Label‐free spectroscopic tissue characterization using fluorescence excitation‐scanning spectral imaging

Spectral imaging approaches provide new possibilities for measuring and discriminating fluorescent molecules in living cells and tissues. These approaches often employ tunable filters and robust image processing algorithms to identify many fluorescent labels in a single image set. Here, we present results from a novel spectral imaging technology that scans the fluorescence excitation spectrum, demonstrating that excitation‐scanning hyperspectral image data can discriminate among tissue types and estimate the molecular composition of tissues. This approach allows fast, accurate quantification of many fluorescent species from multivariate image data without the need of exogenous labels or dyes. We evaluated the ability of the excitation‐scanning approach to identify endogenous fluorescence signatures in multiple unlabeled tissue types. Signatures were screened using multi‐pass principal component analysis. Endmember extraction techniques revealed conserved autofluorescent signatures across multiple tissue types. We further examined the ability to detect known molecular signatures by constructing spectral libraries of common endogenous fluorophores and applying multiple spectral analysis techniques on test images from lung, liver and kidney. Spectral deconvolution revealed structure‐specific morphologic contrast generated from pure molecule signatures. These results demonstrate that excitation‐scanning spectral imaging, coupled with spectral imaging processing techniques, provides an approach for discriminating among tissue types and assessing the molecular composition of tissues. Additionally, excitation scanning offers the ability to rapidly screen molecular markers across a range of tissues without using fluorescent labels. This approach lays the groundwork for translation of excitation‐scanning technologies to clinical imaging platforms.     

Development of multigene expression signature maps at the protein level from digitized immunohistochemistry slides

Molecular classification of diseases based on multigene expression signatures is increasingly used for diagnosis, prognosis, and prediction of response to therapy. Immunohistochemistry (IHC) is an optimal method for validating expression signatures obtained using high-throughput genomics techniques since IHC allows a pathologist to examine gene expression at the protein level within the context of histologically interpretable tissue sections. Additionally, validated IHC assays may be readily implemented as clinical tests since IHC is performed on routinely processed clinical tissue samples. However, methods have not been available for automated n-gene expression profiling at the protein level using IHC data. We have developed methods to compute expression level maps (signature maps) of multiple genes from IHC data digitized on a commercial whole slide imaging system. Areas of cancer for these expression level maps are defined by a pathologist on adjacent, co-registered H&E slides, allowing assessment of IHC statistics and heterogeneity within the diseased tissue. This novel way of representing multiple IHC assays as signature maps will allow the development of n-gene expression profiling databases in three dimensions throughout virtual whole organ reconstructions.     

Mechanisms underlying the induction of regulatory T cells and its relevance in the adaptive immune response in parasitic infections

To fulfill its function, the immune system must detect and interpret a wide variety of signals and adjust the magnitude, duration, and specific traits of each response during the complex host-parasite relationships in parasitic infections. Inflammation must be tightly regulated since uncontrolled inflammation may be as destructive as the triggering stimulus and leads to immune-mediated tissue injury. During recent years, increasing evidence points to regulatory T cells (Tregs) as key anti-inflammatory cells, critically involved in limiting the inflammatory response. Herein, we review the published information on the induction of Tregs and summarize the most recent findings on Treg generation in parasitic diseases.     

An overview of inflammation: mechanism and consequences

Inflammation is an essential response provided by the immune systems that ensures the survival during infection and tissue injury. Inflammatory responses are essential for the maintenance of normal tissue homeostasis. The molecular mechanism of inflammation is quite a complicated process which is initiated by the recognition of specific molecular patterns associated with either infection or tissue injury. The entire process of the inflammatory response is mediated by several key regulators involved in the selective expression of proinflammatory molecules. Prolonged inflammations are often associated with severe detrimental side effects on health. Alterations in inflammatory responses due to persistent inducers or genetic variations are on the rise over the last couple of decades, causing a variety of inflammatory diseases and pathophysiological conditions.     

Circulating microRNAs in the identification of biological fluids: A new approach to standardization of expression-based diagnostics

Molecular profiling of normal tissues is a regular and necessary step when developing systems for expression analyses in biological samples, including diagnostic panels for various diseases and conditions. Yet there are still no rigorous criteria to allow precise typing of normal tissues. A main problem is that the methods employed in diagnostic expression testing are difficult to standardize. While various technologies, instruments, and reagents are available, universal protocols of handling biological material are lacking, thus impairing the reproducibility of data from independent studies. The review describes a new approach to standardizing circulating microRNA studies in forensic biology, which has relatively recently (7–8 years ago) come to employ RNA markers in molecular typing of tissues and biological fluids. Forensic biology is now one of the few disciplines where several panels of tissue mRNA markers have been developed within a short period of time and a number of specific microRNA markers have been established and validated for several biological fluids. To allow their successful use, new protocols have been combined with the available, rigidly standardized system of genetic personal identification. Although a ready diagnostic product has still not been obtained with this well-working approach, the apparent efficiency of the standardization methods clearly demonstrates that the problem is possible to solve in other biomedical fields, including those where RNA-based diagnostic protocols are still under development.     

