In JoVE (2)

Other Publications (115)

Articles by Wolfgang Weninger in JoVE

 JoVE Medicine

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

1The Centenary Institute, 2Sydney Medical School, University of Sydney, 3The University of Queensland Diamantina Institute, Translational Research Institute, The University of Queensland, 4Department of Dermatology, Royal Prince Alfred Hospital, 5Discipline of Dermatology, University of Sydney


JoVE 53486

 JoVE Medicine

Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses

1Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Biopolis, 2Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 3Lee Kong Chian School of Medicine, Nanyang Technological University, 4Centenary Institute for Cancer Medicine and Cell Biology, 5Discipline of Dermatology, University of Sydney, 6Department of Dermatology, Royal Prince Alfred Hospital, 7LSI Immunology Programme, National University of Singapore, 8School of Biological Sciences, Nanyang Technological University


JoVE 54956

Other articles by Wolfgang Weninger on PubMed

Phenotyping Transgenic Embryos: a Rapid 3-D Screening Method Based on Episcopic Fluorescence Image Capturing

Nature Genetics. Jan, 2002  |  Pubmed ID: 11743576

We describe a technique suitable for routine three-dimensional (3-D) analysis of mouse embryos that is based on episcopic fluorescence images captured during serial sectioning of wax-embedded specimens. We have used this procedure to describe the cardiac phenotype and associated blood vessels of trisomic 16 (Ts16) and Cited2-null mutant mice, as well as the expression pattern of an Myf5 enhancer/beta-galactosidase transgene. The consistency of the images and their precise alignment are ideally suited for 3-D analysis using video animations, virtual resectioning or commercial 3-D reconstruction software packages. Episcopic fluorescence image capturing (EFIC) provides a simple and powerful tool for analyzing embryo and organ morphology in normal and transgenic embryos.

Increased and Prolonged Inflammation and Angiogenesis in Delayed-type Hypersensitivity Reactions Elicited in the Skin of Thrombospondin-2--deficient Mice

Blood. Jan, 2002  |  Pubmed ID: 11781236

Angiogenesis and enhanced microvascular permeability are hallmarks of a large number of inflammatory diseases. Although up-regulation of proangiogenic factors such as vascular endothelial growth factor and interleukin-8 have been previously reported in inflamed tissue, the biologic role of endogenous inhibitors of angiogenesis in inflammation has remained unclear. To investigate the biologic role of the potent angiogenesis inhibitor thrombospondin-2 (TSP-2) in the control of cutaneous inflammation, delayed-type hypersensitivity reactions were elicited in the ear skin of wild-type and TSP-2-deficient mice by topical sensitization and challenge with oxazolone. Cutaneous TSP-2 expression was up-regulated in the inflamed skin of wild-type mice, predominantly in dermal fibroblasts and microvessels. Lack of TSP-2 resulted in a significantly enhanced inflammatory response with increased angiogenesis, edema formation, and inflammatory infiltration. Ear swelling and inflammation persisted for more than 2 weeks in TSP-2-deficient mice, as compared with 1 week in wild-type mice. Although baseline vascular permeability was unchanged, significantly enhanced microvascular leakage was found in the inflamed skin of TSP-2-deficient mice. Moreover, the fraction of rolling leukocytes was significantly increased in the untreated skin of TSP-2-deficient mice. These results reveal an important role of TSP-2 in limiting the extent and the duration of edema formation, angiogenesis, and inflammatory cell infiltration during acute and chronic inflammation.

Folic Acid Prevents Exencephaly in Cited2 Deficient Mice

Human Molecular Genetics. Feb, 2002  |  Pubmed ID: 11823447

Cited2 (also Mrg1/p35srj) is a member of a new conserved gene family that is expressed during mouse development and in adult tissues. In order to investigate the function of Cited2 during mouse embryogenesis, we introduced a null mutation into the Cited2 locus. Cited2(-/-) mutants died at late gestation and exhibited heart defects and exencephaly, arising from defective closure of the midbrain (MB) and hindbrain. Initiation of neural tube closure at the forebrain-midbrain (FB-MB) boundary, an essential step for closure of the cranial neural tube, was impaired in the Cited2(-/-) mutants. Gene marker analysis using in situ hybridization revealed that the patterning of the anterior neural plate and head mesenchyme was little affected or normal in the Cited2(-/-) embryos. However, Cited2 was required for the survival of neuroepithelial cells and its absence led to massive apoptosis in dorsal neuroectoderm around the FB-MB boundary and in a restricted transverse domain in the hindbrain. Treatment with folic acid significantly reduced the exencephalic phenotype in the Cited2(-/-) embryos both in vivo and in vitro. However, assessment of folate metabolism revealed no defect in the Cited2(-/-) mutants, and the elevated apoptosis observed in the neuroepithelium of the Cited2(-/-) mutants was apparently not decreased by folic acid supplementation. To our knowledge, the Cited2 mouse represents the first genetic model in which folic acid can prevent a defect in neural tube closure by a mechanism other than the neutralization of a defect in folate homeostasis.

Migration and Differentiation of CD8+ T Cells

Immunological Reviews. Aug, 2002  |  Pubmed ID: 12234374

Antigen-specific responses by CD8+ T cells require direct cell-cell interactions between T cells and antigen-presenting cells (APC). Initially, naïve T cells must communicate with APC in lymphoid organs. Once stimulated, the resulting effector cells interact with APC in peripheral tissues. To this end, T cells must migrate to discrete sites throughout the body where antigen may be found. Recent progress in the field has revealed that the migratory abilities of T cells are critically dependent on their differentiation state, which is shaped by a multitude of factors. Thus, naïve T cells are normally restricted to recirculate between the blood and secondary lymphoid tissues, although in some autoimmune diseases they may also accumulate in chronically inflamed tissues. When CD8+ T cells encounter antigen and differentiate into short-lived effector CTL, they lose the ability to home to lymph nodes but gain access to peripheral tissues and sites of inflammation. Long-lived memory cells exist in (at least) two flavors: central memory cells that migrate to both lymphoid organs and peripheral sites of inflammation, and effector memory cells that are preferentially localized in non-lymphoid tissues. Our current understanding of the interplay of T cell differentiation and migration has been boosted by the development of T-GFP mice, in which transgenic green fluorescent protein is expressed selectively in naïve and central memory T cells, but not in effector cytotoxic T cells (CTL). This review will focus on recent studies in which T-GFP mice were used to dissect the traffic signals for naïve T cell homing to secondary lymphoid organs, the factors that influence the differentiation of naïve CD8+ T cells into cytotoxic and memory cells, as well as the in vivo trafficking routes of antigen-experienced subsets.

Naive T Cell Recruitment to Nonlymphoid Tissues: a Role for Endothelium-expressed CC Chemokine Ligand 21 in Autoimmune Disease and Lymphoid Neogenesis

Journal of Immunology (Baltimore, Md. : 1950). May, 2003  |  Pubmed ID: 12707342

Naive T cells are usually excluded from nonlymphoid tissues. Only when such tertiary tissues are subjected to chronic inflammation, such as in some (but not all) autoimmune diseases, are naive T cells recruited to these sites. We show that the CCR7 ligand CC chemokine ligand (CCL)21 is sufficient for attracting naive T cells into tertiary organs. We performed intravital microscopy of cremaster muscle venules in T-GFP mice, in which naive T cells express green fluorescent protein (GFP). GFP(+) cells underwent selectin-dependent rolling, but no firm adherence (sticking). Superfusion with CCL21, but not CXC chemokine ligand 12, induced integrin-dependent sticking of GFP(+) cells. Moreover, CCL21 rapidly elicited accumulation of naive T cells into sterile s.c. air pouches. Interestingly, a second CCR7 ligand, CCL19, triggered T cell sticking in cremaster muscle venules, but failed to induce extravasation in air pouches. Immunohistochemistry studies implicate ectopic expression of CCL21 as a mechanism for naive T cell traffic in human autoimmune diseases. Most blood vessels in tissue samples from patients with rheumatoid arthritis (85 +/- 10%) and ulcerative colitis (66 +/- 1%) expressed CCL21, and many perivascular CD45RA(+) naive T cells were found in these tissues, but not in psoriasis, where CCL21(+) vessels were rare (17 +/- 1%). These results identify endothelial CCL21 expression as an important determinant for naive T cell migration to tertiary tissues, and suggest the CCL21/CCR7 pathway as a therapeutic target in diseases that are associated with naive T cell recruitment.

Selective Imprinting of Gut-homing T Cells by Peyer's Patch Dendritic Cells

Nature. Jul, 2003  |  Pubmed ID: 12840763

Whereas naive T cells migrate only to secondary lymphoid organs, activation by antigen confers to T cells the ability to home to non-lymphoid sites. Activated effector/memory T cells migrate preferentially to tissues that are connected to the secondary lymphoid organs where antigen was first encountered. Thus, oral antigens induce effector/memory cells that express essential receptors for intestinal homing, namely the integrin alpha4beta7 and CCR9, the receptor for the gut-associated chemokine TECK/CCL25 (refs 6, 8, 9). Here we show that this imprinting of gut tropism is mediated by dendritic cells from Peyer's patches. Stimulation of CD8-expressing T cells by dendritic cells from Peyer's patches, peripheral lymph nodes and spleen induced equivalent activation markers and effector activity in T cells, but only Peyer's patch dendritic cells induced high levels of alpha4beta7, responsiveness to TECK and the ability to home to the small intestine. These findings establish that Peyer's patch dendritic cells imprint gut-homing specificity on T cells, and thus license effector/memory cells to access anatomical sites most likely to contain their cognate antigen.

Visualizing T Cell Migration in Vivo

International Archives of Allergy and Immunology. Dec, 2003  |  Pubmed ID: 14707460

Ever since the realization that T lymphocytes are key players in the defense against pathogens and tumors, a major aim of immunologists has been to understand the relationship between the functional and migratory properties of antigen-specific T cells. The current paradigm proposes that T cells follow organ-specific trafficking pathways to exit from blood into the extravascular compartment. T cell homing is regulated at the level of adhesion molecules and chemokine receptors, whose expression is linked tightly to the differentiation state of the cell. Naïve T lymphocytes follow relatively uniform recirculation routes through secondary lymphoid organs, the molecular cues of which are fairly well understood. As effector and memory T cells must be capable of reaching virtually any site in the body, their migratory behavior is considerably more heterogeneous. During the past few years, innovative approaches for tracking T cells in vivo have emerged. Here, we review recent technical developments in experimental methods for the visualization of T cells both at the population and single cell level in vivo, and discuss what these methods have taught us about T cell trafficking.

Chemokine Regulation of Naïve T Cell Traffic in Health and Disease

Seminars in Immunology. Oct, 2003  |  Pubmed ID: 15001175

A central feature of the immune response is the precise spatio-temporal convergence of T cells and antigen presenting cells (APC) in particular microenvironments within secondary lymphoid organs (SLO). CCR7 and its ligands CCL19 and CCL21 have been identified as the gatekeepers for both naïve T lymphocytes and dendritic cells (DC) to these defined anatomical compartments. A new perception on the regulation of lymphocyte traffic in lymph nodes (LN) has come from observations that sphingosine-1-phosphate (S1P) receptor agonists affect T cell entry and exit from these organs. Recent developments in intravital microscopy (IVM) techniques reveal unexpected autonomous random motion of lymphocytes within secondary lymphoid tissues, and provoke questions about the mechanisms that guide their compartmental navigation.

Sheet Preparations Expose the Dermal Nerve Plexus of Human Skin and Render the Dermal Nerve End Organ Accessible to Extensive Analysis

The Journal of Investigative Dermatology. Jan, 2004  |  Pubmed ID: 14962106

Since vertical tissue sections used for the study of the human cutaneous nervous system inherently allow visualization of only a small part of the mainly horizontally oriented cutaneous nerves, we searched for possibilities to extend this view. We now propose a method based on the immuno-staining of dermal sheet preparations for subsequent analysis by electron-, light- or laser scanning microscopy. Dermal sheet preparations for the first time allowed the imaging of the complex structure of the nerve end organ over several cm2, and facilitated viewing of its topological relationship to other tissue components. We could visualize that the bulk of free ending nerve fibers ramified within 25 microm of the dermo-epidermal junction, whereas below that only larger nerve bundles were present. This method further allowed the detection and quantification of NCAM/CD56+ non-myelinating Schwann cells which envelope terminal axons within the dermis. Depending on the body region, we detected between 140 to over 300 individual terminal Schwann cells per mm2 skin surface. Our method should allow the acquisition of new insights into the highly organized architecture of the skin nerve end organ. Its further application will give new impetus in the investigation of alterations of this skin compartment under pathological conditions.

In Vivo 3D Analysis of the Adipose Tissue in the Orbital Apex and the Compartments of the Parasellar Region

Clinical Anatomy (New York, N.Y.). Mar, 2004  |  Pubmed ID: 14974098

As tissue dissections carried out on formaldehyde-fixed anatomical specimens demonstrate, the parasellar region (PSR), traditionally referred to as the cavernous sinus, is composed of three distinct compartments: orbital, pterygopalatine, and lateral sellar. The aim of our study was to identify and measure these compartments in the living and to describe the topography of their adipose body tissues. For this purpose data sets of 35 patients, who had undergone thin-section multislice computed tomography (CT), were examined using 3D-reconstruction software. The pterygopalatine and orbital compartments of the PSR could be identified in the images by their adipose bodies. We provide more exacting measurements of their size than have been presented in earlier studies. Furthermore, we include data about the uni- and bilateral presence of the single compartments and analyze the topography of the adipose tissue bodies in the orbital apex region. Our quantitative data and topographical descriptions confirm the compartmentalization concept of the PSR, allow for correct interpretation of CT scans of the orbital apex and anterior PSR, and provide baseline information for individual planning of surgical and radiological interventions.

CXCL12 Mediates CCR7-independent Homing of Central Memory Cells, but Not Naive T Cells, in Peripheral Lymph Nodes

The Journal of Experimental Medicine. Apr, 2004  |  Pubmed ID: 15096537

Central memory CD8(+) T cells (T(CM)) confer superior protective immunity against infections compared with other T cell subsets. T(CM) recirculate mainly through secondary lymphoid organs, including peripheral lymph nodes (PLNs). Here, we report that T(CM), unlike naive T cells, can home to PLNs in both a CCR7-dependent and -independent manner. Homing experiments in paucity of lymph node T cells (plt/plt) mice, which do not express CCR7 ligands in secondary lymphoid organs, revealed that T(CM) migrate to PLNs at approximately 20% of wild-type (WT) levels, whereas homing of naive T cells was reduced by 95%. Accordingly, a large fraction of endogenous CD8(+) T cells in plt/plt PLNs displayed a T(CM) phenotype. Intravital microscopy of plt/plt subiliac lymph nodes showed that T(CM) rolled and firmly adhered (sticking) in high endothelial venules (HEVs), whereas naive T cells were incapable of sticking. Sticking of T(CM) in plt/plt HEVs was pertussis toxin sensitive and was blocked by anti-CXCL12 (SDF-1alpha). Anti-CXCL12 also reduced homing of T(CM) to PLNs in WT animals by 20%, indicating a nonredundant role for this chemokine in the presence of physiologic CCR7 agonists. Together, these data distinguish naive T cells from T(CM), whereby only the latter display greater migratory flexibility by virtue of their increased responsiveness to both CCR7 ligands and CXCL12 during homing to PLN.

From Experimental Imaging Techniques to Virtual Embryology

History and Philosophy of the Life Sciences. 2004  |  Pubmed ID: 16302693

Modern embryology increasingly relies on descriptive and functional three dimensional (3D) and four dimensional (4D) analysis of physically, optically, or virtually sectioned specimens. To cope with the technical requirements, new methods for high detailed in vivo imaging, as well as the generation of high resolution digital volume data sets for the accurate visualisation of transgene activity and gene product presence, in the context of embryo morphology, were recently developed and are under construction. These methods profoundly change the scientific applicability, appearance and style of modern embryo representations. In this paper, we present an overview of the emerging techniques to create, visualise and administrate embryo representations (databases, digital data sets, 3-4D embryo reconstructions, models, etc.), and discuss the implications of these new methods on the work of modern embryologists, including, research, teaching, the selection of specific model organisms, and potential collaborators.

