The macrophage-TCRαß is a cholesterol-responsive combinatorial immune receptor and implicated in atherosclerosis.

Recent evidence indicates constitutive expression of a recombinatorial TCRαß immune receptor in mammalian monocytes and macrophages. Here, we demonstrate in vitro that macrophage-TCRß repertoires are modulated by atherogenic low density cholesterol (LDL) and high-density cholesterol (HDL). In vivo, analysis of freshly obtained artery specimens from patients with severe carotid atherosclerosis reveals massive abundance of TCRαß(+) macrophages within the atherosclerotic lesions. Experimental atherosclerosis in mouse carotids induces accumulation of TCR bearing macrophages in the vascular wall and TCR deficient rag(-/-) mice have an altered macrophage-dependent inflammatory response. We find that the majority of TCRαß bearing macrophages are localized in the hot spot regions of the atherosclerotic lesions. Advanced carotid artery lesions express highly restricted TCRαß repertoires that are characterized by a striking usage of the Vß22 and Vß16 chains. This together with a significant degree of interindividual lesion repertoire sharing suggests the existence of atherosclerosis-associated TCRαß signatures. Our results implicate the macrophage-TCRαß combinatorial immunoreceptor in atherosclerosis and thus identify an as yet unknown adaptive component in the innate response-to-injury process that underlies this macrophage-driven disease.

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) αß based recombinatorial immune receptor in subpopulations of human and mouse monocytes and macrophages. In vitro, we find that the macrophage-TCRαß induces the release of CCL2 and modulates phagocytosis. TNF blockade suppresses macrophage-TCRαß expression. Infection of macrophages from healthy individuals with mycobacteria triggers formation of clusters that express restricted TCR Vß repertoires. In vivo, TCRαß 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-TCRαß 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.

Opsonization-independent antigen-specific recognition by myeloid phagocytes expressing monoclonal antibodies.

This report demonstrates a novel class of innate immune cells designated "variable immunoreceptor-expressing myeloids" (VIREMs). Using single-cell transcriptomics and genome-wide epigenetic profiling, we establish that VIREMs are myeloid cells unrelated to lymphocytes. We visualize the phenotype of B-VIREMs that are capable of genetically recombining and expressing antibody genes, the exclusive hallmark function of B lymphocytes. These cells, designated B-VIREMs, display monoclonal antibody cell surface signatures and regularly circulate in the blood of healthy individuals. Single-cell data reveal clonal expansion of circulating B-VIREMs as a dynamic response to disease stimuli. Live-cell imaging models suggest that B-VIREMs load their own Fc receptors with endogenous antibodies during vesicle transport to the cell surface. A first cloned B-VIREM-derived antibody (Vab1) specifically binds stomatin, a ubiquitous scaffold protein that is strictly expressed intracellularly, allowing Vab1-bearing macrophages to phagocytose cell debris without requiring prior opsonization. Our results suggest important antigen-specific tissue maintenance functionalities in these innate immune cells.

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) αß-based recombinatorial immune receptors in phagocytes. Here, we performed a systematic survey of the variable ß-chain repertoires of the neutrophil TCR-like αß immunoreceptor (referred to as TCRL(n)αß) 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) Vß-chains from the total pool of 25 human Vß-chains. Neutrophils from aged subjects (age 76 ± 6 years, n=12) also consitutively express the TCRL(n), however, only a small number of Vß-chains is used (4 ± 2). Consistent with this, the average number of expressed CDR3 Vß 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) Vß-chains. In contrast, >70years individuals displayed a striking repertoire polarization towards the TCRL(n) Vß1 and Vß5b chains and a high degree of Vß5b 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.

Murine RAW Macrophages Are a Suitable Model to Study the CD3 Signaling in Myeloid Cells.

In recent years, a growing body of evidence has shown the presence of a subpopulation of macrophages that express CD3, especially in the context of mycobacterial infections. Despite these findings, the function of these cells has been poorly understood. Furthermore, the low frequency of CD3+ macrophages in humans limits the study of this subpopulation. This work aimed to evaluate the expression of CD3 in a murine macrophage cell line and its potential for the study of CD3 signaling. The murine macrophage cell line RAW was used to evaluate CD3 expression at the transcriptional and protein levels and the effect of in vitro infection with the Mycobacterium bovis Bacillus Calmette-Guérin (BCG) on these. Our data showed that RAW macrophages express CD3, both the ε and ζ chains, and it is further increased at the transcriptional level after BCG infection. Furthermore, our data suggest that CD3 can be found on the cell surface and intracellularly. However, this molecule is internalized constantly, mainly after activation with anti-CD3 stimulus, but interestingly, it is stably maintained at the transcriptional level. Finally, signaling proteins such as NFAT1, c-Jun, and IKK-α are highly expressed in RAW macrophages. They may play a role in the CD3-controlled signaling pathway to deliver inflammatory cytokines such as TNF and IL-6. Our study provides evidence to support that RAW cells are a suitable model to study the function and signaling of the CD3 complex in myeloid cells.