Evaluation of effector cell fate and function by in vivo bioluminescence imaging

The effector functions of immune cells have typically been examined using assays that require sampling of tissues or cells to reveal specific aspects of an immune response (e.g., antigen-specificity, cytokine expression or killing of target cells). The outcome of an immune response in vivo, however, is not solely determined by a single effector function of a specific cell population, but is the result of numerous cellular and molecular interactions that occur in the complex environment of intact organ systems. These interactions influence survival, migration, and activation, as well as final effector function of a given population of cells. Efforts to reveal the cellular and molecular basis of biological processes have resulted in a number of technologies that combine molecular biology and imaging sciences that are collectively termed as Molecular Imaging. This emerging field has developed to reveal functional aspects of cells, genes, and proteins in real time in living animals and humans and embraces multiple modalities, including established clinical imaging methods such as magnetic resonance imaging, single photon emission computed tomography, and positron emission tomography, as well as novel methodologies specifically designed for research animals. Here, we highlight one of the newer modalities, in vivo bioluminescence imaging, as a method for evaluating effector T cell proliferation, migration, and function in model systems of malignant and non-malignant diseases.     

NF-κB in the regulation of epithelial homeostasis and inflammation

The IκB kinase/NF-κB signaling pathway has been implicated in the pathogenesis of several inflammatory diseases. Increased activation of NF-κB is often detected in both immune and non-immune cells in tissues affected by chronic inflammation, where it is believed to exert detrimental functions by inducing the expression of proinflammatory mediators that orchestrate and sustain the inflammatory response and cause tissue damage. Thus, increased NF-κB activation is considered an important pathogenic factor in many acute and chronic inflammatory disorders, raising hopes that NF-κB inhibitors could be effective for the treatment of inflammatory diseases. However, ample evidence has accumulated that NF-κB inhibition can also be harmful for the organism, and in some cases trigger the development of inflammation and disease. These findings suggested that NF-κB signaling has important functions for the maintenance of physiological immune homeostasis and for the prevention of inflammatory diseases in many tissues. This beneficial function of NF-κB has been predominantly observed in epithelial cells, indicating that NF-κB signaling has a particularly important role for the maintenance of immune homeostasis in epithelial tissues. It seems therefore that NF-κB displays two faces in chronic inflammation: on the one hand increased and sustained NF-κB activation induces inflammation and tissue damage, but on the other hand inhibition of NF-κB signaling can also disturb immune homeostasis, triggering inflammation and disease. Here, we discuss the mechanisms that control these apparently opposing functions of NF-κB signaling, focusing particularly on the role of NF-κB in the regulation of immune homeostasis and inflammation in the intestine and the skin.     

An overview of inflammation: mechanism and consequences

Inflammation is an essential response provided by the immune systems that ensures the survival during infection and tissue injury.Inflammatory responses are essential for the maintenance of normal tissue homeostasis.The molecular mechanism of inflammation is quite a complicated process which is initiated by the recognition of specific molecular patterns associated with either infection or tissue injury.The entire process of the inflammatory response is mediated by several key regulators involved in the selective expression of proinflammatory molecules.Prolonged inflammations are often associated with severe detrimental side effects on health.Alterations in inflammatory responses due to persistent inducers or genetic variations are on the rise over the last couple of decades,causing a variety of inflammatory diseases and pathophysiological conditions.     

Resolvins: natural agonists for resolution of pulmonary inflammation

Inappropriate or excessive pulmonary inflammation can contribute to chronic lung diseases. In health, the resolution of inflammation is an active process that terminates inflammatory responses. The recent identification of endogenous lipid-derived mediators of resolution has provided a window to explore the pathobiology of inflammatory disease and structural templates for the design of novel pro-resolving therapeutics. Resolvins (resolution-phase interaction products) are a family of pro-resolving mediators that are enzymatically generated from essential omega-3 polyunsaturated fatty acids. Two molecular series of resolvins have been characterised, namely E- and D-series resolvins which possess distinct structural, biochemical and pharmacological properties. Acting as agonists at specific receptors (CMKLR1, BLT1, ALX/FPR2 and GPR32), resolvins can signal for potent counter-regulatory effects on leukocyte functions, including preventing uncontrolled neutrophil swarming, decreasing the generation of cytokines, chemokines and reactive oxygen species and promoting clearance of apoptotic neutrophils from inflamed tissues. Hence, resolvins provide mechanisms for cytoprotection of host tissues to the potentially detrimental effects of unresolved inflammation. This review highlights recent experimental findings in resolvin research, and the impact of these stereospecific molecules on the resolution of pulmonary inflammation and tissue catabasis.     