Bone Marrow is a Major Reservoir and Site of Recruitment for Central Memory CD8+ T Cells

Immunity. Feb, 2005  |  Pubmed ID: 15723813

Normal bone marrow (BM) contains T cells whose function and origin are poorly understood. We observed that CD8+ T cells in BM consist chiefly of CCR7+ L-selectin+ central memory cells (TCMs). Adoptively transferred TCMs accumulated more efficiently in the BM than naive and effector T cells. Intravital microscopy (IVM) showed that TCMs roll efficiently in BM microvessels via L-, P-, and E-selectin, whereas firm arrest required the VCAM-1/alpha4beta1 pathway. alpha4beta1 integrin activation did not depend on pertussis toxin (PTX)-sensitive Galphai proteins but was reduced by anti-CXCL12. In contrast, TCM diapedesis did not require CXCL12 but was blocked by PTX. After extravasation, TCMs displayed agile movement within BM cavities, remained viable, and mounted potent antigen-specific recall responses for at least two months. Thus, the BM functions as a major reservoir for TCMs by providing specific recruitment signals that act in sequence to mediate the constitutive recruitment of TCMs from the blood.

Cited2 is Required Both for Heart Morphogenesis and Establishment of the Left-right Axis in Mouse Development

Development (Cambridge, England). Mar, 2005  |  Pubmed ID: 15750185

Establishment of the left-right axis is a fundamental process of vertebrate embryogenesis. Failure to develop left-right asymmetry leads to incorrect positioning and morphogenesis of numerous internal organs, and is proposed to underlie the etiology of several common cardiac malformations. The transcriptional modulator Cited2 is essential for embryonic development: Cited2-null embryos die during gestation with profound developmental abnormalities, including cardiac malformations, exencephaly and adrenal agenesis. Cited2 is also required for normal establishment of the left-right axis; we demonstrate that abnormal heart looping and right atrial and pulmonary isomerism are consistent features of the left-right-patterning defect. We show by gene expression analysis that Cited2 acts upstream of Nodal, Lefty2 and Pitx2 in the lateral mesoderm, and of Lefty1 in the presumptive floor plate. Although abnormal left-right patterning has a major impact on the cardiac phenotype in Cited2-null embryos, laterality defects are only observed in a proportion of these embryos. We have therefore used a combination of high-resolution imaging and three-dimensional (3D) modeling to systematically document the full spectrum of Cited2-associated cardiac defects. Previous studies have focused on the role of Cited2 in cardiac neural crest cell development, as Cited2 can bind the transcription factor Tfap2, and thus affect the expression of Erbb3 in neural crest cells. However, we have identified Cited2-associated cardiac defects that cannot be explained by laterality or neural crest abnormalities. In particular, muscular ventricular septal defects and reduced cell density in the atrioventricular (AV) endocardial cushions are evident in Cited2-null embryos. As we found that Cited2 expression tightly correlated with these sites, we believe that Cited2 plays a direct role in development of the AV canal and cardiac septa. We therefore propose that, in addition to the previously described reduction of cardiac neural crest cells, two other distinct mechanisms contribute to the spectrum of complex cardiac defects in Cited2-null mice; disruption of normal left-right patterning and direct loss of Cited2 expression in cardiac tissues.

Activation of Bone Marrow-resident Memory T Cells by Circulating, Antigen-bearing Dendritic Cells

Nature Immunology. Oct, 2005  |  Pubmed ID: 16155571

Dendritic cells (DCs) carry antigen from peripheral tissues via lymphatics to lymph nodes. We report here that differentiated DCs can also travel from the periphery into the blood. Circulating DCs migrated to the spleen, liver and lung but not lymph nodes. They also homed to the bone marrow, where they were retained better than in most other tissues. Homing of DCs to the bone marrow depended on constitutively expressed vascular cell adhesion molecule 1 and endothelial selectins in bone marrow microvessels. Two-photon intravital microscopy in bone marrow cavities showed that DCs formed stable antigen-dependent contacts with bone marrow-resident central memory T cells. Moreover, using this previously unknown migratory pathway, antigen-pulsed DCs were able to trigger central memory T cell-mediated recall responses in the bone marrow.

Dendritic Cell Maturation, but Not CD8+ T Cell Induction, is Dependent on Type I IFN Signaling During Vaccination with Adenovirus Vectors

Journal of Immunology (Baltimore, Md. : 1950). Nov, 2005  |  Pubmed ID: 16237098

To understand how vaccines initiate adaptive immune responses, it is necessary to study how they interact with APCs such as dendritic cells (DCs). In this study, we analyzed interactions between recombinant adenovirus (Ad) vectors and mouse DCs. Mouse bone marrow-derived DCs transduced with Ad vectors produced type I IFN, which promoted the maturation of both transduced and bystander DCs. DCs transduced with a vector derived from a chimpanzee Ad serotype (AdC68) produced more type I IFN and matured more efficiently compared with DCs transduced with a vector derived from a human Ad serotype (AdHu5). Both vectors stimulated type I IFN production independently of viral transcription, replication, and TLR signaling. However, each vector induced type I IFN through distinct pathways; whereas AdHu5 vectors required phosphoinositide-3-OH kinase for type I IFN induction, AdC68 vectors did not. Both vectors induced strong transgene product-specific CD8+ T cell responses in wild-type mice. DCs isolated from mice that have a defect in type I IFN signaling failed to undergo full maturation after Ad vaccination, but surprisingly, these mice mounted strong transgene product-specific CD8+ T cell responses. In these mice, we were able to detect a small number of transduced DCs that expressed high levels of costimulatory molecules, and these DCs were able to stimulate transgene product-specific CD8+ T cells. Thus, type I IFN signaling is an important component of Ad-mediated DC maturation but is dispensable during the generation of transgene product-specific CD8+ T cell responses.

High-resolution Episcopic Microscopy: a Rapid Technique for High Detailed 3D Analysis of Gene Activity in the Context of Tissue Architecture and Morphology

Anatomy and Embryology. Jun, 2006  |  Pubmed ID: 16429276

We describe a new methodology for rapid 2D and 3D computer analysis and visualisation of gene expression and gene product pattern in the context of anatomy and tissue architecture. It is based on episcopic imaging of embryos and tissue samples, as they are physically sectioned, thereby producing inherently aligned digital image series and volume data sets, which immediately permit the generation of 3D computer representations. The technique uses resin as embedding medium, eosin for unspecific tissue staining, and colour reactions (beta-galactosidase/Xgal or BCIP/NBT) for specific labelling of gene activity and mRNA pattern. We tested the potential of the method for producing high-resolution volume data sets of adult human and porcine tissue samples and of specifically and unspecifically stained mouse, chick, quail, frog, and zebrafish embryos. The quality of the episcopic images resembles the quality of digital images of true histological sections with respect to resolution and contrast. Specifically labelled structures can be extracted using simple thresholding algorithms. Thus, the method is capable of quickly and precisely detecting molecular signals simultaneously with anatomical details and tissue architecture. It has no tissue restrictions and can be applied for analysis of human tissue samples as well as for analysis of all developmental stages of embryos of a wide variety of biomedically relevant species.

Regulatory T Cells Reversibly Suppress Cytotoxic T Cell Function Independent of Effector Differentiation

Immunity. Jul, 2006  |  Pubmed ID: 16860762

Mechanisms of dominant tolerance have evolved within the mammalian immune system to prevent inappropriate immune responses. CD4(+)CD25(+) regulatory T (T(reg)) cells have emerged as central constituents of this suppressive activity. By using multiphoton intravital microscopy in lymph nodes (LNs) of anesthetized mice, we have analyzed how cytotoxic T lymphocytes (CTLs) interact with antigen-presenting target cells in the presence or absence of activated T(reg) cells. Nonregulated CTLs killed their targets at a 6.6-fold faster rate than regulated CTLs. In spite of this compromised effector activity, regulated CTLs exhibited no defect in proliferation, induction of cytotoxic effector molecules and secretory granules, in situ motility, or ability to form antigen-dependent conjugates with target cells. Only granule exocytosis by CTLs was markedly impaired in the presence of T(reg) cells. This selective form of regulation did not require prolonged contact between CTLs and T(reg) cells but depended on CTL responsiveness to transforming growth factor-beta. CTLs quickly regained full killing capacity in LNs upon selective removal of T(reg) cells. Thus, T(reg) cells reversibly suppress CTL-mediated immunity by allowing acquisition of full effector potential but withholding the license to kill.

Immune Cell Migration As a Means to Control Immune Privilege: Lessons from the CNS and Tumors

Immunological Reviews. Oct, 2006  |  Pubmed ID: 16972905

Certain organs, such as the brain, eye, and gonads, are particularly sensitive to damage by inflammation. Therefore, these tissues have developed unique immunological properties that curtail inflammatory responses, a phenomenon termed immune privilege. In addition, by co-opting some of the regulatory cues operant in immune privilege in normal organs, tumors can evade immunosurveillance. While many different mechanisms contribute to immune privilege, there is evidence that leukocyte migration is an important checkpoint in its control. This hypothesis is based on the fact that leukocyte entry into these organs is restricted by physical barriers and that the collapse of these obstacles marks a critical step in the development of inflammatory/autoimmune disease at these sites. Numerous studies in a variety of experimental systems have characterized the molecular and cellular mechanisms involved in leukocyte homing to immune-privileged organs. Recently, two-photon microscopy has revealed critical insights into the events occurring in the extravascular space of immune-privileged organs, including locomotion patterns and interactive behavior of leukocytes in the interstitial space. Here, we review our current understanding of immune cell migration to and within immune-privileged organs and highlight how this knowledge may be exploited for immunotherapeutic purposes.

CCN3 Controls 3D Spatial Localization of Melanocytes in the Human Skin Through DDR1

The Journal of Cell Biology. Nov, 2006  |  Pubmed ID: 17101694

Melanocytes reside within the basal layer of the human epidermis, where they attach to the basement membrane and replicate at a rate proportionate to that of keratinocytes, maintaining a lifelong stable ratio. In this study, we report that coculturing melanocytes with keratinocytes up-regulated CCN3, a matricellular protein that we subsequently found to be critical for the spatial localization of melanocytes to the basement membrane. CCN3 knockdown cells were dissociated either upward to the suprabasal layers of the epidermis or downward into the dermis. The overexpression of CCN3 increased adhesion to collagen type IV, the major component of the basement membrane. As the receptor responsible for CCN3-mediated melanocyte localization, we identified discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase that acts as a collagen IV adhesion receptor. DDR1 knockdown decreased melanocyte adhesion to collagen IV and shifted melanocyte localization in a manner similar to CCN3 knockdown. These results demonstrate an intricate and necessary communication between keratinocytes and melanocytes in maintaining normal epidermal homeostasis.

Random Migration Precedes Stable Target Cell Interactions of Tumor-infiltrating T Cells

The Journal of Experimental Medicine. Nov, 2006  |  Pubmed ID: 17116735

The tumor microenvironment is composed of an intricate mixture of tumor and host-derived cells that engage in a continuous interplay. T cells are particularly important in this context as they may recognize tumor-associated antigens and induce tumor regression. However, the precise identity of cells targeted by tumor-infiltrating T lymphocytes (TILs) as well as the kinetics and anatomy of TIL-target cell interactions within tumors are incompletely understood. Furthermore, the spatiotemporal conditions of TIL locomotion through the tumor stroma, as a prerequisite for establishing contact with target cells, have not been analyzed. These shortcomings limit the rational design of immunotherapeutic strategies that aim to overcome tumor-immune evasion. We have used two-photon microscopy to determine, in a dynamic manner, the requirements leading to tumor regression by TILs. Key observations were that TILs migrated randomly throughout the tumor microenvironment and that, in the absence of cognate antigen, they were incapable of sustaining active migration. Furthermore, TILs in regressing tumors formed long-lasting (>or=30 min), cognate antigen-dependent contacts with tumor cells. Finally, TILs physically interacted with macrophages, suggesting tumor antigen cross-presentation by these cells. Our results demonstrate that recognition of cognate antigen within tumors is a critical determinant of optimal TIL migration and target cell interactions, and argue against TIL guidance by long-range chemokine gradients.

Asymmetric T Lymphocyte Division in the Initiation of Adaptive Immune Responses

Science (New York, N.Y.). Mar, 2007  |  Pubmed ID: 17332376

A hallmark of mammalian immunity is the heterogeneity of cell fate that exists among pathogen-experienced lymphocytes. We show that a dividing T lymphocyte initially responding to a microbe exhibits unequal partitioning of proteins that mediate signaling, cell fate specification, and asymmetric cell division. Asymmetric segregation of determinants appears to be coordinated by prolonged interaction between the T cell and its antigen-presenting cell before division. Additionally, the first two daughter T cells displayed phenotypic and functional indicators of being differentially fated toward effector and memory lineages. These results suggest a mechanism by which a single lymphocyte can apportion diverse cell fates necessary for adaptive immunity.

MicroMRI-HREM Pipeline for High-throughput, High-resolution Phenotyping of Murine Embryos

Journal of Anatomy. Jul, 2007  |  Pubmed ID: 17532797

Rapid and precise phenotyping analysis of large numbers of wild-type and mutant mouse embryos is essential for characterizing the genetic and epigenetic factors regulating embryogenesis. We present a novel methodology that permits precise high-throughput screening of the phenotype of embryos with both targeted and randomly generated mutations. To demonstrate the potential of this methodology we show embryo phenotyping results produced in a large-scale ENU-mutagenesis study. In essence this represents an analysis pipeline, which starts with simultaneous micro-magentic resonance imaging (microMRI) screening (voxel size: 25.4 x 25.4 x 24.4 microm) of 32 embryos in one run. Embryos with an indistinct phenotype are then cut into parts and suspect organs and structures are analysed with HREM (high-resolution episcopic microscopy). HREM is an imaging technique that employs 'positive' eosin staining and episcopic imaging for generating three-dimensional (3D) high-resolution (voxel size: 1.07 x 1.07 x 2 microm) digital data of near histological contrast and quality. The results show that our method guarantees the rapid availability of comprehensive phenotype information for high numbers of embryos in, if necessary, histological quality and detail. The combination of high-throughput microMRI with HREM provides an alternative screening pipeline with advantages over existing 3D phenotype screening methods as well as traditional histology. Thus, the microMRI-HREM phenotype analysis pipeline recommends itself as a routine tool for analysing the phenotype of transgenic and mutant embryos.

Negative Regulation of TLR-signaling Pathways by Activating Transcription Factor-3

Journal of Immunology (Baltimore, Md. : 1950). Sep, 2007  |  Pubmed ID: 17785797

Activating transcription factor-3 (ATF3) is rapidly induced by LPS in mouse macrophages and regulates TLR4 responses. We show that ATF3 is rapidly induced by various TLRs in mouse macrophages and plasmacytoid dendritic cells (DCs), as well as plasmacytoid and myeloid subsets of human DCs. In primary macrophages from mice with a targeted deletion of the atf3 gene (ATF3-knockout (KO)), TLR-stimulated levels of IL-12 and IL-6 were elevated relative to responses in wild-type macrophages. Similarly, targeted deletion of atf3 correlated with enhanced responsiveness of myeloid DCs to TLR activation as measured by IL-12 secretion. Ectopic expression of ATF3 antagonized TLR-stimulated IL-12p40 activation in a reporter assay. In vivo, CpG-oligodeoxynucleotide, a TLR9 agonist, given i.p. to ATF3-KO mice resulted in enhanced cytokine production from splenocytes. Furthermore, while ATF3-KO mice challenged with a sublethal dose of PR8 influenza virus were delayed in body weight recovery in comparison to wild type, the ATF3-KO mice showed higher titers of serum neutralizing Ab against PR8 5 mo postinfection. Thus, ATF3 behaves as a negative regulatory transcription factor in TLR pathways and, accordingly, deficiency in atf3 alters responses to immunological challenges in vivo. ATF3 dysregulation merits further exploration in diseases such as type I diabetes and cancer, where altered innate immunity has been implicated in their pathogenesis.

