, tuberculosis and atherosclerosis. Inflammation is also a well-established attribute of cancer progression and macrophages are one of the major immune cells that infiltrate tumors. Here, we demonstrate that the macrophage-TCRαβ is expressed in the tumor microenvironment of human and murine malignancies. We identify TCRαβ+ macrophages in each case of four randomly selected distinct human tumor entities. In human tumor tissues, the TCRαβ expressed by macrophages in the tumor microenvironment is a combinatorial and individual-specific immune receptor. Furthermore, we routinely find TCRαβ+ macrophage subpopulations in experimental tumors (TS/A, mammary adenocarcinoma) which we induced both in normal mice and mice deficient in the macrophage receptor stabilin-1. Expression of the combinatorial murine tumor macrophage TCRαβ is individual-specific and independent of stabilin-1. These results demonstrate that TCRαβ expression is a characteristic feature of macrophages in the tumor microenvironment and identify an as yet unrecognized flexible element in the macrophage-based host response to tumors.

[ABC A-subclass transporters--key regulators of molecular lipid transport].

ZusammenfassungDie kontrollierte Aufnahme und Freisetzung von Lipidsubstanzen ist eine wesentliche Eigenschaft lebender Zellen. Eine Vielzahl von Faktoren ist in diese komplexen Transportprozesse involviert.Die kürzliche Identifizierung einer neuen Klasse von Transportmolekülen, der A-Subfamilie der „ATP-binding cassette“-(ABC-)Transporter, hat grundlegende neue Erkenntnisse auf dem Gebiet des molekularen Lipidtransports erbracht und zugleich deren Bedeutung als Krankheitsgene in wichtigen physiologischen Systemen aufgedeckt.Vererbte Defekte in ABC-A-Transportergenen führen zu ausgeprägten Krankheitsbildern im kardiovaskulären, respiratorischen und visuellen System sowie in der Haut. Darüber hinaus wird zunehmend deutlich, dass ABC-A-Transporter relevante Pathogenitätsfaktoren für komplexe Erkrankungen mit hoher Inzidenz wie die Atherosklerose, die Alzheimer-Demenz und die altersabhängige Makuladegeneration darstellen.Dieser Beitrag stellt die neue Genfamilie der ABC-A-Transporter vor und beleuchtet deren Bedeutung für die klinische Medizin.AbstractThe controlled uptake and release of lipid compounds is a hallmark feature of living cells. Numerous factors have been implicated in these complex transmembrane transport processes.The recent discovery of a novel class of transporter molecules, the group of A-subclass ATP-binding cassette (ABC) transporters, has brought new insights into the molecular basis of cellular lipid transport. Available evidence indicates that individual ABC A-subclass transporters function as key components of highly specialized cellular lipid export machineries in major physiological systems and, when defective, cause hereditary diseases in the cardiovascular, respiratory, visual and integumentary systems, respectively.Intriguingly, a steadily growing body of evidence suggests that ABC A lipid transporters play important roles in the pathogenesis of complex disorders with high incidence including atherosclerosis, Alzheimers disease and age-related macula degeneration.The present article reviews the biology of this emerging group of proteins and their implication in human pathologies.

Makrophagen exprimieren variable Immunrezeptoren und sind bei der Arteriosklerose von Bedeutung

Background: Traditionally, specific immune recognition is thought to be restricted to lymphoid cells. Recent evidence of T cell receptor (TCR) expression in neutrophil and eosinophil granulocytes, however, indicates that variable immunoreceptors are not restricted to lymphocytes. In this study, we tested whether monocytes/macrophages, the second major population of professional phagocytes next to neutrophils, express a variable immunoreceptor. Methods and Findings: We demonstrate that subpopulations of monocytes, monocyte-derived macrophages and tissue macrophages express a TCRab immunoreceptor and essential components of the TCR signaling complex. Consistent with V(D)J rearrangement of the TCRb locus, we find individual-specific expression of TCR repertoires in human monocytes/macrophages. In vitro, Th1/Th2 cytokines (IFNg, IL-4) and forced import and export of cholesterol elicit expression of differential TCR Vab-repertoires in monocyte derived macrophages from healthy individuals. This indicates that normal macrophages have the capacity to respond to various stimuli in a flexible manner. Engagement of the macrophage-TCR stimulates secretion of the major monocyte chemoattractant MCP-1. In vivo, analysis of freshly obtained artery specimens from patients (n = 10) with severe carotid atherosclerosis consistently revealed the excessive presence of TCRab+ macrophages that express highly restricted Vb repertoires including Vb16 and Vb22. Similar results were obtained for advanced coronary artery disease. Experimental atherosclerosis in mouse carotids induces accumulation of TCRab bearing macrophages in the vascular wall and TCR deficient rag-/- mice have a reduced inflammatory response in their media relative to wildtype controls. Conclusions: We have identified an as yet undiscovered myeloid machinery for variable immune recognition in macrophages which is implicated in a major inflammatory disease as paradigmatically shown for atherosclerosis. These results may further our understanding of macrophage-dependent clinical diseases.