Imaging of cell trafficking in Crohn's disease

Inflammatory bowel diseases are represented by ulcerative colitis and Crohn's disease, both consisting of a chronic, uncontrolled inflammation of the intestinal mucosa of any part of the gastrointestinal tract with patchy or continuous inflammation. Ileo‐colonoscopy is considered the current gold standard imaging technique for the diagnosis. However, as the majority of patients need a long‐term follow‐up it would be ideal to rely on a non‐invasive technique with good compliance. This review focuses on nuclear medicine imaging techniques in Crohn's disease. Different scintigraphic methods of imaging cells involved in the pathogenesis are described. The radiopharmaceuticals can be divided into non‐specific radiopharmaceuticals for inflammation and specific radiopharmaceuticals that directly image lymphocytes involved in the process. This non‐invasive molecular imaging approach can be useful also because it images the small bowel or other areas––where colonoscopy is not useful—and that it may play a role for constant follow‐up, because relapses are frequent. Finally, an update on other imaging modalities, and particularly MRI, in the evaluation of Crohn's disease activity, is provided. Although MRI cannot directly detect inflammatory cells, it has shown a high sensitivity in detecting the macroscopic signs of inflammation at the level of the intestinal wall affected by Crohn's disease and Ulcerative colitis. The current diagnostic value of MRI in the detection of inflamed bowel segment and in the assessment of CD activity, as well the potentials MR spectroscopy, MR diffusion imaging and MR molecular imaging, is briefly discussed. J. Cell. Physiol. 223:562–571, 2010. © 2010 Wiley‐Liss, Inc.     

Immunobiology of mesenchymal stem cells

Mesenchymal stem cells (MSCs) can be isolated from almost all tissues and effectively expanded in vitro. Although their true in situ properties and biological functions remain to be elucidated, these in vitro expanded cells have been shown to possess potential to differentiate into specific cell lineages. It is speculated that MSCs in situ have important roles in tissue cellular homeostasis by replacing dead or dysfunctional cells. Recent studies have demonstrated that in vitro expanded MSCs of various origins have great capacity to modulate immune responses and change the progression of different inflammatory diseases. As tissue injuries are often accompanied by inflammation, inflammatory factors may provide cues to mobilize MSCs to tissue sites with damage. Before carrying out tissue repair functions, MSCs first prepare the microenvironment by modulating inflammatory processes and releasing various growth factors in response to the inflammation status. In this review, we focus on the crosstalk between MSCs and immune responses and their potential clinical applications, especially in inflammatory diseases.     

Communications via the Small Leucine-rich Proteoglycans: Molecular Specificity in Inflammation and Autoimmune Diseases

Inflammation is a highly regulated biological response of the immune system that is triggered by assaulting pathogens or endogenous alarmins. It is now well established that some soluble extracellular matrix constituents, such as small leucine-rich proteoglycans (SLRPs), can act as danger signals and trigger aseptic inflammation by interacting with innate immune receptors. SLRP inflammatory signaling cascade goes far beyond its canonical function. By choosing specific innate immune receptors, coreceptors, and adaptor molecules, SLRPs promote a switch between pro- and anti-inflammatory signaling, thereby determining disease resolution or chronification. Moreover, by orchestrating signaling through various receptors, SLRPs fine-tune inflammation and, despite their structural homology, regulate inflammatory processes in a molecule-specific manner. Hence, the overarching theme of this review is to highlight the molecular and functional specificity of biglycan-, decorin-, lumican-, and fibromodulin-mediated signaling in inflammatory and autoimmune diseases     

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.     