Three-dimensional Analysis of Molecular Signals with Episcopic Imaging Techniques

Methods in Molecular Biology (Clifton, N.J.). 2007  |  Pubmed ID: 18287637

This chapter describes two episcopic imaging methods, episcopic fluorescence image capturing (EFIC) and high-resolution episcopic microscopy (HREM). These allow analysis of molecular signals in a wide variety of biological samples such as tissues or embryos, in their precise anatomical and histological context. Both methods are designed to work with histologically prepared and whole-mount stained material, and both provide high-resolution data sets that lend themselves to 3D visualization and modeling. Specimens are embedded in wax (EFIC) or resin (HREM) and sectioned on a microtome. During the sectioning process, a series of digital images of each freshly cut block surface is captured, using a microscope and CCD camera aligned with the position at which the microtome block holder comes to rest after each cutting cycle. The resulting stacks of serial images retain virtually exact alignment and are readily converted to volume data sets. The two methods differ in how tissue architecture is visualized and hence how specific molecular signals are detected. EFIC uses endogenous, broad-range, tissue autofluorescence to reveal specimen structure. Addition of dyes to the wax embedding medium suppresses detection of any signal except that originating from the block surface. EFIC can be used to detect specific signals (such as LacZ) by virtue of their ability to suppress such fluorescence. In contrast, the plastic embedding medium used in HREM is strongly fluorescent, and tissue architecture is detected at the surface because of the ability of cellular and subcellular structures to suppress this signal. Specific signals generated as a result of chromogenic reactions can be visualized using band-pass filters that suppress the appearance of morphological data. In both methods, the digital volume data show high contrast; for HREM, such data achieve true cellular resolution. Their intrinsic alignment greatly facilitates their use for 3D analysis of transgene activity that can be visualized in the context of complex cellular and tissue morphology. Both methods are relatively simple and can be set up using common laboratory apparatuses. Together, they provide powerful tools for analyzing gene function in embryogenesis or tissue remodeling and for investigating developmental malformations.

Two Distinct Activation States of Plasmacytoid Dendritic Cells Induced by Influenza Virus and CpG 1826 Oligonucleotide

Journal of Leukocyte Biology. Mar, 2008  |  Pubmed ID: 18029397

There is growing evidence that plasmacytoid dendritic cells (pDC) are involved in the innate recognition of various microbes. However, the precise consequences of pathogen recognition on pDC activation and function are incompletely understood. Using a novel transgenic mouse model that facilitates the isolation of highly pure pDC populations, we found that influenza virus PR/8, a TLR7 ligand, and CpG 1826 oligonucleotide, a TLR9 ligand, induced surprisingly divergent activation programs in these cells. pDC stimulated with PR/8 produced large amounts of type I IFNs, and CpG 1826-stimulated pDC expressed higher levels of costimulatory molecules and proinflammatory cytokines and induced stronger proliferation of T cells. Transcriptome analysis uncovered the differential regulation in pDC of 178 and 1577 genes by PR/8 and CpG 1826, respectively. These differences may relate to the activation of discrete signaling pathways, as evidenced by distinct ERK1/2 and p38 MAPK phosphorylation kinetics. Finally, pDC isolated ex vivo during PR/8 infection or after i.v. CpG 1826 injection resembled their in vitro counterparts, corroborating that these cells can adopt specialized phenotypes in vivo. Thus, pDC display remarkable functional flexibility, which emphasizes their versatile functions in antimicrobial immunity and inflammatory processes.

Two-photon Imaging of Effector T-cell Behavior: Lessons from a Tumor Model

Immunological Reviews. Feb, 2008  |  Pubmed ID: 18275480

Recent advances in two-photon microscopy have provided a new way of visualizing the behavior of fluorescently tagged cells within their natural microenvironment. This technology has allowed for generating a detailed picture of the cellular interaction dynamics operant in the activation of T cells and B cells during primary immune responses within secondary lymphoid organs. In contrast, relatively little is known about the migratory and interactive behavior of effector T cells within peripheral organs. We have recently developed a two-photon microscopy model that enables tracking of cytotoxic T cells within tumors. We have demonstrated that tumor-infiltrating T lymphocytes (TILs) follow random migratory paths and that their migratory properties depend on signals from the T-cell receptor. We further showed that TILs underwent short- and long-term interactions with tumor cells as well as macrophages. Recently, we succeeded in dynamic imaging of the distribution of fluorescently tagged molecules within TILs at subcellular resolution, which will be instrumental for defining the composition of the lytic synapse as well as the targeted release of cytotoxic granules by these cells. The purpose of this review is to put our findings into the context of the current literature and to point out the molecular cues mediating effector T-cell function as candidates for future investigation.

Three-dimensional Description and Mathematical Characterization of the Parasellar Internal Carotid Artery in Human Infants

Journal of Anatomy. May, 2008  |  Pubmed ID: 18397239

Inside the 'cavernous sinus' or 'parasellar region' the human internal carotid artery takes the shape of a siphon that is twisted and torqued in three dimensions and surrounded by a network of veins. The parasellar section of the internal carotid artery is of broad biological and medical interest, as its peculiar shape is associated with temperature regulation in the brain and correlated with the occurrence of vascular pathologies. The present study aims to provide anatomical descriptions and objective mathematical characterizations of the shape of the parasellar section of the internal carotid artery in human infants and its modifications during ontogeny. Three-dimensional (3D) computer models of the parasellar section of the internal carotid artery of infants were generated with a state-of-the-art 3D reconstruction method and analysed using both traditional morphometric methods and novel mathematical algorithms. We show that four constant, demarcated bends can be described along the infant parasellar section of the internal carotid artery, and we provide measurements of their angles. We further provide calculations of the curvature and torsion energy, and the total complexity of the 3D skeleton of the parasellar section of the internal carotid artery, and compare the complexity of this in infants and adults. Finally, we examine the relationship between shape parameters of the parasellar section of the internal carotid artery in infants, and the occurrence of intima cushions, and evaluate the reliability of subjective angle measurements for characterizing the complexity of the parasellar section of the internal carotid artery in infants. The results can serve as objective reference data for comparative studies and for medical imaging diagnostics. They also form the basis for a new hypothesis that explains the mechanisms responsible for the ontogenetic transformation in the shape of the parasellar section of the internal carotid artery.

Fibroblast Migration is Mediated by CD44-dependent TGF Beta Activation

Journal of Cell Science. May, 2008  |  Pubmed ID: 18397995

CD44 contributes to inflammation and fibrosis in response to injury. As fibroblast recruitment is critical to wound healing, we compared cytoskeletal architecture and migration of wild-type (CD44WT) and CD44-deficient (CD44KO) fibroblasts. CD44KO fibroblasts exhibited fewer stress fibers and focal adhesion complexes, and their migration was characterized by increased velocity but loss of directionality, compared with CD44WT fibroblasts. Mechanistically, we demonstrate that CD44WT cells generated more active TGFbeta than CD44KO cells and that CD44 promotes the activation of TGFbeta via an MMP-dependent mechanism. Reconstitution of CD44 expression completely rescued the phenotype of CD44KO cells whereas exposure of CD44KO cells to exogenous active TGFbeta rescued the defect in stress fibers and migrational velocity, but was not sufficient to restore directionality of migration. These results resolve the TGFbeta-mediated and TGFbeta-independent effects of CD44 on fibroblast migration and suggest that CD44 may be critical for the recruitment of fibroblasts to sites of injury and the function of fibroblasts in tissue remodeling and fibrosis.

Dendritic Cell Behaviour in Vivo: Lessons Learned from Intravital Two-photon Microscopy

Immunology and Cell Biology. Jul, 2008  |  Pubmed ID: 18431356

Dendritic cells (DC) are central regulators of immune responses. Their functional characterization has, thus far, mainly relied on the analysis of ex vivo isolated cells or immunohistology, which provides information in a static manner. While these approaches have enabled an excellent understanding of the role of DC in antigen uptake, processing and presentation, there has been a clear need to investigate the behaviour of DC in the context of intact tissues in real time. This demand has recently been met by the availability of intravital two-photon microscopy, which allows for the visualization of single cells deep within intact organs over time. Thus, during the past few years, exciting new data have been generated as to how DC behave within secondary lymphoid and peripheral tissues both under homoeostatic and inflammatory conditions. Here, we will review what two-photon microscopy studies have taught us about the migration of DC in the interstitial space as well as their interactions with adaptive immune cells.

Objective Characterization of the Course of the Parasellar Internal Carotid Artery Using Mathematical Tools

Surgical and Radiologic Anatomy : SRA. Aug, 2008  |  Pubmed ID: 18523720

Along the internal carotid artery (ICA), atherosclerotic plaques are often located in its cavernous sinus (parasellar) segments (pICA). Studies indicate that the incidence of pre-atherosclerotic lesions is linked with the complexity of the pICA; however, the pICA shape was never objectively characterized. Our study aims at providing objective mathematical characterizations of the pICA shape.

Transforming Growth Factor-beta Receptor Blockade Augments the Effectiveness of Adoptive T-cell Therapy of Established Solid Cancers

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Jun, 2008  |  Pubmed ID: 18559619

Adoptive cellular immunotherapy is a promising approach to eradicate established tumors. However, a significant hurdle in the success of cellular immunotherapy involves recently identified mechanisms of immune suppression on cytotoxic T cells at the effector phase. Transforming growth factor-beta (TGF-beta) is one of the most important of these immunosuppressive factors because it affects both T-cell and macrophage functions. We thus hypothesized that systemic blockade of TGF-beta signaling combined with adoptive T-cell transfer would enhance the effectiveness of the therapy.

Visualizing Dendritic Cell Migration Within the Skin

Histochemistry and Cell Biology. Dec, 2008  |  Pubmed ID: 18987873

Dendritic cells (DCs) within the skin are a heterogeneous population of cells, including Langerhans cells of the epidermis and at least three subsets of dermal DCs. Collectively, these DCs play important roles in the initiation of adaptive immune responses following antigen challenge of the skin as well as being mediators of tolerance to self-antigen. A key functional aspect of cutaneous DCs is their migration both within the skin and into lymphatic vessels, resulting in their emigration to draining lymph nodes. Here, we discuss our current understanding of the requirements for successful DC migration in and from the skin, and introduce some of the microscopic techniques developed in our laboratory to facilitate a better understanding of this process. In particular, we detail our current use of multi-photon excitation (MPE) microscopy of murine skin to dissect the migratory behavior of DCs in vivo.

Migratory Dermal Dendritic Cells Act As Rapid Sensors of Protozoan Parasites

PLoS Pathogens. Nov, 2008  |  Pubmed ID: 19043558

Dendritic cells (DC), including those of the skin, act as sentinels for intruding microorganisms. In the epidermis, DC (termed Langerhans cells, LC) are sessile and screen their microenvironment through occasional movements of their dendrites. The spatio-temporal orchestration of antigen encounter by dermal DC (DDC) is not known. Since these cells are thought to be instrumental in the initiation of immune responses during infection, we investigated their behavior directly within their natural microenvironment using intravital two-photon microscopy. Surprisingly, we found that, under homeostatic conditions, DDC were highly motile, continuously crawling through the interstitial space in a Galpha(i) protein-coupled receptor-dependent manner. However, within minutes after intradermal delivery of the protozoan parasite Leishmania major, DDC became immobile and incorporated multiple parasites into cytosolic vacuoles. Parasite uptake occurred through the extension of long, highly dynamic pseudopods capable of tracking and engulfing parasites. This was then followed by rapid dendrite retraction towards the cell body. DDC were proficient at discriminating between parasites and inert particles, and parasite uptake was independent of the presence of neutrophils. Together, our study has visualized the dynamics and microenvironmental context of parasite encounter by an innate immune cell subset during the initiation of the immune response. Our results uncover a unique migratory tissue surveillance program of DDC that ensures the rapid detection of pathogens.

CD44 Mediates Successful Interstitial Navigation by Killer T Cells and Enables Efficient Antitumor Immunity

Immunity. Dec, 2008  |  Pubmed ID: 19100702

Although T lymphocytes are constitutively nonadherent cells, they undergo facultative polarity during migration and upon interaction with cells presenting cognate antigen, suggesting that cell polarity might be critical for target cell destruction. Using two-photon imaging of tumor-infiltrating T lymphocytes, we found that CD44, a receptor for extracellular matrix proteins and glycosaminoglycans, was crucial for interstitial T cell navigation and, consequently, efficient tumor cell screening. CD44 functioned as a critical regulator of intratumoral movement by stabilizing cell polarity in migrating T cells, but not during target cell interactions. Stable anterior-posterior asymmetry was maintained by CD44 independently of its extracellular domain. Instead, migratory polarity depended on the recruitment of ezrin, radixin, moesin (ERM) proteins by the intracellular domain of CD44 to the posterior cellular protrusion. Our results formally demonstrate that CD44-dependent T lymphocyte locomotion within target sites represents an essential immunologic checkpoint that determines the potency of T cell effector functions.

Episcopic 3D Imaging Methods: Tools for Researching Gene Function

Current Genomics. Jun, 2008  |  Pubmed ID: 19452045

This work aims at describing episcopic 3D imaging methods and at discussing how these methods can contribute to researching the genetic mechanisms driving embryogenesis and tissue remodelling, and the genesis of pathologies. Several episcopic 3D imaging methods exist. The most advanced are capable of generating high-resolution volume data (voxel sizes from 0.5x0.5x1 microm upwards) of small to large embryos of model organisms and tissue samples. Beside anatomy and tissue architecture, gene expression and gene product patterns can be three dimensionally analyzed in their precise anatomical and histological context with the aid of whole mount in situ hybridization or whole mount immunohistochemical staining techniques. Episcopic 3D imaging techniques were and are employed for analyzing the precise morphological phenotype of experimentally malformed, randomly produced, or genetically engineered embryos of biomedical model organisms. It has been shown that episcopic 3D imaging also fits for describing the spatial distribution of genes and gene products during embryogenesis, and that it can be used for analyzing tissue samples of adult model animals and humans. The latter offers the possibility to use episcopic 3D imaging techniques for researching the causality and treatment of pathologies or for staging cancer. Such applications, however, are not yet routine and currently only preliminary results are available. We conclude that, although episcopic 3D imaging is in its very beginnings, it represents an upcoming methodology, which in short terms will become an indispensable tool for researching the genetic regulation of embryo development as well as the genesis of malformations and diseases.

Plasmacytoid Dendritic Cells Are Dispensable During Primary Influenza Virus Infection

Journal of Immunology (Baltimore, Md. : 1950). Jan, 2009  |  Pubmed ID: 19124730

Plasmacytoid dendritic cells (pDC) are thought to be pivotal in the first line of defense against viral infections. Although previous studies have suggested that pDC regulate the immune response against respiratory syncytial virus, their role in pulmonary infection with influenza virus has remained unclear. Using mice with GFP-tagged pDC, we observed a marked increase in pDC numbers in the lung airways 3 days after intranasal infection with influenza virus A/PR/8/34. To further investigate their potential involvement in the disease, we made use of pDC-deficient IkarosL/L mice. In the absence of pDC, the recruitment of T cells to the bronchoalveolar space was delayed, which could be reversed by the adoptive transfer of pDC before infection. Surprisingly, however, when compared with wild-type animals, IkarosL/L mice revealed a similar course of disease, as determined by weight loss, viral titers, levels of neutralizing Ab, and lung pathology. Moreover, the activation and differentiation of influenza-specific CD8+ effector T cells was unaltered in the absence of pDC, as was the generation of CD8+ memory T cells. Taken together, our study suggests that pDC regulate the accumulation of T cells in the bronchoalveolar space during early influenza virus infection, but are dispensable for the control of this disease.

Behavior of Parasite-specific Effector CD8+ T Cells in the Brain and Visualization of a Kinesis-associated System of Reticular Fibers

Immunity. Feb, 2009  |  Pubmed ID: 19167248

To understand lymphocyte behavior in the brain, we used two-photon microscopy to visualize effector CD8(+) T cells during toxoplasmic encephalitis. These cells displayed multiple behaviors with two distinct populations of cells apparent: one with a constrained pattern of migration and one with a highly migratory subset. The proportion of these populations varied over time associated with changes in antigen availability as well as T cell expression of the inhibitory receptor PD1. Unexpectedly, the movement of infiltrating cells was closely associated with an infection-induced reticular system of fibers. This observation suggests that, whereas in other tissues pre-existing scaffolds exist that guide lymphocyte migration, in the brain specialized structures are induced by inflammation that guide migration of T cells in this immune-privileged environment.