Evaluation is Key: Providing Appropriate Evaluation Measures for Participatory and User-Centred Design Processes of Healthcare IT.

INTRODUCTION: The increasing availability of healthcare IT has the potential to improve the integration of health services. Existing projects developing healthcare IT mostly disregard the potential and importance of incorporating user feedback and proper evaluation measures to gain user feedback throughout the development process. We therefore provide methodological guidance for evaluation in a stepwise user-centred design process. METHODS: Based on a literature review we propose adequate methods for data collection in each phase of participatory and user-centred healthcare IT development. In order to provide an orientation within the plethora of development processes used in practice, we consolidate a generic blueprint process from the literature review. The applicability of our methodological guidance is shown in three diverse use cases from the field of integrated care. RESULTS: From 14 literature items, we identified common evaluation methods including, among others, interviews, focus groups, and surveys. These methods can be associated to six typical development phases that could be derived from research: State of the Art Research, Requirement Analysis, Conceptual Prototype, Preliminary Prototype, Full Prototype, Full Application. The use cases demonstrate the value of qualitative methods and mixed methods designs. DISCUSSION: Our methodological guidance has proven applicable for designing healthcare IT solutions from scratch - both for patient and professional settings - and to develop a platform for combining existing component-based solutions. In integrated care settings, where a wide range of stakeholders with multiple needs exist, we thus provide methodological guidance on how to involve users in the development process. CONCLUSION: Our stepwise methodological guidance helps to design and properly evaluate healthcare IT solutions, which meet the user needs and requirements, for integrated care settings.

Trilineage Sequencing Reveals Complex TCRß Transcriptomes in Neutrophils and Monocytes Alongside T Cells.

Recent findings indicate the presence of T cell receptor (TCR)-based combinatorial immune receptors beyond T cells in neutrophils and monocytes/macrophages. In this study, using a semiquantitative trilineage immune repertoire sequencing approach as well as under rigorous bioinformatic conditions, we identify highly complex TCRß transcriptomes in human circulating monocytes and neutrophils that separately encode repertoire diversities one and two orders of magnitude smaller than that of T cells. Intraindividual transcriptomic analyses reveal that neutrophils, monocytes, and T cells express distinct TCRß repertoires with less than 0.1% overall trilineage repertoire sharing. Interindividual comparison shows that in all three leukocyte lineages, the vast majority of the expressed TCRß variants are private. We also find that differentiation of monocytes into macrophages induces dramatic individual-specific repertoire shifts, revealing a surprising degree of immune repertoire plasticity in the monocyte lineage. These results uncover the remarkable complexity of the two phagocyte-based flexible immune systems which until now has been hidden in the shadow of T cells.

The Role of a Decision Support System in Back Pain Diagnoses: A Pilot Study.

It is the main goal of this study to investigate the concordance of a decision support system and the recommendation of spinal surgeons regarding back pain. 111 patients had to complete the decision support system. Furthermore, their illness was diagnosed by a spinal surgeon. The results showed significant medium relation between the DSS and the diagnosis of the medical doctor. Besides, in almost 50% of the cases the recommendation for the treatment was concordant and overestimation occurred more often than underestimation. The results are discussed in relation to the "symptom checker" literature and the claim of further evaluations.

Circulating monocytes and tumor-associated macrophages express recombined immunoglobulins in glioblastoma patients.