Intestinal inflammation responds to microbial tissue load independent of pathogen/non-pathogen discrimination

The intestinal immune system mounts inflammatory responses to pathogens but tolerates harmless commensal microbiota. Various mechanisms for pathogen/non-pathogen discrimination have been proposed but their general relevance for inflammation control is unclear. Here, we compared intestinal responses to pathogenic Salmonella and non-pathogenic E. coli. Both microbes entered intestinal Peyer's patches and, surprisingly, induced qualitatively and quantitatively similar initial inflammatory responses revealing a striking discrimination failure. Diverging inflammatory responses only occurred when Salmonella subsequently proliferated and induced escalating neutrophil infiltration, while harmless E. coli was rapidly cleared from the tissue and inflammation resolved. Transient intestinal inflammation induced by harmless E. coli tolerized against subsequent exposure thereby preventing chronic inflammation during repeated exposure. These data revealed a striking failure of the intestinal immune system to discriminate pathogens from harmless microbes based on distinct molecular signatures. Instead, appropriate intestinal responses to gut microbiota might be ensured by immediate inflammatory responses to any rise in microbial tissue loads, and desensitization after bacterial clearance.     

Imaging molecular expression on vascular endothelial cells by in vivo immunofluorescence microscopy

Molecular expression on the vascular endothelium is critical in regulating the interaction of circulating cells with the blood vessel wall. Leukocytes as well as circulating cancer cells gain entry into tissue by interacting with adhesion molecules on the endothelial cells (EC). Molecular targets on the EC are increasingly being explored for tissue-specific delivery of therapeutic and imaging agents. Here we use in vivo immunofluorescence microscopy to visualize the endothelial molecular expression in the vasculature of live animals. High-resolution images are obtained by optical sectioning through the intact skin using in vivo confocal and multiphoton microscopy after in situ labeling of EC surface markers with fluorescent antibodies. Other vascular beds such as the bone marrow and ocular blood vessels can be imaged with little or no tissue manipulation. Live imaging is particularly useful for following the dynamic expression of inducible molecules such as E-selectin during an inflammatory response.     

Systems analysis of eleven rodent disease models reveals an inflammatome signature and key drivers

Common inflammatome gene signatures as well as disease‐specific signatures were identified by analyzing 12 expression profiling data sets derived from 9 different tissues isolated from 11 rodent inflammatory disease models. The inflammatome signature significantly overlaps with known drug targets and co‐expressed gene modules linked to metabolic disorders and cancer. A large proportion of genes in this signature are tightly connected in tissue‐specific Bayesian networks (BNs) built from multiple independent mouse and human cohorts. Both the inflammatome signature and the corresponding consensus BNs are highly enriched for immune response‐related genes supported as causal for adiposity, adipokine, diabetes, aortic lesion, bone, muscle, and cholesterol traits, suggesting the causal nature of the inflammatome for a variety of diseases. Integration of this inflammatome signature with the BNs uncovered 151 key drivers that appeared to be more biologically important than the non‐drivers in terms of their impact on disease phenotypes. The identification of this inflammatome signature, its network architecture, and key drivers not only highlights the shared etiology but also pinpoints potential targets for intervention of various common diseases.     

Dendritic cells,chemokine receptors and autoimmune inflammatory diseases

Dendritic cells (DC) have been implicated in the induction of autoimmune diseases and have been identified in lesions associated with several autoimmune inflammatory diseases. Since DC are regarded as the professional antigen-presenting cell (APC) of the immune system and the only APC capable of activating na?ve T cells, they are likely to play a significant role in breaking tolerance of self-reactive lymphocytes and in supporting autoimmune responses in these diseases. A number of studies have revealed that small molecular weight chemotactic proteins known as chemokines are present within the autoimmune lesions and may contribute to the recruitment not only of DC populations, but also of immune cells such as T cells, B cells, neutrophils and monocytes into the site, and to the formation of organized lymphoid tissue structures within the target organ. The focus of this review will be a discussion of the role of chemokines in the recruitment of DC in human autoimmune inflammatory disorders, specifically the trafficking of DC into the inflammatory sites and the subsequent migration of differentiated DC from the inflammatory sites into the draining lymph nodes. Once DC are properly positioned within the lymph nodes, circulating antigen specific na?ve T cells can interact with DC and become activated, clonally expanded and stimulated to undergo differentiation into antigen-experienced memory T cells. Subsequent reactivation of memory T cells that enter the autoimmune lesions by DC present in the inflammatory lesion is thought to play a central role in tissue inflammation.     

The molecular biology of inflammatory bowel diseases

IBDs (inflammatory bowel diseases) are a group of diseases affecting the gastrointestinal tract. The diseases are multifactorial and cover genetic aspects: susceptibility genes, innate and adaptive responses to inflammation, and structure and efficacy of the mucosal protective barrier. Animal models of IBD have been developed to gain further knowledge of the disease mechanisms. These topics form an overlapping background to enable an improved understanding of the molecular features of these diseases. A series of articles is presented based on the topics covered at the Biochemical Society Focused Meeting The Molecular Biology of Inflammatory Bowel Diseases.