In Vivo Imaging of Cutaneous T-cell Lymphoma Migration to the Skin

Cancer Research. Apr, 2009  |  Pubmed ID: 19318575

Cutaneous T-cell lymphoma (CTCL) is characterized by the accumulation of malignant CD4(+) T cells in the skin. Although the expression of adhesion molecules and chemokine receptors on CTCL cells has been studied extensively on ex vivo isolated cells, very little is known about the dynamics and mechanisms of CTCL trafficking in vivo. However, detailed knowledge of the molecular cues mediating CTCL migration may be used to interfere with their homing to the skin. We made use of real-time intravital epifluorescence video and two-photon microscopy to visualize malignant T cells from Sezary syndrome (SS), a leukemic variant of CTCL, in dermal microvessels in mouse ear skin. We found that SS cells rolled along dermal venules in a P-selectin- and E-selectin-dependent manner at ratios similar to CD4(+) memory T cells from normal donors. We furthermore show that the chemokine CCL17/TARC, but not CCL27/CTACK, was sufficient to induce the arrest of SS cells in the microvasculature. However, a combination of both chemokines was required to induce extravasation of SS cells. Together, our experiments delineate the molecular adhesion cascade operant in SS cell homing to the skin in vivo.

Distribution and Three-dimensional Appearance of the Interstitial Cells of Cajal in the Rat Stomach and Duodenum

Microscopy Research and Technique. Dec, 2009  |  Pubmed ID: 19455682

The relationship between the interstitial cells of Cajal (ICC) and enteric nerves or smooth muscles cells is not fully defined. Presently, distribution and appearance of ICC in the rat stomach and duodenum was studied by immunohistochemistry, electron microscopy, and three-dimensional reconstruction. c-kit expressing ICC were regularly observed in the Auerbach's myenteric plexus (AP) of the stomach and duodenum. ICC in stomach and duodenum muscle layers was dissimilarly distributed. c-kit immunoreactive cells were sparsely distributed in the stomach circular muscle layer but were abundant in the duodenum deep muscular plexus (DMP). Electron microscopy revealed that stomach ICC-AP were irregular ovals with few cytoplasmic processes, and possessed an electron-dense cytoplasm, numerous mitochondria, intermediate filaments, and caveolae. Duodenum and stomach ICC-AP were similar in appearance. Ultrastructure observations and three-dimensional reconstructions revealed ICC-AP processes wrapping the nerve fibers and projecting into the space between smooth muscle cells. While ICC-AP was occasionally close to enteric nerves or smooth muscle cells, no connections were observed. ICC-DMP in duodenum was elongated and adopted the same cell axis orientation as the circular muscle cells. Unlike ICC-AP, ICC-DMP formed gap junctions with smooth muscle cells and had close contact with nerves. These results indicate that ICC-AP is regularly distributed in stomach and duodenum, while ICC-DMP is exclusively located in the duodenum. ICC-DMP, which possess gap junctions and closely contacts nerves, may participate in neuromuscular transmission.

Dynamic Imaging of CD8(+) T Cells and Dendritic Cells During Infection with Toxoplasma Gondii

PLoS Pathogens. Jul, 2009  |  Pubmed ID: 19578440

To better understand the initiation of CD8(+) T cell responses during infection, the primary response to the intracellular parasite Toxoplasma gondii was characterized using 2-photon microscopy combined with an experimental system that allowed visualization of dendritic cells (DCs) and parasite specific CD8(+) T cells. Infection with T. gondii induced localization of both these populations to the sub-capsular/interfollicular region of the draining lymph node and DCs were required for the expansion of the T cells. Consistent with current models, in the presence of cognate antigen, the average velocity of CD8(+) T cells decreased. Unexpectedly, infection also resulted in modulation of the behavior of non-parasite specific T cells. This TCR-independent process correlated with the re-modeling of the lymph node micro-architecture and changes in expression of CCL21 and CCL3. Infection also resulted in sustained interactions between the DCs and CD8(+) T cells that were visualized only in the presence of cognate antigen and were limited to an early phase in the response. Infected DCs were rare within the lymph node during this time frame; however, DCs presenting the cognate antigen were detected. Together, these data provide novel insights into the earliest interaction between DCs and CD8(+) T cells and suggest that cross presentation by bystander DCs rather than infected DCs is an important route of antigen presentation during toxoplasmosis.

Matrix Crosslinking Forces Tumor Progression by Enhancing Integrin Signaling

Cell. Nov, 2009  |  Pubmed ID: 19931152

Tumors are characterized by extracellular matrix (ECM) remodeling and stiffening. The importance of ECM remodeling to cancer is appreciated; the relevance of stiffening is less clear. We found that breast tumorigenesis is accompanied by collagen crosslinking, ECM stiffening, and increased focal adhesions. Induction of collagen crosslinking stiffened the ECM, promoted focal adhesions, enhanced PI3 kinase (PI3K) activity, and induced the invasion of an oncogene-initiated epithelium. Inhibition of integrin signaling repressed the invasion of a premalignant epithelium into a stiffened, crosslinked ECM and forced integrin clustering promoted focal adhesions, enhanced PI3K signaling, and induced the invasion of a premalignant epithelium. Consistently, reduction of lysyl oxidase-mediated collagen crosslinking prevented MMTV-Neu-induced fibrosis, decreased focal adhesions and PI3K activity, impeded malignancy, and lowered tumor incidence. These data show how collagen crosslinking can modulate tissue fibrosis and stiffness to force focal adhesions, growth factor signaling and breast malignancy.

Visualizing Vertebrate Embryos with Episcopic 3D Imaging Techniques

TheScientificWorldJournal. 2009  |  Pubmed ID: 20024516

The creation of highly detailed, three-dimensional (3D) computer models is essential in order to understand the evolution and development of vertebrate embryos, and the pathogenesis of hereditary diseases. A still-increasing number of methods allow for generating digital volume data sets as the basis of virtual 3D computer models. This work aims to provide a brief overview about modern volume data-generation techniques, focusing on episcopic 3D imaging methods. The technical principles, advantages, and problems of episcopic 3D imaging are described. The strengths and weaknesses in its ability to visualize embryo anatomy and labeled gene product patterns, specifically, are discussed.

Western Lifestyle and Increased Prevalence of Atopic Diseases: an Example from a Small Papua New Guinean Island

The World Allergy Organization Journal. Jul, 2009  |  Pubmed ID: 23283062

: Allergic diseases represent an increasing problem in public health in most modern societies as their prevalence has risen markedly during recent decades. Nevertheless, the causes of this increase are not yet fully explained.

Trafficking of Immune Cells in the Central Nervous System

The Journal of Clinical Investigation. May, 2010  |  Pubmed ID: 20440079

The CNS is an immune-privileged environment, yet the local control of multiple pathogens is dependent on the ability of immune cells to access and operate within this site. However, inflammation of the distinct anatomical sites (i.e., meninges, cerebrospinal fluid, and parenchyma) associated with the CNS can also be deleterious. Therefore, control of lymphocyte entry and migration within the brain is vital to regulate protective and pathological responses. In this review, several recent advances are highlighted that provide new insights into the processes that regulate leukocyte access to, and movement within, the brain.

Herpes Simplex Virus Infects Skin Gamma Delta T Cells Before Langerhans Cells and Impedes Migration of Infected Langerhans Cells by Inducing Apoptosis and Blocking E-cadherin Downregulation

Journal of Immunology (Baltimore, Md. : 1950). Jul, 2010  |  Pubmed ID: 20519652

The role individual skin dendritic cell (DC) subsets play in the immune response to HSV remains unclear. We investigated the effect of HSV on DC virus uptake, viability, and migration after cutaneous infection in vitro and in vivo. HSV increased the emigration of skin DCs from whole skin explants over 3 d postinfection (p.i.) compared with mock controls, but the kinetics of emigration was influenced by the skin DC subset. Uninfected (bystander) Langerhans cells (LCs) were the major emigrant DC subset at 24 h p.i., but thereafter, large increases in infected CD103(+)langerin(+) dermal DC (dDC) and uninfected langerin(-) dDC emigration were also observed. LC infection was confirmed by the presence of HSV glycoprotein D (gD) and was associated with impaired migration from cultured skin. Langerin(+) dDC also expressed HSV gD, but infection did not impede migration. We then followed the virus in live MacGreen mice in which LCs express GFP using a fluorescent HSV-1 strain by time-lapse confocal microscopy. We observed a sequential infection of epidermal cells, first in keratinocytes and epidermal gammadelta T cells at 6 h p.i., followed by the occurrence of HSVgD(+) LCs at 24 h p.i. HSV induced CCR7 upregulation on all langerin(+) DC, including infected LCs, and increased production of skin TNF-alpha and IL-1beta. However, a large proportion of infected LCs that remained within the skin was apoptotic and failed to downregulate E-cadherin compared with bystander LCs or mock controls. Thus, HSV infection of LCs is preceded by infection of gammadelta T cells and delays migration.

Bone Marrow Chimeric Mice Reveal a Role for CX₃CR1 in Maintenance of the Monocyte-derived Cell Population in the Olfactory Neuroepithelium

Journal of Leukocyte Biology. Oct, 2010  |  Pubmed ID: 20610801

Macrophages in the olfactory neuroepithelium are thought to play major roles in tissue homeostasis and repair. However, little information is available at present about possible heterogeneity of these monocyte-derived cells, their turnover rates, and the role of chemokine receptors in this process. To start addressing these issues, this study used Cx₃cr1(gfp) mice, in which the gene sequence for eGFP was knocked into the CX₃CR1 gene locus in the mutant allele. Using neuroepithelial whole-mounts from Cx₃cr1(gfp/+) mice, we show that eGFP(+) cells of monocytic origin are distributed in a loose network throughout this tissue and can be subdivided further into two immunophenotypically distinct subsets based on MHC-II glycoprotein expression. BM chimeric mice were created using Cx₃cr1(gfp/+) donors to investigate turnover of macrophages (and other monocyte-derived cells) in the olfactory neuroepithelium. Our data indicate that the monocyte-derived cell population in the olfactory neuroepithelium is actively replenished by circulating monocytes and under the experimental conditions, completely turned over within 6 months. Transplantation of Cx₃cr1(gfp/gfp) (i.e., CX₃CR1-deficient) BM partially impaired the replenishment process and resulted in an overall decline of the total monocyte-derived cell number in the olfactory epithelium. Interestingly, replenishment of the CD68(low)MHC-II(+) subset appeared minimally affected by CX₃CR1 deficiency. Taken together, the established baseline data about heterogeneity of monocyte-derived cells, their replenishment rates, and the role of CX₃CR1 provide a solid basis to further examine the importance of different monocyte subsets for neuroregeneration at this unique frontier with the external environment.

Cell-autonomous and Environmental Contributions to the Interstitial Migration of T Cells

Seminars in Immunopathology. Sep, 2010  |  Pubmed ID: 20623124

A key to understanding the functioning of the immune system is to define the mechanisms that facilitate directed lymphocyte migration to and within tissues. The recent development of improved imaging technologies, most prominently multi-photon microscopy, has enabled the dynamic visualization of immune cells in real-time directly within intact tissues. Intravital imaging approaches have revealed high spontaneous migratory activity of T cells in secondary lymphoid organs and inflamed tissues. Experimental evidence points towards both environmental and cell-intrinsic cues involved in the regulation of lymphocyte motility in the interstitial space. Based on these data, several conceptually distinct models have been proposed in order to explain the coordination of lymphocyte migration both at the single cell and population level. These range from "stochastic" models, where chance is the major driving force, to "deterministic" models, where the architecture of the microenvironment dictates the migratory trajectory of cells. In this review, we focus on recent advances in understanding naïve and effector T cell migration in vivo. In addition, we discuss some of the contradictory experimental findings in the context of theoretical models of migrating leukocytes.

Advances in Imaging the Innate and Adaptive Immune Response to Toxoplasma Gondii

Future Microbiology. Sep, 2010  |  Pubmed ID: 20860479

Toxoplasma gondii is an intracellular protozoan parasite that infects a wide variety of warm-blooded hosts and can have devastating effects in the developing fetus as well as the immunocompromised host. An appreciation of how this organism interacts with the host immune system is crucial to understanding the pathogenesis of this disease. The last decade has been marked by the application of various imaging techniques, such as bioluminescent imaging as well as confocal and multiphoton microscopy to study toxoplasmosis. The ability to manipulate parasites to express fluorescent/bioluminescent markers or model antigens/enzymes combined with the development of reporter mice that allow the detection of distinct immune populations have been crucial to the success of many of these studies. These approaches have permitted the visualization of parasites and immune cells in real-time and provided new insights into the nature of host-pathogen interactions. This article highlights some of the advances in imaging techniques, their strengths and weaknesses, and how these techniques have impacted our understanding of the interaction between parasites and various immune populations during toxoplasmosis.

How Nickel Turns on Innate Immune Cells

Immunology and Cell Biology. Jan, 2011  |  Pubmed ID: 20938456

Targeted Induction of Antigen Expression Within Dendritic Cells Modulates Antigen-specific Immunity Afforded by Recombinant BCG

Vaccine. Feb, 2011  |  Pubmed ID: 21211591

The targeted modulation of antigen expression by recombinant vaccine vehicles would significantly aid development of effective immunotherapeutic strategies. In this report we demonstrate that the Mycobacterium tuberculosis hspX promoter can be used to regulate in vivo induction of antigens expressed by recombinant Bacille Calmette Guérin (rBCG). HspX promoter induction occurred rapidly upon entry of rBCG into cultured dendritic cells (DCs), as evidenced by GFP levels in DCs when infected with BCG:P(hspX)-GFP, in which P(hspX) controlled GFP expression. Vaccination of mice with BCG:P(hspX)-GFP led to rapid in vivo induction of GFP associated with an influx of GFP(+) DCs at the infection site. P(hspX)-driven antigen expression resulted in an improved capacity of DCs to prime antigen-specific T cells, as infection of DCs with BCG:P(hspX)-85B, where the hspX promoter controls expression of M. tuberculosis Ag85B, led to enhanced proliferation of Ag85B-reactive CD4(+) T cells compared to BCG overexpressing Ag85B using the strong Mycobacterium bovis hsp60 promoter. This enhancement of rBCG-induced immunity was also evident in vivo; mice vaccinated with BCG:P(hspX)-85B displayed markedly improved generation of Ag85B-reactive IFN-γ-secreting T cells compared to control BCG-vaccinated mice, which was most pronounced at extended times points post-vaccination. These data reveal a novel strategy to enhance the development and maintenance of vaccine-specific T cell responses.

Cutaneous Immunosurveillance by Self-renewing Dermal Gammadelta T Cells

The Journal of Experimental Medicine. Mar, 2011  |  Pubmed ID: 21339323

The presence of γδ T cell receptor (TCR)-expressing cells in the epidermis of mice, termed dendritic epidermal T cells (DETCs), is well established. Because of their strict epidermal localization, it is likely that DETCs primarily respond to epithelial stress, such as infections or the presence of transformed cells, whereas they may not participate directly in dermal immune responses. In this study, we describe a prominent population of resident dermal γδ T cells, which differ from DETCs in TCR usage, phenotype, and migratory behavior. Dermal γδ T cells are radioresistant, cycle in situ, and are partially depend on interleukin (IL)-7, but not IL-15, for their development and survival. During mycobacterial infection, dermal γδ T cells are the predominant dermal cells that produce IL-17. Absence of dermal γδ T cells is associated with decreased expansion in skin draining lymph nodes of CD4(+) T cells specific for an immunodominant Mycobacterium tuberculosis epitope. Decreased CD4(+) T cell expansion is related to a reduction in neutrophil recruitment to the skin and decreased BCG shuttling to draining lymph nodes. Thus, dermal γδ T cells are an important part of the resident cutaneous immunosurveillance program. Our data demonstrate functional specialization of T cells in distinct microcompartments of the skin.