BACKGROUND: Glioblastoma is the most common and malignant brain tumor in adults. Glioblastoma is usually fatal 12-15 months after diagnosis and the current possibilities in therapy are mostly only palliative. Therefore, new forms of diagnosis and therapy are urgently needed. Since tumor-associated macrophages are key players in tumor progression and survival there is large potential in investigating their immunological characteristics in glioblastoma patients. Recent evidence shows the expression of variable immunoglobulins and TCRαß in subpopulations of monocytes, in vitro polarized macrophages and macrophages in the tumor microenvironment. We set out to investigate the immunoglobulin sequences of circulating monocytes and tumor-associated macrophages from glioblastoma patients to evaluate their potential as novel diagnostic or therapeutic targets. RESULTS: We routinely find consistent expression of immunoglobulins in tumor-associated macrophages (TAM) and circulating monocytes from all glioblastoma patients analyzed in this study. However, the immunoglobulin repertoires of circulating monocytes and TAM are generally more restricted compared to B cells. Furthermore, the immunoglobulin expression in the macrophage populations negatively correlates with the tumor volume. Interestingly, the comparison of somatic mutations, V-chain usage, CDR3-length and the distribution of used heavy chain genes on the locus of chromosome 14 of the immunoglobulins from myeloid to B cells revealed virtually no differences. CONCLUSIONS: The investigation of the immunoglobulin repertoires from TAM and circulating monocytes in glioblastoma-patients revealed a negative correlation to the tumor volume, which could not be detected in the immunoglobulin repertoires of the patients' B lymphocytes. Furthermore, the immunoglobulin repertoires of monocytes were more diverse than the repertoires of the macrophages in the tumor microenvironment from the same patients suggesting a tumor-specific immune response which could be advantageous for the use as diagnostic or therapeutic target.

Immediate Neutrophil-Variable-T Cell Receptor Host Response in Bacterial Meningitis.

Bacterial meningitis is a life-threatening disease that evokes an intense neutrophil-dominated host response to microbes invading the subarachnoid space. Recent evidence indicates the existence of combinatorial V(D)J immune receptors in neutrophils that are based on the T cell receptor (TCR). Here, we investigated expression of the novel neutrophil TCRαß-based V(D)J receptors in cerebrospinal fluid (CSF) from human patients with acute-phase bacterial meningitis using immunocytochemical, genetic immunoprofiling, cell biological, and mass spectrometric techniques. We find that the human neutrophil combinatorial V(D)J receptors are rapidly induced in CSF neutrophils during the first hours of bacterial meningitis. Immune receptor repertoire diversity is consistently increased in CSF neutrophils relative to circulating neutrophils and phagocytosis of baits directed to the variable immunoreceptor is enhanced in CSF neutrophils during acute-phase meningitis. Our results reveal that a flexible immune response involving neutrophil V(D)J receptors which enhance phagocytosis is immediately initiated at the site of acute bacterial infection.

Expression of combinatorial immunoglobulins in macrophages in the tumor microenvironment.

Recent evidence indicates the presence of macrophage subpopulations that express the TCRαß in chronic inflammatory diseases such as tuberculosis and atherosclerosis and in the tumor microenvironment. Here, we demonstrate that a second subpopulation of macrophages expresses rearranged heavy and light chain immunoglobulins. We identify immunoglobulin expression in human and murine monocytes, in ex vivo differentiated macrophages and macrophages from the tumor microenvironment of five randomly selected distinct human tumor entities. The immunoglobulin heavy and light chains are expressed in a small macrophage subfraction (~3-5%) as combinatorial and individual-specific immune receptors. Using Sanger sequencing and deep sequencing, we routinely find markedly restricted Ig repertoires in monocytes/macrophages compared to normal B cells. Furthermore, we report the complete Ig heavy and light chain sequences of a fully functional immunoglobulin from a single tumor-associated macrophage. These results demonstrate that Ig expression is a defining feature of monocytes and also macrophages in the tumor microenvironment and thus reveal an as yet unrecognized modus operandi of host defense in professional phagocytes.

Activated protein C reverses epigenetically sustained p66Shc expression in plaque-associated macrophages in diabetes.

Impaired activated protein C (aPC) generation is associated with atherosclerosis and diabetes mellitus. Diabetes-associated atherosclerosis is characterized by the hyperglycaemic memory, e.g., failure of disease improvement despite attenuation of hyperglycaemia. Therapies reversing the hyperglycaemic memory are lacking. Here we demonstrate that hyperglycaemia, but not hyperlipidaemia, induces the redox-regulator p66Shc and reactive oxygen species (ROS) in macrophages. p66Shc expression, ROS generation, and a pro-atherogenic phenotype are sustained despite restoring normoglycemic conditions. Inhibition of p66Shc abolishes this sustained pro-atherogenic phenotype, identifying p66Shc-dependent ROS in macrophages as a key mechanism conveying the hyperglycaemic memory. The p66Shc-associated hyperglycaemic memory can be reversed by aPC via protease-activated receptor-1 signalling. aPC reverses glucose-induced CpG hypomethylation within the p66Shc promoter by induction of the DNA methyltransferase-1 (DNMT1). Thus, epigenetically sustained p66Shc expression in plaque macrophages drives the hyperglycaemic memory, which-however-can be reversed by aPC. This establishes that reversal of the hyperglycaemic memory in diabetic atherosclerosis is feasible.