Active Immunotherapy Combined with Blockade of a Coinhibitory Pathway Achieves Regression of Large Tumor Masses in Cancer-prone Mice

Molecular Therapy : the Journal of the American Society of Gene Therapy. Sep, 2011  |  Pubmed ID: 21587210

Vaccines that aim to expand tumor-specific CD8(+) T cells have yielded disappointing results in cancer patients although they showed efficacy in transplantable tumor mouse models. Using a system that more faithfully mimics a progressing cancer and its immunoinhibitory microenvironment, we here show that in transgenic mice, which gradually develop adenocarcinomas due to expression of HPV-16 E7 within their thyroid, a highly immunogenic vaccine expressing E7 only induces low E7-specific CD8(+) T-cell responses, which fail to affect the size of the tumors. In contrast, the same type of vaccine expressing E7 fused to herpes simplex virus (HSV)-1 glycoprotein D (gD), an antagonist of the coinhibitory B- and T-lymphocyte attenuator (BTLA)/CD160-herpes virus entry mediator (HVEM) pathways, stimulates potent E7-specific CD8(+) T-cell responses, which can be augmented by repeated vaccination, resulting in initial regression of even large tumor masses in all mice with sustained regression in more than half of them. These results indicate that active immunization concomitantly with blockade of the immunoinhibitory HVEM-BTLA/CD160 pathways through HSV-1 gD may result in sustained tumor regression.

Visualizing the Neutrophil Response to Sterile Tissue Injury in Mouse Dermis Reveals a Three-phase Cascade of Events

The Journal of Investigative Dermatology. Oct, 2011  |  Pubmed ID: 21697893

Neutrophil granulocytes traffic into sites of organ injury in which they may not only participate in tissue repair and pathogen clearance but may also contribute to collateral cell damage through the release of noxious mediators. The dynamics and mechanisms of neutrophil migration in the extravascular space toward loci of tissue damage are not well understood. Here, we have used intravital multi-photon microscopy to dissect the behavior of neutrophils in response to tissue injury in the dermis of mice. We found that, following confined physical injury, initially rare scouting neutrophils migrated in a directional manner toward the damage focus. This was followed by the attraction of waves of additional neutrophils, and finally stabilization of the neutrophil cluster around the injury. Although neutrophil migration in the steady state and during the scouting phase depended on pertussis toxin-sensitive signals, the amplification phase was sensitive to interference with the cyclic adenosine diphosphate ribose pathway. We finally demonstrated that neutrophil scouts also transit through the non-inflamed dermis, suggesting immunosurveillance function by these cells. Together, our data unravel a three-step cascade of events that mediates the specific accumulation of neutrophils at sites of sterile tissue injury in the interstitial space.

Imaging Heart Development Using High-resolution Episcopic Microscopy

Current Opinion in Genetics & Development. Oct, 2011  |  Pubmed ID: 21893408

Development of the heart in vertebrate embryos is a complex process in which the organ is continually remodelled as chambers are formed, valves sculpted and connections established to the developing vascular system. Investigating the genetic programmes driving these changes and the environmental factors that may influence them is critical for our understanding of congenital heart disease. A recurrent challenge in this work is how to integrate studies as diverse as those of cardiac gene function and regulation with an appreciation of the localised interactions between cardiac tissues not to mention the manner in which both may be affected by cardiac function itself. Meeting this challenge requires an accurate way to analyse the changes in 3D morphology of the developing heart, which can be swift or protracted and both dramatic or subtle in consequence. Here we review the use of high-resolution episcopic microscopy as a simple and effective means to examine organ structure and one that allows modern computing methods pioneered by clinical imaging to be applied to the embryonic heart.

Analysis of Behavior and Trafficking of Dendritic Cells Within the Brain During Toxoplasmic Encephalitis

PLoS Pathogens. Sep, 2011  |  Pubmed ID: 21949652

Under normal conditions the immune system has limited access to the brain; however, during toxoplasmic encephalitis (TE), large numbers of T cells and APCs accumulate within this site. A combination of real time imaging, transgenic reporter mice, and recombinant parasites allowed a comprehensive analysis of CD11c+ cells during TE. These studies reveal that the CNS CD11c+ cells consist of a mixture of microglia and dendritic cells (DCs) with distinct behavior associated with their ability to interact with parasites or effector T cells. The CNS DCs upregulated several chemokine receptors during TE, but none of these individual receptors tested was required for migration of DCs into the brain. However, this process was pertussis toxin sensitive and dependent on the integrin LFA-1, suggesting that the synergistic effect of signaling through multiple chemokine receptors, possibly leading to changes in the affinity of LFA-1, is involved in the recruitment/retention of DCs to the CNS and thus provides new insights into how the immune system accesses this unique site.

Langerhans Cells Are Precommitted to Immune Tolerance Induction

Proceedings of the National Academy of Sciences of the United States of America. Nov, 2011  |  Pubmed ID: 22006331

Antigen-dependent interactions between T lymphocytes and dendritic cells (DCs) can produce two distinct outcomes: tolerance and immunity. It is generally considered that all DC subsets are capable of supporting both tolerogenic and immunogenic responses, depending on their exposure to activating signals. Here, we tested whether epidermal Langerhans cells (LCs) can support immunogenic responses in vivo in the absence of antigen presentation by other DC subsets. CD4 T cells responding to antigen presentation by activated LCs initially proliferated but then failed to differentiate into effector/memory cells or to survive long term. The tolerogenic function of LCs was maintained after exposure to potent adjuvants and occurred despite up-regulation of the costimulatory molecules CD80, CD86, and IL-12, but was consistent with their failure to translocate the NF-κB family member RelB from the cytoplasm to the nucleus. Commitment of LCs to tolerogenic function may explain why commensal microorganisms expressing Toll-like receptor (TLR) ligands but confined to the skin epithelium are tolerated, whereas invading pathogens that breach the epithelial basement membrane and activate dermal DCs stimulate a strong immune response.

High-resolution Episcopic Microscopy Data-based Measurements of the Arteries of Mouse Embryos: Evaluation of Significance and Reproducibility Under Routine Conditions

Cells, Tissues, Organs. 2012  |  Pubmed ID: 21997436

Defining the role of genes in the genesis of congenital cardiovascular defects involves comparisons of the diameters of arteries measured in wild-type and genetically engineered mouse embryos. This study aims at evaluating the significance and reproducibility of measurements of the diameters of the great intrathoracic arteries of mouse embryos, as produced under routine conditions, by employing a recently suggested measuring method. Using high-resolution episcopic microscopy, we generated digital volume data of 60 mouse embryos (voxel size 1.07 × 1.07 × 2 μm(3)) of developmental stage 23 according to Theiler. We randomly split the 60 data sets into two groups of 30 and assigned each group to a diploma student. In addition, an experienced scientist received 12 randomly selected specimens of each group. Independently, the researchers created three-dimensional models of the intrathoracic arteries and identified comparable measurement positions along the ascending aorta, pulmonary trunk and descending aorta. At each position, they defined virtual resections cutting through the volume data perpendicular to the longitudinal axis of the artery. In the virtual resections, the researchers measured the perimeter of the lumen of the artery. The diameter was calculated from the perimeter. Then, we performed statistic comparisons of the diameters measured in micrometres and of the ratio of each measured diameter and the diameter of the ascending aorta. Comparisons of the ratios did not reveal statistically significant differences between the measurements created by the different scientists. We assume that the used measuring protocol is highly robust and produces reproducible and significant results under routine conditions.

Transendothelial Migration of Lymphocytes Mediated by Intraendothelial Vesicle Stores Rather Than by Extracellular Chemokine Depots

Nature Immunology. Jan, 2012  |  Pubmed ID: 22138716

Chemokines presented by the endothelium are critical for integrin-dependent adhesion and transendothelial migration of naive and memory lymphocytes. Here we found that effector lymphocytes of the type 1 helper T cell (T(H)1 cell) and type 1 cytotoxic T cell (T(C)1 cell) subtypes expressed adhesive integrins that bypassed chemokine signals and established firm arrests on variably inflamed endothelial barriers. Nevertheless, the transendothelial migration of these lymphocytes strictly depended on signals from guanine nucleotide-binding proteins of the G(i) type and was promoted by multiple endothelium-derived inflammatory chemokines, even without outer endothelial surface exposure. Instead, transendothelial migration-promoting endothelial chemokines were stored in vesicles docked on actin fibers beneath the plasma membranes and were locally released within tight lymphocyte-endothelial synapses. Thus, effector T lymphocytes can cross inflamed barriers through contact-guided consumption of intraendothelial chemokines without surface-deposited chemokines or extraendothelial chemokine gradients.

Modulation of NOXA and MCL-1 As a Strategy for Sensitizing Melanoma Cells to the BH3-mimetic ABT-737

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Feb, 2012  |  Pubmed ID: 22173547

Drug resistance in melanoma is commonly attributed to ineffective apoptotic pathways. Inhibiting antiapoptotic BCL-2 and its relatives is an attractive strategy for sensitizing lymphoid malignancies to drugs but it has been largely unsuccessful for melanoma and other solid tumors. ABT-737, a small-molecule BH3-mimetic, selectively inhibits BCL-2, BCL-XL, and BCL-w and shows promise for treating leukemia, lymphoma, and small-cell lung cancer. Melanoma cells are insensitive to ABT-737, but MCL-1 inhibition reportedly increases the sensitivity of other tumors to the compound.

Phenotype and Functions of Conventional Dendritic Cells Are Not Compromised in Aged Mice

Immunology and Cell Biology. Aug, 2012  |  Pubmed ID: 22231652

Aging has profound effects on the immune system, including thymic involution, reduced diversity of the T cell receptor repertoire, reduced effector T cell and B cell function and chronic increase of proinflammatory cytokine production by innate immune cells. The precise effects of aging on conventional dendritic cells (cDC), the main antigen presenting cells of the immune system, however, are not well understood. We found that in aged mice the number of cDC in the spleen and lymph nodes remained stable, whereas the number of cDC in the lungs increased with age. Whereas cDC in mice showed similar cycling kinetics in all organs tested, cDC reconstitution by aged bone marrow precursors was relatively higher than that of their young counterparts. With the exception of CD86, young and aged cDC did not differ in their expression of co-stimulatory molecules at steady state. Most toll-like receptor (TLR) ligands induced comparable upregulation of co-stimulatory molecules CD40, CD86 and B7H1 on young and aged cDC, whereas TLR2 and TLR5 stimulation resulted in reduced upregulation of CD80 and CD86 on aged cDC in vitro. In vivo, influenza infection-induced upregulation of CD86, but not other co-stimulatory molecules, was lower in aged DC. Young and aged DC were equally capable of direct and cross presentation of antigens in vitro. Transcriptome analysis did not reveal any significant difference between young and aged cDC. These data show that unlike T and B cells, the maintenance of cDC throughout the life of a healthy animal is relatively robust during the aging process.

Intravital Multiphoton Imaging of Immune Responses in the Mouse Ear Skin

Nature Protocols. Feb, 2012  |  Pubmed ID: 22240584

Multiphoton (MP) microscopy enables the direct in vivo visualization, with high spatial and temporal resolution, of fluorescently tagged immune cells, extracellular matrix and vasculature in tissues. This approach, therefore, represents a powerful alternative to traditional methods of assessing immune cell function in the skin, which are mainly based on flow cytometry and histology. Here we provide a step-by-step protocol describing experimental procedures for intravital MP imaging of the mouse ear skin, which can be easily adapted to address many specific skin-related biological questions. We demonstrate the use of this procedure by characterizing the response of neutrophils during cutaneous inflammation, which can be used to perform in-depth analysis of neutrophil behavior in the context of the skin microanatomy, including the epidermis, dermis and blood vessels. Such experiments are typically completed within 1 d, but as the procedures are minimally invasive, it is possible to perform longitudinal studies through repeated imaging.

Some Mice Feature 5th Pharyngeal Arch Arteries and Double-lumen Aortic Arch Malformations

Cells, Tissues, Organs. 2012  |  Pubmed ID: 22287557

A 5th pair of pharyngeal arch arteries (PAAs) has never been identified with certainty in mice. Murines in general are considered to not develop a 5th pair. If true, the significance of the mouse as a model for researching the genesis of malformations of the great intrathoracic arteries is limited. We aimed to investigate whether mouse embryos develop a 5th pair of PAAs and to identify malformations known to be caused by defective remodelling of the 5th PAAs. We employed the high-resolution episcopic microscopy method for creating digital volume data and three-dimensional (3D) computer models of the great intrathoracic arteries of 30 mouse embryos from days 12-12.5 post conception and 180 mouse fetuses from days 14.5 and 15.5 post conception. The 3D models of the fetuses were screened for the presence of a double-lumen aortic arch malformation. We identified such a malformation in 1 fetus. The 3D models of the embryos were analysed for the presence of 5th PAAs. Six of the 30 embryos (20%) showed a 5th PAA bilaterally, and an additional 9 (30%) showed a 5th PAA unilaterally. Our results prove that some mice do develop a 5th pair of PAAs. They also show that malformations which occur rarely in humans and result from defective remodelling of the left 5th PAA can be identified in mice as well. Thus, the mouse does represent an excellent model for researching the mechanisms driving PAA remodelling and the genesis of malformations of the great intrathoracic arteries.

A Mouse Model of Vitiligo with Focused Epidermal Depigmentation Requires IFN-γ for Autoreactive CD8⁺ T-cell Accumulation in the Skin

The Journal of Investigative Dermatology. Jul, 2012  |  Pubmed ID: 22297636

Vitiligo is an autoimmune disease of the skin causing disfiguring patchy depigmentation of the epidermis and, less commonly, hair. Therapeutic options for vitiligo are limited, reflecting in part limited knowledge of disease pathogenesis. Existing mouse models of vitiligo consist of hair depigmentation but lack prominent epidermal involvement, which is the hallmark of human disease. They are thus unable to provide a platform to fully investigate disease mechanisms and treatment. CD8(+) T cells have been implicated in the pathogenesis of vitiligo, and expression of IFN-γ is increased in the lesional skin of patients, however, it is currently unknown what role IFN-γ has in disease. Here, we have developed an adoptive transfer mouse model of vitiligo using melanocyte-specific CD8(+) T cells, which recapitulates the human condition by inducing epidermal depigmentation while sparing the hair. Like active lesions in human vitiligo, histology of depigmenting skin reveals a patchy mononuclear infiltrate and single-cell infiltration of the epidermis. Depigmentation is accompanied by accumulation of autoreactive CD8(+) T cells in the skin, quantifiable loss of tyrosinase transcript, and local IFN-γ production. Neutralization of IFN-γ with antibody prevents CD8(+) T-cell accumulation and depigmentation, suggesting a therapeutic potential for this approach.

Episcopic Three-dimensional Imaging of Embryos

Cold Spring Harbor Protocols. Jun, 2012  |  Pubmed ID: 22661435

Episcopic fluorescence image capturing (EFIC) and high-resolution episcopic microscopy (HREM) are related techniques that are used to generate digital volume data and create three-dimensional (3D) images. Both techniques require specimens that are embedded in an appropriate medium, and images are captured from successive sections before removal from the embedded tissue block. EFIC detects autofluorescence emitted from the embedded tissue, whereas HREM requires the tissue to be stained with a fluorescent dye such as eosin. Different procedures are therefore necessary for embedding tissue for EFIC or HREM imaging. This article describes episcopic imaging and gives the advantages and disadvantages of the EFIC and HREM techniques. It also describes the imaging setup required to collect volume data and generate 3D images.

Embedding Embryos for Episcopic Fluorescence Image Capturing (EFIC)

Cold Spring Harbor Protocols. Jun, 2012  |  Pubmed ID: 22661436

Episcopic fluorescence image capturing (EFIC) and high-resolution episcopic microscopy (HREM) are related techniques that are used to generate digital volume data and create three-dimensional (3D) images. Both techniques require specimens that are embedded in an appropriate medium, and images are captured from successive sections before removal from the embedded tissue block. EFIC detects autofluorescence emitted from the embedded tissue, whereas HREM requires the tissue to be stained with a fluorescent dye such as eosin. Different procedures are therefore necessary for embedding tissue for EFIC or HREM imaging. This protocol describes the procedure for embedding E11.5 mouse embryos for EFIC imaging. Processing times should be adapted for the size and nature of other specimens.

Embedding Embryos for High-resolution Episcopic Microscopy (HREM)

Cold Spring Harbor Protocols. Jun, 2012  |  Pubmed ID: 22661437

Episcopic fluorescence image capturing (EFIC) and high-resolution episcopic microscopy (HREM) are related techniques that are used to generate digital volume data and create three-dimensional (3D) images. Both techniques require specimens that are embedded in an appropriate medium, and images are captured from successive sections before removal from the embedded tissue block. EFIC detects autofluorescence emitted from the embedded tissue, whereas HREM requires the tissue to be stained with a fluorescent dye such as eosin. Different procedures are therefore necessary for embedding tissue for EFIC or HREM imaging. For HREM, the choice of fixative appears to be of little consequence. If gene expression patterns are to be visualized in addition to tissue architecture, whole-mount staining for gene expression patterns using the 4-nitro blue tetrazolium chloride (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) or LacZ detection system must be performed before embedding of samples. This protocol describes the procedure for embedding E11.5 mouse embryos for HREM imaging. Processing times should be adapted for the size and nature of other specimens.

Generation of Volume Data by Episcopic Three-dimensional Imaging of Embryos

Cold Spring Harbor Protocols. Jun, 2012  |  Pubmed ID: 22661438

Episcopic fluorescence image capturing (EFIC) and high-resolution episcopic microscopy (HREM) are related techniques that are used to generate digital volume data and create three-dimensional (3D) images. Both techniques require specimens that are embedded in an appropriate medium, and images are captured from successive sections before removal from the embedded tissue block. EFIC detects autofluorescence emitted from the embedded tissue, whereas HREM requires the tissue to be stained with a fluorescent dye such as eosin. Volume data are generated as the successive sections are imaged and removed using a microtome. This protocol describes the procedure for digital data processing, visualization, and archiving for both EFIC and HREM.

Models in Researching Cardiovascular Morphogenesis

Birth Defects Research. Part C, Embryo Today : Reviews. Jun, 2012  |  Pubmed ID: 22692889

Several types of "models" are used in modern biological and biomedical research and teaching. This article attempts to provide a brief explanation of the termini model organism, theoretic and mathematic model, three-dimensional (3D) model, and 4D model, and an overview about the generation and the fields of applications of these types of models. It will then focus on the application of theoretic and mathematic models, 3D models, 4D models, and simulation for researching cardiovascular morphogenesis.

Generalized Lévy Walks and the Role of Chemokines in Migration of Effector CD8+ T Cells

Nature. Jun, 2012  |  Pubmed ID: 22722867

Chemokines have a central role in regulating processes essential to the immune function of T cells, such as their migration within lymphoid tissues and targeting of pathogens in sites of inflammation. Here we track T cells using multi-photon microscopy to demonstrate that the chemokine CXCL10 enhances the ability of CD8+ T cells to control the pathogen Toxoplasma gondii in the brains of chronically infected mice. This chemokine boosts T-cell function in two different ways: it maintains the effector T-cell population in the brain and speeds up the average migration speed without changing the nature of the walk statistics. Notably, these statistics are not Brownian; rather, CD8+ T-cell motility in the brain is well described by a generalized Lévy walk. According to our model, this unexpected feature enables T cells to find rare targets with more than an order of magnitude more efficiency than Brownian random walkers. Thus, CD8+ T-cell behaviour is similar to Lévy strategies reported in organisms ranging from mussels to marine predators and monkeys, and CXCL10 aids T cells in shortening the average time taken to find rare targets.

Inflammasome-dependent IFN-γ Drives Pathogenesis in Streptococcus Pneumoniae Meningitis

Journal of Immunology (Baltimore, Md. : 1950). Nov, 2012  |  Pubmed ID: 23071286

The pathology associated with Streptococcus pneumoniae meningitis results largely from activation of immune-associated pathways. We systematically investigated the production of IFN subtypes, as well as their influence on pathology, in a mouse model of S. pneumoniae meningitis. Despite the occurrence of a mixed IFN type I/II gene signature, no evidence for production or involvement of type I IFNs in disease progression was found. In contrast, type II IFN (IFN-γ) was strongly induced, and IFN-γ(-/-) mice were significantly protected from severe disease. Using intracellular cytokine staining and targeted cell-depletion approaches, NK cells were found to be the dominant source of IFN-γ. Furthermore, production of IFN-γ was found to be dependent upon ASC and IL-18, indicating that an ASC-dependent inflammasome pathway was responsible for mediating IFN-γ induction. The influence of IFN-γ gene deletion on a range of processes known to be involved in bacterial meningitis pathogenesis was examined. Although neutrophil numbers in the brain were similar in infected wild-type and IFN-γ(-/-) mice, both monocyte recruitment and CCL2 production were less in infected IFN-γ(-/-) mice compared with infected wild-type controls. Additionally, gene expression of NO synthase was strongly diminished in infected IFN-γ(-/-) mice compared with infected controls. Finally, bacterial clearance was enhanced in IFN-γ(-/-) mice, although the underlying mechanism remains unclear. Together, these data suggest that inflammasome-dependent IFN-γ contributes via multiple pathways to pathology during S. pneumoniae meningitis.

Visualizing Leukocyte Trafficking in the Living Brain with 2-photon Intravital Microscopy

Frontiers in Cellular Neuroscience. 2012  |  Pubmed ID: 23316136

Intravital imaging of the superficial brain tissue in mice represents a powerful tool for the dissection of the cellular and molecular cues underlying inflammatory and infectious central nervous system (CNS) diseases. We present here a step-by-step protocol that will enable a non-specialist to set up a two-photon brain-imaging model. The protocol offers a two-part approach that is specifically optimized for imaging leukocytes but can be easily adapted to answer varied CNS-related biological questions. The protocol enables simultaneous visualization of fluorescently labeled immune cells, the pial microvasculature and extracellular structures such as collagen fibers at high spatial and temporal resolution. Intracranial structures are exposed through a cranial window, and physiologic conditions are maintained during extended imaging sessions via continuous superfusion of the brain surface with artificial cerebrospinal fluid (aCSF). Experiments typically require 1-2 h of preparation, which is followed by variable periods of immune cell tracking. Our methodology converges the experience of two laboratories over the past 10 years in diseased animal models such as cerebral ischemia, lupus, cerebral malaria, and toxoplasmosis. We exemplify the utility of this protocol by tracking leukocytes in transgenic mice in the pial vessels under steady-state conditions.

Metric Characterization of the Aortic Arch of Early Mouse Fetuses and of a Fetus Featuring a Double Lumen Aortic Arch Malformation

Annals of Anatomy = Anatomischer Anzeiger : Official Organ of the Anatomische Gesellschaft. Mar, 2013  |  Pubmed ID: 23123185

This study aimed at providing an objective metric characterization of the aortic arch of a mouse fetus featuring a double lumen aortic arch malformation. As a side effect it provides reference data defining the length and the diameters of the aortic arch segments of normally developed mouse fetuses at developmental stage 22 according to Theiler (TS22). We analyzed a total of 22 TS22 mouse fetuses of the Him:OF1 strain. We produced high-resolution three-dimensional (3D) computer models and measured the diameters and cross sectional areas of the aortic arch segments and of the ascending and descending aorta. In addition, we defined 3D skeletons of the arteries and measured the length of the aortic arch segments. We provide statistics on the measurements obtained from the normally developed TS22 fetuses and detailed characterizations of the double lumen aortic arch. Our data suggest that: firstly, in Him:OF1 fetuses of TS22, the formation of the aortic arch is not yet finished. The left subclavian artery still receives a significant amount of blood from the right ventricle. Secondly, persistence of the 5th pharyngeal arch artery does not affect remodeling of the arteries distal to the junction of 5th pharyngeal arch artery and dorsal aorta. Thirdly, hemodynamic forces define the dimensions of the aortic arch between the left common carotid and the left subclavian artery. Fourthly, the blood volume streaming through the 4th pharyngeal arch artery influences its enlargement between TS20 and TS22.

Shedding Light on Cutaneous Innate Immune Responses: the Intravital Microscopy Approach

Immunology and Cell Biology. Apr, 2013  |  Pubmed ID: 23459295

The skin is under constant assault by environmental factors and microbes. Innate immune cells in epidermis and dermis regulate immune responses against pathogens while maintaining tolerance against commensal bacteria and autoantigens. The introduction of intravital imaging approaches, in particular multiphoton microscopy, has enabled studying the cellular and molecular regulation of cutaneous immunity in real time within intact skin. Here, we discuss recent advances in our understanding of innate immune cell behaviour in the skin, as unravelled by intravital microscopy, with emphasis on the function of myeloid cells, including dendritic cells, neutrophils and monocytes.

Deciphering the Mechanisms of Developmental Disorders (DMDD): a New Programme for Phenotyping Embryonic Lethal Mice

Disease Models & Mechanisms. May, 2013  |  Pubmed ID: 23519034

International efforts to test gene function in the mouse by the systematic knockout of each gene are creating many lines in which embryonic development is compromised. These homozygous lethal mutants represent a potential treasure trove for the biomedical community. Developmental biologists could exploit them in their studies of tissue differentiation and organogenesis; for clinical researchers they offer a powerful resource for investigating the origins of developmental diseases that affect newborns. Here, we outline a new programme of research in the UK aiming to kick-start research with embryonic lethal mouse lines. The 'Deciphering the Mechanisms of Developmental Disorders' (DMDD) programme has the ambitious goal of identifying all embryonic lethal knockout lines made in the UK over the next 5 years, and will use a combination of comprehensive imaging and transcriptomics to identify abnormalities in embryo structure and development. All data will be made freely available, enabling individual researchers to identify lines relevant to their research. The DMDD programme will coordinate its work with similar international efforts through the umbrella of the International Mouse Phenotyping Consortium [see accompanying Special Article (Adams et al., 2013)] and, together, these programmes will provide a novel database for embryonic development, linking gene identity with molecular profiles and morphology phenotypes.

Cutaneous Immunosurveillance and Regulation of Inflammation by Group 2 Innate Lymphoid Cells

Nature Immunology. Apr, 2013  |  Pubmed ID: 23603794

Type 2 immunity is critical for defense against cutaneous infections but also underlies the development of allergic skin diseases. We report the identification in normal mouse dermis of an abundant, phenotypically unique group 2 innate lymphoid cell (ILC2) subset that depended on interleukin 7 (IL-7) and constitutively produced IL-13. Intravital multiphoton microscopy showed that dermal ILC2 cells specifically interacted with mast cells, whose function was suppressed by IL-13. Treatment of mice deficient in recombination-activating gene 1 (Rag1(-/-)) with IL-2 resulted in the population expansion of activated, IL-5-producing dermal ILC2 cells, which led to spontaneous dermatitis characterized by eosinophil infiltrates and activated mast cells. Our data show that ILC2 cells have both pro- and anti-inflammatory properties and identify a previously unknown interactive pathway between two innate populations of cells of the immune system linked to type 2 immunity and allergic diseases.

A Novel Class of Anticancer Compounds Targets the Actin Cytoskeleton in Tumor Cells

Cancer Research. Aug, 2013  |  Pubmed ID: 23946473

The actin cytoskeleton is a potentially vulnerable property of cancer cells, yet chemotherapeutic targeting attempts have been hampered by unacceptable toxicity. In this study, we have shown that it is possible to disrupt specific actin filament populations by targeting isoforms of tropomyosin, a core component of actin filaments, that are selectively upregulated in cancers. A novel class of anti-tropomyosin compounds has been developed that preferentially disrupts the actin cytoskeleton of tumor cells, impairing both tumor cell motility and viability. Our lead compound, TR100, is effective in vitro and in vivo in reducing tumor cell growth in neuroblastoma and melanoma models. Importantly, TR100 shows no adverse impact on cardiac structure and function, which is the major side effect of current anti-actin drugs. This proof-of-principle study shows that it is possible to target specific actin filament populations fundamental to tumor cell viability based on their tropomyosin isoform composition. This improvement in specificity provides a pathway to the development of a novel class of anti-actin compounds for the potential treatment of a wide variety of cancers.

Mesenchymal Cells Hold the Key to Immune Cell Recruitment to and Migration Within Melanoma

The Journal of Investigative Dermatology. Sep, 2013  |  Pubmed ID: 23949769

Samaniego et al. (this issue) report on distinct tumor-associated mesenchymal cell (MC) populations in human melanomas. FAP(-)CD90(+) peritumoral MCs may be involved in immune cell recruitment from the bloodstream. FAP(+)CD90(-) intratumoral MCs were associated with extracellular matrix fiber deposition, and their numbers correlated with high immune cell infiltration. Thus, different MC subsets modulate the cellular composition of the intratumoral and peritumoral melanoma microenvironment.

A Coming of Age: Advanced Imaging Technologies for Characterising the Developing Mouse

Trends in Genetics : TIG. Dec, 2013  |  Pubmed ID: 24035368

The immense challenge of annotating the entire mouse genome has stimulated the development of cutting-edge imaging technologies in a drive for novel information. These techniques promise to improve understanding of the genes involved in embryo development, at least one third of which have been shown to be essential. Aligning advanced imaging technologies with biological needs will be fundamental to maximising the number of phenotypes discovered in the coming years. International efforts are underway to meet this challenge through an integrated and sophisticated approach to embryo phenotyping. We review rapid advances made in the imaging field over the past decade and provide a comprehensive examination of the relative merits of current and emerging techniques. The aim of this review is to provide a guide to state-of-the-art embryo imaging that will enable informed decisions as to which technology to use and fuel conversations between expert imaging laboratories, researchers, and core mouse production facilities.

Perivascular Macrophages Mediate Neutrophil Recruitment During Bacterial Skin Infection

Nature Immunology. Jan, 2014  |  Pubmed ID: 24270515

Transendothelial migration of neutrophils in postcapillary venules is a key event in the inflammatory response against pathogens and tissue damage. The precise regulation of this process is incompletely understood. We report that perivascular macrophages are critical for neutrophil migration into skin infected with the pathogen Staphylococcus aureus. Using multiphoton intravital microscopy we showed that neutrophils extravasate from inflamed dermal venules in close proximity to perivascular macrophages, which are a major source of neutrophil chemoattractants. The virulence factor α-hemolysin produced by S. aureus lyses perivascular macrophages, which leads to decreased neutrophil transmigration. Our data illustrate a previously unrecognized role for perivascular macrophages in neutrophil recruitment to inflamed skin and indicate that S. aureus uses hemolysin-dependent killing of these cells as an immune evasion strategy.

A Promiscuous Lipid-binding Protein Diversifies the Subcellular Sites of Toll-like Receptor Signal Transduction

Cell. Feb, 2014  |  Pubmed ID: 24529375

The Toll-like receptors (TLRs) of the innate immune system are unusual in that individual family members are located on different organelles, yet most activate a common signaling pathway important for host defense. It remains unclear how this common signaling pathway can be activated from multiple subcellular locations. Here, we report that, in response to natural activators of innate immunity, the sorting adaptor TIRAP regulates TLR signaling from the plasma membrane and endosomes. TLR signaling from both locations triggers the TIRAP-dependent assembly of the myddosome, a protein complex that controls proinflammatory cytokine expression. The actions of TIRAP depend on the promiscuity of its phosphoinositide-binding domain. Different lipid targets of this domain direct TIRAP to different organelles, allowing it to survey multiple compartments for the presence of activated TLRs. These data establish how promiscuity, rather than specificity, can be a beneficial means of diversifying the subcellular sites of innate immune signal transduction.

Targeting Rho-GTPases in Immune Cell Migration and Inflammation

British Journal of Pharmacology. Dec, 2014  |  Pubmed ID: 24571448

Leukocytes are unmatched migrators capable of traversing barriers and tissues of remarkably varied structural composition. An effective immune response relies on the ability of its constituent cells to infiltrate target sites. Yet, unwarranted mobilization of immune cells can lead to inflammatory diseases and tissue damage ranging in severity from mild to life-threatening. The efficacy and plasticity of leukocyte migration is driven by the precise spatiotemporal regulation of the actin cytoskeleton. The small GTPases of the Rho family (Rho-GTPases), and their immediate downstream effector kinases, are key regulators of cellular actomyosin dynamics and are therefore considered prime pharmacological targets for stemming leukocyte motility in inflammatory disorders. This review describes advances in the development of small-molecule inhibitors aimed at modulating the Rho-GTPase-centric regulatory pathways governing motility, many of which stem from studies of cancer invasiveness. These inhibitors promise the advent of novel treatment options with high selectivity and potency against immune-mediated pathologies.