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

Recent evidence indicates the presence of macrophage subpopulations that express the TCRαß 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-TCRαß is expressed in the tumor microenvironment of human and murine malignancies. We identify TCRαß+ macrophages in each case of four randomly selected distinct human tumor entities. In human tumor tissues, the TCRαß expressed by macrophages in the tumor microenvironment is a combinatorial and individual-specific immune receptor. Furthermore, we routinely find TCRαß+ 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 TCRαß is individual-specific and independent of stabilin-1. These results demonstrate that TCRαß 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.

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)αß-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 TCRαß/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.

Prognostic impact of extracellular matrix metalloprotease inducer: immunohistochemical analyses of colorectal tumors and immunocytochemical screening of disseminated tumor cells in bone marrow from patients with gastrointestinal cancer.

BACKGROUND: Extracellular matrix metalloprotease inducer (EMMPRIN) induces matrix metalloproteinase (MMP) expression, tumor-stroma cell interaction, and invasion/angiogenesis. The objectives of the current study were to find the first evidence of a prognostic impact of total and relative EMMPRIN expression in colorectal cancer cells and to analyze EMMPRIN in bone marrow-disseminated tumor cells and normal cells from 2 different gastrointestinal cancer entities. METHODS: Tumors and normal tissues from 40 patients with colorectal cancer who were followed prospectively (median follow-up, 31 months) were analyzed for EMMPRIN by immunohistochemistry. Bone marrow from 51 patients (13 patients with gastric cancer and 38 patients with colorectal cancer) with evidence of disseminated tumor cells was screened for EMMPRIN in tumor cells and normal cells (cytokeratin 18/EMMPRIN double immunocytochemistry). RESULTS: A significant correlation between poor disease-specific survival (P=.037; Kaplan-Meier method; Mantel-Cox log-rank tests) and an increased ratio of EMMPRIN in tumor cells versus corresponding normal epithelial cells were observed. Furthermore, the relative increase of EMMPRIN was associated with a trend toward poor overall and recurrence-free survival. High relative EMMPRIN expression was associated significantly with positive metastasis status (M1) (P=.001) and with a trend towards advanced pathologic tumor classification. Sixteen percent of disseminated tumor cells in bone marrow samples from patients with colorectal cancer and 48.5% of disseminated tumor cells in bone marrow samples from patients with gastric cancer stained positive for EMMPRIN, and EMMPRIN on micrometastatic cells was associated significantly with parameters of tumor progression (M status, noncurative resectability). A minority of normal bone marrow cells were stained for EMMPRIN, suggesting their suitability for molecular targeting. CONCLUSIONS: To the authors' knowledge, this study was the first to indicate that increased relative EMMPRIN protein in tumor-specific cells compared with normal cells predicts poor disease-specific survival in patients with colorectal cancer and that EMMPRIN in primary and bone marrow-disseminated tumor cells is associated with clinical markers of tumor progression in patients with colorectal/gastric cancer.

Transcriptional regulation of the urokinase receptor (u-PAR)--a central molecule of invasion and metastasis.

The phenomenon of tumor-associated proteolysis has been acknowledged as a decisive step in the progression of cancer. This short review focuses on the urokinase receptor (u-PAR), a central molecule involved in tumor-associated invasion and metastasis, and summarizes the transcriptional regulation of u-PAR. The urokinase receptor (u-PAR) is a heavily glycosylated cell surface protein and binds the serine protease urokinase specifically and with high affinity. It consists of three similar cysteine-rich repeats and is anchored to the cell membrane via a GPI-anchor. The u-PAR gene comprises 7 exons and is located on chromosome 19q13. Transcriptional activation of the u-PAR promoter region can be induced by binding of transcription factors (Sp1, AP-1, AP-2, NF-kappa B). One current study gives an example for transcriptional downregulation of u-PAR through a PEA3/ets transcriptional silencing element. Knowledge of the molecular regulation of this molecule in tumor cells could be very important for diagnosis and therapy in the near future.