The Skin-Resident Immune Network

Current Dermatology Reports. 2014  |  Pubmed ID: 24587975

The skin provides an effective physical and biological barrier against environmental and pathogenic insults whilst ensuring tolerance against commensal microbes. This protection is afforded by the unique anatomy and cellular composition of the skin, particularly the vast network of skin-associated immune cells. These include the long-appreciated tissue-resident macrophages, dendritic cells, and mast cells, as well as the more recently described dermal γδ T cells and innate lymphoid cells. Collectively, these cells orchestrate the defense against a wide range of pathogens and environmental challenges, but also perform a number of homeostatic functions. Here, we review recent developments in our understanding of the various roles that leukocyte subsets play in cutaneous immunobiology, and introduce the newer members of the skin immune system. Implications for human disease are discussed.

Leukocyte Migration in the Interstitial Space of Non-lymphoid Organs

Nature Reviews. Immunology. Apr, 2014  |  Pubmed ID: 24603165

Leukocyte migration through interstitial tissues is essential for mounting a successful immune response. Interstitial motility is governed by a vast array of cell-intrinsic and cell-extrinsic factors that together ensure the proper positioning of immune cells in the context of specific microenvironments. Recent advances in imaging modalities, in particular intravital confocal and multi-photon microscopy, have helped to expand our understanding of the cellular and molecular mechanisms that underlie leukocyte navigation in the extravascular space. In this Review, we discuss the key factors that regulate leukocyte motility within three-dimensional environments, with a focus on neutrophils and T cells in non-lymphoid organs.

Anatomical and Ultrasound Correlation of the Superficial Branch of the Radial Nerve

Muscle & Nerve. Dec, 2014  |  Pubmed ID: 24604158

This anatomical study evaluates the role and correlation of ultrasound (US) with anatomy in depicting the superficial branch of the radial nerve (SBRN) and to evaluate the feasibility of US guided perineural infiltration as a potential therapeutic option in Wartenberg syndrome.

Real-time Cell Cycle Imaging During Melanoma Growth, Invasion, and Drug Response

Pigment Cell & Melanoma Research. Sep, 2014  |  Pubmed ID: 24902993

Solid cancers are composed of heterogeneous zones containing proliferating and quiescent cells. Despite considerable insight into the molecular mechanisms underlying aberrant cell cycle progression, there is limited understanding of the relationship between the cell cycle on the one side, and melanoma cell motility, invasion, and drug sensitivity on the other side. Utilizing the fluorescent ubiquitination-based cell cycle indicator (FUCCI) to longitudinally monitor proliferation and migration of melanoma cells in 3D culture and in vivo, we found that invading melanoma cells cycle actively, while G1-arrested cells showed decreased invasion. Melanoma cells in a hypoxic environment or treated with mitogen-activated protein kinase pathway inhibitors remained G1-arrested for extended periods of time, with proliferation and invasion resuming after re-exposure to a more favorable environment. We challenge the idea that the invasive and proliferative capacity of melanoma cells are mutually exclusive and further demonstrate that a reversibly G1-arrested subpopulation survives in the presence of targeted therapies.

T Cell Migration in Intact Lymph Nodes in Vivo

Current Opinion in Cell Biology. Oct, 2014  |  Pubmed ID: 24907445

In the lymph node, T cells migrate rapidly and with striking versatility in a continuous scan for antigen presenting dendritic cells. The scanning process is greatly facilitated by the lymph node structure and composition. In vivo imaging has been instrumental in deciphering the spatiotemporal dynamics of intranodal T cell migration in both health and disease. Here we review recent developments in uncovering the migration modes employed by T cells in the lymph node, the underlying molecular mechanisms, and the scanning strategies utilised by T cells to ensure a timely response to antigenic stimuli.

Antigen Expression Level Threshold Tunes the Fate of CD8 T Cells During Primary Hepatic Immune Responses

Proceedings of the National Academy of Sciences of the United States of America. Jun, 2014  |  Pubmed ID: 24927525

CD8 T-cell responses to liver-expressed antigens range from deletional tolerance to full effector differentiation resulting in overt hepatotoxicity. The reasons for these heterogeneous outcomes are not well understood. To identify factors that govern the fate of CD8 T cells activated by hepatocyte-expressed antigen, we exploited recombinant adenoassociated viral vectors that enabled us to vary potential parameters determining these outcomes in vivo. Our findings reveal a threshold of antigen expression within the liver as the dominant factor determining T-cell fate, irrespective of T-cell receptor affinity or antigen cross-presentation. Thus, when a low percentage of hepatocytes expressed cognate antigen, high-affinity T cells developed and maintained effector function, whereas, at a high percentage, they became functionally exhausted and silenced. Exhaustion was not irreversibly determined by initial activation, but was maintained by high intrahepatic antigen load during the early phase of the response; cytolytic function was restored when T cells primed under high antigen load conditions were transferred into an environment of low-level antigen expression. Our study reveals a hierarchy of factors dictating the fate of CD8 T cells during hepatic immune responses, and provides an explanation for the different immune outcomes observed in a variety of immune-mediated liver pathologic conditions.

Monocyte Homeostasis and the Plasticity of Inflammatory Monocytes

Cellular Immunology. Sep-Oct, 2014  |  Pubmed ID: 24962351

Monocytes are mononuclear myeloid cells that develop in the bone marrow and circulate within the bloodstream. Although they have long been argued to play a role in the repopulation of tissue-resident macrophages, this has been questioned by numerous recent studies, which has forced a reappraisal of their biology. Here we discuss monocyte development, as well as the homeostatic control of monocyte subpopulations within the blood. We also outline the known functions of monocyte subsets. Finally, we highlight the plastic nature of monocytes, which are capable of a remarkable range of phenotypic and functional changes that depend on signals from local microenvironments.

Real-time Imaging Reveals the Dynamics of Leukocyte Behaviour During Experimental Cerebral Malaria Pathogenesis

PLoS Pathogens. Jul, 2014  |  Pubmed ID: 25033406

During experimental cerebral malaria (ECM) mice develop a lethal neuropathological syndrome associated with microcirculatory dysfunction and intravascular leukocyte sequestration. The precise spatio-temporal context in which the intravascular immune response unfolds is incompletely understood. We developed a 2-photon intravital microscopy (2P-IVM)-based brain-imaging model to monitor the real-time behaviour of leukocytes directly within the brain vasculature during ECM. Ly6C(hi) monocytes, but not neutrophils, started to accumulate in the blood vessels of Plasmodium berghei ANKA (PbA)-infected MacGreen mice, in which myeloid cells express GFP, one to two days prior to the onset of the neurological signs (NS). A decrease in the rolling speed of monocytes, a measure of endothelial cell activation, was associated with progressive worsening of clinical symptoms. Adoptive transfer experiments with defined immune cell subsets in recombinase activating gene (RAG)-1-deficient mice showed that these changes were mediated by Plasmodium-specific CD8(+) T lymphocytes. A critical number of CD8(+) T effectors was required to induce disease and monocyte adherence to the vasculature. Depletion of monocytes at the onset of disease symptoms resulted in decreased lymphocyte accumulation, suggesting reciprocal effects of monocytes and T cells on their recruitment within the brain. Together, our studies define the real-time kinetics of leukocyte behaviour in the central nervous system during ECM, and reveal a significant role for Plasmodium-specific CD8(+) T lymphocytes in regulating vascular pathology in this disease.

CD326(lo)CD103(lo)CD11b(lo) Dermal Dendritic Cells Are Activated by Thymic Stromal Lymphopoietin During Contact Sensitization in Mice

Journal of Immunology (Baltimore, Md. : 1950). Sep, 2014  |  Pubmed ID: 25057004

The cytokine thymic stromal lymphopoietin (TSLP) is produced by epithelia exposed to the contact sensitizer dibutyl phthalate (DBP), and it is critical for the induction of Th2 immune responses by DBP-FITC. TSLP is thought to act on dendritic cells (DC), but the precise DC subsets involved in the response to TSLP remain to be fully characterized. In this study we show that a subset of CD326(lo)CD103(lo)CD11b(lo) dermal DC, which we termed "triple-negative (TN) DC," is highly responsive to TSLP. In DBP-FITC-treated mice, TN DC upregulated expression of CD86 and rapidly migrated to the draining lymph node to become the most abundant skin-derived DC subset at 24 and 48 h after sensitization. None of these responses was observed in TSLPR-deficient mice. In contrast, TN DC numbers were not increased after treatment with the allergen house dust mite or the bacteria Escherichia coli and bacillus Calmette-Guérin, which increased other DC subsets. In vivo, treatment with rTSLP preferentially increased the numbers of TN DC in lymph nodes. In vitro, TN DC responded to rTSLP treatment with a higher level of STAT5 phosphorylation compared with other skin-derived DC subsets. The TN DC subset shared the morphology, phenotype, and developmental requirements of conventional DC, depending on FLT3 expression for their optimal development from bone marrow precursors, and CCR7 for migration to the draining lymph node. Thus, TN DC represent a dermal DC subset that should be considered in future studies of TSLP-dependent contact sensitization and skin immune responses.

Intrahepatic Activation of Naive CD4+ T Cells by Liver-resident Phagocytic Cells

Journal of Immunology (Baltimore, Md. : 1950). Sep, 2014  |  Pubmed ID: 25070847

Naive T cell activation is normally restricted to the lymphoid organs, in part because of their limited ability to migrate into the parenchyma of peripheral tissues. The liver vasculature is unique, however, and circulating leukocytes within the hepatic sinusoids have direct access to liver-resident cells, which include an abundant population of Kupffer cells. It is well accepted that recognition of cognate Ag within the liver leads to naive CD8(+) T cell activation in situ, but it is unclear whether the liver also supports naive CD4(+) T cell activation. In this study, we show that naive CD4(+) T cells can be activated to proliferate in the liver when cognate Ag expression is induced in hepatocytes by recombinant adeno-associated viral vectors. Ag-specific retention and activation of naive CD4(+) T cells within the liver are independent of lymphoid tissues but dependent on a clodronate liposome-sensitive population of liver-resident phagocytic cells. To our knowledge, this study provides the first unequivocal evidence that naive CD4(+) T cells can be activated in a nonlymphoid organ. It also gives critical insight into how CD4(+) T cells specific for Ag expressed in the liver are recruited to participate in protective or pathological responses during hepatotropic infections and autoimmune liver disease.

High-resolution Episcopic Microscopy (HREM): a Tool for Visualizing Skin Biopsies

Microscopy and Microanalysis : the Official Journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada. Oct, 2014  |  Pubmed ID: 25198556

We evaluate the usefulness of digital volume data produced with the high-resolution episcopic microscopy (HREM) method for visualizing the three-dimensional (3D) arrangement of components of human skin, and present protocols designed for processing skin biopsies for HREM data generation. A total of 328 biopsies collected from normally appearing skin and from a melanocytic nevus were processed. Cuboidal data volumes with side lengths of ~2×3×6 mm3 and voxel sizes of 1.07×1.07×1.5 µm3 were produced. HREM data fit ideally for visualizing the epidermis at large, and for producing highly detailed volume and surface-rendered 3D representations of the dermal and hypodermal components at a structural level. The architecture of the collagen fiber bundles and the spatial distribution of nevus cells can be easily visualized with volume-rendering algorithms. We conclude that HREM has great potential to serve as a routine tool for researching and diagnosing skin pathologies.

Phenotyping Structural Abnormalities in Mouse Embryos Using High-resolution Episcopic Microscopy

Disease Models & Mechanisms. Oct, 2014  |  Pubmed ID: 25256713

The arrival of simple and reliable methods for 3D imaging of mouse embryos has opened the possibility of analysing normal and abnormal development in a far more systematic and comprehensive manner than has hitherto been possible. This will not only help to extend our understanding of normal tissue and organ development but, by applying the same approach to embryos from genetically modified mouse lines, such imaging studies could also transform our knowledge of gene function in embryogenesis and the aetiology of developmental disorders. The International Mouse Phenotyping Consortium is coordinating efforts to phenotype single gene knockouts covering the entire mouse genome, including characterising developmental defects for those knockout lines that prove to be embryonic lethal. Here, we present a pilot study of 34 such lines, utilising high-resolution episcopic microscopy (HREM) for comprehensive 2D and 3D imaging of homozygous null embryos and their wild-type littermates. We present a simple phenotyping protocol that has been developed to take advantage of the high-resolution images obtained by HREM and that can be used to score tissue and organ abnormalities in a reliable manner. Using this approach with embryos at embryonic day 14.5, we show the wide range of structural abnormalities that are likely to be detected in such studies and the variability in phenotypes between sibling homozygous null embryos.

Simultaneous Dermal Matrix and Autologous Split-thickness Skin Graft Transplantation in a Porcine Wound Model: a Three-dimensional Histological Analysis of Revascularization

Wound Repair and Regeneration : Official Publication of the Wound Healing Society [and] the European Tissue Repair Society. Nov-Dec, 2014  |  Pubmed ID: 25358670

Despite the popularity of a simultaneous application of dermal matrices and split-thickness skin grafts, scarce evidence exists about the process of revascularization involved. In this study, we aimed at analyzing the progression of revascularization by high-resolution episcopic microscopy (HREM) in a porcine excisional wound model. Following the surgical procedure creating 5 × 5 cm(2) full-thickness defects on the back, one area was covered with an autologous split-thickness skin graft alone (control group), the other with a collagen-elastin dermal matrix plus split-thickness skin graft (dermal matrix group). Two skin biopsies per each group and location were performed on day 5, 10, 15, and 28 postoperatively and separately processed for H&E as well as HREM. The dermal layer was thicker in the dermal matrix group vs. control on day 5 and 28. No differences were found for revascularization by conventional histology. In HREM, the dermal matrix did not appear to decelerate the revascularization process. The presence of the dermal matrix could be distinguished until day 15. By day 28, the structure of the dermal matrix could no longer be delineated and was replaced by autologous tissue. As assessed by conventional histology and confirmed by HREM, the revascularization process was comparable in both groups, notably with regard to the vertical ingrowth of sprouting vessels. The presented technique of HREM is a valuable addition for analyzing small vessel sprouting in dermal matrices in the future.

Dual Modality Optical Coherence and Whole-body Photoacoustic Tomography Imaging of Chick Embryos in Multiple Development Stages

Biomedical Optics Express. Sep, 2014  |  Pubmed ID: 25401028

Chick embryos are an important animal model for biomedical studies. The visualization of chick embryos, however, is limited mostly to postmortem sectional imaging methods. In this work, we present a dual modality optical imaging system that combines swept-source optical coherence tomography and whole-body photoacoustic tomography, and apply it to image chick embryos at three different development stages. The explanted chick embryos were imaged in toto with complementary contrast from both optical scattering and optical absorption. The results serve as a prelude to the use of the dual modality system in longitudinal whole-body monitoring of chick embryos in ovo.

Dermal Group 2 Innate Lymphoid Cells in Atopic Dermatitis and Allergy

Current Opinion in Immunology. Dec, 2014  |  Pubmed ID: 25459002

Type 2 immune responses in the skin cause a variety of pathologies, including urticaria and atopic dermatitis. Traditionally, CD4(+) helper T cells have been implicated in the pathogenesis of these conditions. However, recently a new player, the group 2 innate lymphoid (ILC2) cell, has emerged as an important contributor to skin allergies. In this review, we summarize our current knowledge of the role ILC2 cells play in the physiology and pathology of mouse and human skin.

Granzyme B Promotes Cytotoxic Lymphocyte Transmigration Via Basement Membrane Remodeling

Immunity. Dec, 2014  |  Pubmed ID: 25526309

Granzyme B (GzmB) is a protease with a well-characterized intracellular role in targeted destruction of compromised cells by cytotoxic lymphocytes. However, GzmB also cleaves extracellular matrix components, suggesting that it influences the interplay between cytotoxic lymphocytes and their environment. Here, we show that GzmB-null effector T cells and natural killer (NK) cells exhibited a cell-autonomous homing deficit in mouse models of inflammation and Ectromelia virus infection. Intravital imaging of effector T cells in inflamed cremaster muscle venules revealed that GzmB-null cells adhered normally to the vessel wall and could extend lamellipodia through it but did not cross it efficiently. In vitro migration assays showed that active GzmB was released from migrating cytotoxic lymphocytes and enabled chemokine-driven movement through basement membranes. Finally, proteomic analysis demonstrated that GzmB cleaved basement membrane constituents. Our results highlight an important role for GzmB in expediting cytotoxic lymphocyte diapedesis via basement membrane remodeling.

The Skin Immune Atlas: Three-dimensional Analysis of Cutaneous Leukocyte Subsets by Multiphoton Microscopy

The Journal of Investigative Dermatology. Jan, 2015  |  Pubmed ID: 25007044

Site-specific differences in skin response to pathogens and in the course of cutaneous inflammatory diseases are well appreciated. The composition and localization of cutaneous leukocytes has been studied extensively using histology and flow cytometry. However, the precise three-dimensional (3D) distribution of distinct immune cell subsets within skin at different body sites requires visualization of intact living skin. We used intravital multiphoton microscopy in transgenic reporter mice in combination with quantitative flow cytometry to generate a 3D immune cell atlas of mouse skin. The 3D location of innate and adaptive immune cells and site-specific differences in the densities of macrophages, T cells, and mast cells at four defined sites (ear, back, footpad, and tail) is presented. The combinatorial approach further demonstrates an as yet unreported age-dependent expansion of dermal gamma-delta T cells. Localization of dermal immune cells relative to anatomical structures was also determined. Although dendritic cells were dispersed homogeneously within the dermis, mast cells preferentially localized to the perivascular space. Finally, we show the functional relevance of site-specific mast cell disparities using the passive cutaneous anaphylaxis model. These approaches are applicable to assessing immune cell variations and potential functional consequences in the setting of infection, as well as the pathogenesis of inflammatory skin conditions.

Ultrasound and Anatomical Correlation of the Radial Nerve at the Arcade of Frohse

Muscle & Nerve. Jun, 2015  |  Pubmed ID: 25297493

In this anatomical study we evaluated the feasibility of ultrasound (US) guided perineural injection of the deep branch of the radial nerve (DBRN) at the arcade of Frohse as potential therapy for nerve entrapment at this site.

Real-time Imaging of Dendritic Cell Responses to Sterile Tissue Injury

The Journal of Investigative Dermatology. Apr, 2015  |  Pubmed ID: 25431854

High-resolution Episcopic Microscopy (HREM): a Useful Technique for Research in Wound Care

Annals of Anatomy = Anatomischer Anzeiger : Official Organ of the Anatomische Gesellschaft. Jan, 2015  |  Pubmed ID: 25466930

Analysing the three-dimensional (3D) texture of skin substitute materials and evaluating their performance after covering skin defects is essential for improving their design and for optimising surgical procedures and post implantation wound treatment regimes. Here we explore the capacities of the recently developed High-resolution episcopic microscopy (HREM) method for generating digital volume data that permit structural 3D analysis of native and implanted collagen-elastin matrices. We employed HREM to visualise native collagen matrices and collagen matrices seeded with keratinocytes. In a second step, we visualised the appearance and the revascularisation of the matrices after their implantation beneath split skin grafts used for covering skin defects in the porcine model. For this, HREM data were generated from biopsies harvested 5, 10, and 15 days after surgery. In all instances, the high quality and resolution of the HREM data in combination with the relative large field of view proved to be sufficient for visualizing the exact fibre architecture by employing quick volume rendering algorithms. Precise analysis of the 3D distribution of keratinocytes in the matrices populated with keratinocytes and of the detailed topology of the sprouting blood vessels in the implanted matrices was feasible. Our results show that high-resolution episcopic microscopy can be adapted to serve as a tool for evaluating collagen-elastin materials ex- and in vivo.

Imaging the Zebrafish Dentition: from Traditional Approaches to Emerging Technologies

Zebrafish. Feb, 2015  |  Pubmed ID: 25560992

The zebrafish, a model organism for which a plethora of molecular and genetic techniques exists, has a lifelong replacing dentition of 22 pharyngeal teeth. This is in contrast to the mouse, which is the key organism in dental research but whose teeth are never replaced. Employing the zebrafish as the main organism to elucidate the mechanisms of continuous tooth replacement, however, poses at least one major problem, related to the fact that all teeth are located deep inside the body. Investigating tooth replacement thus relies on conventional histological methods, which are often laborious, time-consuming and can cause tissue deformations. In this review, we investigate the advantages and limitations of adapting current visualization techniques to dental research in zebrafish. We discuss techniques for fast sectioning, such as vibratome sectioning and high-resolution episcopic microscopy, and methods for in toto visualization, such as Alizarin red staining, micro-computed tomography, and optical projection tomography. Techniques for in vivo imaging, such as two-photon excitation fluorescence and second harmonic generation microscopy, are also covered. Finally, the possibilities of light sheet microscopy are addressed.

Group 2 Innate Lymphoid Cells in the Regulation of Immune Responses

Advances in Immunology. 2015  |  Pubmed ID: 25591466

Type 2 cytokine-driven immune responses are important against parasite infections but also underlie the development of inflammatory allergic diseases. Type 2 CD4(+) T (Th2) cells have long been believed to act as central regulators of allergic conditions via the production of the signature cytokines IL-4, IL-5, and IL-13. However, the more recent identification of group 2 innate lymphoid cells ILC (ILC2) cells, which also produce the same cytokines, necessitates a reevaluation of the relative roles these two populations play during type 2 inflammation. ILC2 cells preferentially localize to the interface between the host and the environment (lung, intestine, skin) and respond to epithelium-derived cytokines associated with barrier disruption, such as IL-25, IL-33, and thymic stromal lymphopoietin. ILC2 cells are a major source of IL-5 and IL-13 in vivo but may also produce IL-4 and IL-9 under more defined conditions. ILC2 cells regulate local inflammatory responses to environmental challenges, and this in turn enables them to influence downstream adaptive immune responses. Here, we discuss our current understanding of ILC2 cell phenotype, development and function, and detail the expanding array of cell surface receptor and signaling pathways that enable ILC2 cells to perform a variety of biological functions in vivo. We give special attention to the most recently described and poorly understood member of the ILC2 cell family, the dermal ILC2 cells, and discuss their role in regulating skin inflammation.

IRGM3 Contributes to Immunopathology and is Required for Differentiation of Antigen-specific Effector CD8+ T Cells in Experimental Cerebral Malaria

Infection and Immunity. Apr, 2015  |  Pubmed ID: 25644000

Gamma interferon (IFN-γ) drives antiparasite responses and immunopathology during infection with Plasmodium species. Immunity-related GTPases (IRGs) are a class of IFN-γ-dependent proteins that are essential for cell autonomous immunity to numerous intracellular pathogens. However, it is currently unknown whether IRGs modulate responses during malaria. We have used the Plasmodium berghei ANKA (PbA) model in which mice develop experimental cerebral malaria (ECM) to study the roles of IRGM1 and IRGM3 in immunopathology. Induction of mRNA for Irgm1 and Irgm3 was found in the brains and spleens of infected mice at times of peak IFN-γ production. Irgm3-/- but not Irgm1-/- mice were completely protected from the development of ECM, and this protection was associated with the decreased induction of inflammatory cytokines, as well as decreased recruitment and activation of CD8+ T cells within the brain. Although antigen-specific proliferation of transferred CD8+ T cells was not diminished compared to that of wild-type recipients following PbA infection, T cells transferred into Irgm3-/- recipients showed a striking impairment of effector differentiation. Decreased induction of several inflammatory cytokines and chemokines (interleukin-6, CCL2, CCL3, and CCL4), as well as enhanced mRNA expression of type-I IFNs, was found in the spleens of Irgm3-/- mice at day 4 postinfection. Together, these data suggest that protection from ECM pathology in Irgm3-/- mice occurs due to impaired generation of CD8+ effector function. This defect is nonintrinsic to CD8+ T cells. Instead, diminished T cell responses most likely result from defective initiation of inflammatory responses in myeloid cells.

Real-time Tracking of Cell Cycle Progression During CD8+ Effector and Memory T-cell Differentiation

Nature Communications. 2015  |  Pubmed ID: 25709008

The precise pathways of memory T-cell differentiation are incompletely understood. Here we exploit transgenic mice expressing fluorescent cell cycle indicators to longitudinally track the division dynamics of individual CD8(+) T cells. During influenza virus infection in vivo, naive T cells enter a CD62L(intermediate) state of fast proliferation, which continues for at least nine generations. At the peak of the anti-viral immune response, a subpopulation of these cells markedly reduces their cycling speed and acquires a CD62L(hi) central memory cell phenotype. Construction of T-cell family division trees in vitro reveals two patterns of proliferation dynamics. While cells initially divide rapidly with moderate stochastic variations of cycling times after each generation, a slow-cycling subpopulation displaying a CD62L(hi) memory phenotype appears after eight divisions. Phenotype and cell cycle duration are inherited by the progeny of slow cyclers. We propose that memory precursors cell-intrinsically modulate their proliferative activity to diversify differentiation pathways.

The Role of Chemokines in Cutaneous Immunosurveillance

Immunology and Cell Biology. Apr, 2015  |  Pubmed ID: 25776847

The skin serves as a critical barrier against pathogen entry. This protection is afforded by an array of skin-resident immune cells, which act as first-line responders against barrier breach and infection. The recruitment and positioning of these cells is controlled at multiple levels by endothelial cells, pericytes, perivascular macrophages and mast cells, and by the fibroblasts in the dermis and keratinocytes in the epidermis. Chemokine signalling through chemokine receptors expressed by the various leukocyte subsets is critical for their trafficking into and within the skin. The role of chemokines in the skin is complex, and remains incompletely understood despite three decades of investigation. Here, we review the roles that different chemokine pathways play in the skin, and highlight the recent developments in the field.

ILC2s and T Cells Cooperate to Ensure Maintenance of M2 Macrophages for Lung Immunity Against Hookworms

Nature Communications. 2015  |  Pubmed ID: 25912172

Defining the immune mechanisms underlying protective immunity to helminth infection remains an important challenge. Here we report that lung CD4(+) T cells and Group 2 innate lymphoid cells (ILC2s) work in concert to block Nippostrongylus brasiliensis (Nb) development in the parenchyma within 48 h in mice. Immune-damaged larvae have a striking morphological defect that is dependent on the expansion of IL-13-producing ILC2 and CD4(+) T cells, and the activation of M2 macrophages. This T-cell requirement can be bypassed by administration of IL-2 or IL-33, resulting in expansion of IL-13-producing ILC2s and larval killing. Depletion of ILC2s inhibits larval killing in IL-2-treated mice. Our results broaden understanding of ILC2's role in immunity to helminths by demonstrating that they not only act as alarmin sensors, but can also be sustained by CD4(+) T cells, ensuring both the prompt activation and the maintenance of IL-13-dependent M2 macrophage immunity in the lung.

Ultrasound-Guided Perineural Injection at Guyon's Tunnel: An Anatomic Feasibility Study

Ultrasound in Medicine & Biology. Aug, 2015  |  Pubmed ID: 25937521

Compression of the ulnar nerve (UN) at the wrist causes neuropathy in the ulnar tunnel (UT), or Guyon's tunnel. In the absence of trauma and motor syndromes, primarily conservative treatment is considered. As in carpal tunnel syndrome, a perineural injection of corticosteroids may be beneficial. The aim of this study was to investigate the feasibility of ultrasound-guided injections at the UT. We performed ultrasound-guided injections of ink at the UN within the UT in 21 limbs from 11 non-embalmed cadavers. In all cases, we stained the perineural sheath of the superficial branch of the ulnar nerve within the UT. No ink was found inside the nerve or in adjacent structures such as blood vessels and tendons. In conclusion, perineural injection of the UN in the UT seems to be a technically feasible procedure. On the basis of these anatomic data, clinical trials are needed to prove the concept for routine use.

IL-2 is a Critical Regulator of Group 2 Innate Lymphoid Cell Function During Pulmonary Inflammation

The Journal of Allergy and Clinical Immunology. Dec, 2015  |  Pubmed ID: 26025126

Group 2 innate lymphoid cells (ILC2) have been implicated in the pathogenesis of allergic lung diseases. However, the upstream signals that regulate ILC2 function during pulmonary inflammation remain poorly understood. ILC2s have been shown to respond to exogenous IL-2, but the importance of endogenous IL-2 in ILC2 function in vivo remains unclear.

Shedding Light on Cell Cycle Control by T and B Lymphocytes

Cell Cycle (Georgetown, Tex.). Aug, 2015  |  Pubmed ID: 26133027

The Dynamic Anatomy and Patterning of Skin

Experimental Dermatology. Aug, 2015  |  Pubmed ID: 26284579

The skin is often viewed as a static barrier that protects the body from the outside world. Emphasis on studying the skin's architecture and biomechanics in the context of restoring skin movement and function is often ignored. It is fundamentally important that if skin is to be modelled or developed, we do not only focus on the biology of skin but also aim to understand its mechanical properties and structure in living dynamic tissue. In this review, we describe the architecture of skin and patterning seen in skin as viewed from a surgical perspective and highlight aspects of the microanatomy that have never fully been realized and provide evidence or concepts that support the importance of studying living skin's dynamic behaviour. We highlight how the structure of the skin has evolved to allow the body dynamic form and function, and how injury, disease or ageing results in a dramatic changes to the microarchitecture and changes physical characteristics of skin. Therefore, appreciating the dynamic microanatomy of skin from the deep fascia through to the skin surface is vitally important from a dermatological and surgical perspective. This focus provides an alternative perspective and approach to addressing skin pathologies and skin ageing.

Longitudinal Gliding of the Median Nerve in the Carpal Tunnel: Ultrasound Cadaveric Evaluation of Conventional and Novel Concepts of Nerve Mobilization

Archives of Physical Medicine and Rehabilitation. Dec, 2015  |  Pubmed ID: 26315068

To evaluate median nerve excursion during conventional nerve gliding exercises and newly developed exercises, primarily comprising abduction and adduction of the fingers.

Apolipoprotein A-I Limits the Negative Effect of Tumor Necrosis Factor on Lymphangiogenesis

Arteriosclerosis, Thrombosis, and Vascular Biology. Nov, 2015  |  Pubmed ID: 26359513

Lymphatic endothelial dysfunction underlies the pathogenesis of many chronic inflammatory disorders. The proinflammatory cytokine tumor necrosis factor (TNF) is known for its role in disrupting the function of the lymphatic vasculature. This study investigates the ability of apolipoprotein (apo) A-I, the principal apolipoprotein of high-density lipoproteins, to preserve the normal function of lymphatic endothelial cells treated with TNF.

Neutrophils Self-Regulate Immune Complex-Mediated Cutaneous Inflammation Through CXCL2

The Journal of Investigative Dermatology. Oct, 2015  |  Pubmed ID: 26480280

Deposition of immune complexes (ICs) in tissues triggers acute inflammatory pathology characterized by massive neutrophil influx leading to edema and hemorrhage, and is especially associated with vasculitis of the skin, but the mechanisms that regulate this Type III hypersensitivity process remain poorly understood. Here, using a combination of multiphoton intravital microscopy and genomic approaches, we re-examined the cutaneous reverse passive Arthus (RPA) reaction and observed that IC-activated neutrophils underwent transmigration, triggered further IC formation and transported these ICs into the interstitium, whereas neutrophil depletion drastically reduced IC formation and ameliorated vascular leakage in vivo. Thereafter, we show that these neutrophils expressed high levels of CXCL2, which further amplified neutrophil recruitment and activation in an autocrine/paracrine manner. Notably, CXCL1 expression was restricted to tissue-resident cell types, but IC-activated neutrophils may also indirectly, via soluble factors, modulate macrophage CXCL1 expression. Consistent with their distinct cellular origins and localization, only neutralization of CXCL2 but not CXCL1 in the interstitium effectively reduced neutrophil recruitment. In summary, our study establishes that neutrophils are able to self-regulate their own recruitment and responses during IC-mediated inflammation through a CXCL2-driven feed forward loop.Journal of Investigative Dermatology accepted article preview online, 19 October 2015. doi:10.1038/jid.2015.410.

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