Assessment of 2-Year Storage Conditions on Protein, RNA, and DNA in Unstained Human Tissue Sections, Including a Novel Multiplex Digital Gene Expression Profiling Method with Implications for Biobanking.

Background: Formalin-fixed, paraffin-embedded (FFPE) tissues are a valuable resource for clinical and basic science research. Paraffin blocks and the resulting unstained sections (USS) are often stored for years before being used. Previous studies have evaluated the effects of time, temperature, humidity, and inert gases on preservation of USS; however, no study has examined all four variables together. Methods: In the current work, we prospectively and blindly assessed time points from 0 to 24 months, room versus refrigerated temperature, and presence of a desiccant and/or nitrogen atmosphere on a variety of benign and malignant tissues from North America and Africa. End points included immunohistochemistry (IHC), in situ hybridization (ISH), extracted RNA and DNA quantity and quality, and messenger RNA performance in a novel, multiplexed digital gene expression profiling assay of both housekeeping and tumor-specific genes. Results: We found that using current methods of antigen retrieval, staining, and extraction, the end points of IHC, ISH, RNA, and DNA were well preserved under the various conditions tested, with implications that pre-embedding factors contribute to variability in subsequent tissue integrity. We also document that spectrophotometric estimations of nucleic acid concentrations were in general estimated to be higher than with fluorimetric methods, which may be pertinent to end assay development. We further describe a new multiplex assay, the PlexSet digital gene expression assay, suitable for evaluating RNA quality in FFPE tissues. Conclusion: Altogether, these results may provide helpful guidance with regard to approaches for long-term storage conditions for USS.

Lifitegrast ophthalmic solution for treatment of ocular chronic graft-versus-host disease.

Ocular chronic graft-versus-host disease (oGVHD) is a relatively common complication that occurs following allogeneic hematopoietic cell transplantation. Keratoconjunctivitis sicca (KCS) is the most common manifestation of oGVHD. Lifitegrast is a lymphocyte function-associated antigen-1 antagonist approved to reduce inflammation and symptoms in patients with dry eye disease. We evaluated the efficacy and safety of lifitegrast (5% ophthalmic solution) in patients with ocular GVHD in a single-institution retrospective cohort study of eighteen allogeneic transplant recipients who received topical lifitegrast for oGVHD treatment. The outcome of interest was improvement in oGVHD severity score by National Institutes of Health (NIH) criteria. Lifitegrast was well-tolerated and no serious adverse events were observed. Lifitegrast significantly improved NIH severity scores in 8 (44%) patients. The findings of this study suggest lifitegrast is safe, well-tolerated and is an effective option for oGVHD manifesting as KCS. Prospective evaluation is warranted to confirm efficacy of lifitegrast in this population.HighlightsIn this single-institution retrospective cohort study of eighteen patients who received allogeneic transplant between 2013 and 2018, and received topical lifitegrast for treatment of ocular GVHD, the results demonstrate that lifitegrast eye drops were well-tolerated and led to improvement in symptoms of KCS in 8 (44%) patients.Lifitegrast has the potential to fulfill an unmet need in allogeneic transplant patients with ocular GVHD. Further prospective study is warranted for confirmation.

Eosinophil-platelet interactions promote atherosclerosis and stabilize thrombosis with eosinophil extracellular traps.

Clinical observations implicate a role of eosinophils in cardiovascular diseases because markers of eosinophil activation are elevated in atherosclerosis and thrombosis. However, their contribution to atherosclerotic plaque formation and arterial thrombosis remains unclear. In these settings, we investigated how eosinophils are recruited and activated through an interplay with platelets. Here, we provide evidence for a central importance of eosinophil-platelet interactions in atherosclerosis and thrombosis. We show that eosinophils support atherosclerotic plaque formation involving enhanced von Willebrand factor exposure on endothelial cells and augmented platelet adhesion. During arterial thrombosis, eosinophils are quickly recruited in an integrin-dependent manner and engage in interactions with platelets leading to eosinophil activation as we show by intravital calcium imaging. These direct interactions induce the formation of eosinophil extracellular traps (EETs), which are present in human thrombi and constitute a substantial part of extracellular traps in murine thrombi. EETs are decorated with the granule protein major basic protein, which causes platelet activation by eosinophils. Consequently, targeting of EETs diminished thrombus formation in vivo, which identifies this approach as a novel antithrombotic concept. Finally, in our clinical analysis of coronary artery thrombi, we identified female patients with stent thrombosis as the population that might derive the greatest benefit from an eosinophil-inhibiting strategy. In summary, eosinophils contribute to atherosclerotic plaque formation and thrombosis through an interplay with platelets, resulting in mutual activation. Therefore, eosinophils are a promising new target in the prevention and therapy of atherosclerosis and thrombosis.

Vesicle-associated membrane protein 7-mediated eosinophil degranulation promotes allergic airway inflammation in mice.

Eosinophil degranulation is a determining factor in allergy-mediated airway pathology. Receptor-mediated degranulation in eosinophils requires vesicle-associated membrane protein 7 (VAMP-7), a principal component of the SNARE fusion machinery. The specific contribution of eosinophil degranulation to allergen-induced airway responses remains poorly understood. We generated mice with VAMP-7 gene deficiency exclusively in eosinophils (eoCRE/V7) from a cross using eosinophil-specific Cre recombinase-expressing mice crossed with VAMP-7 f/f mice. Eosinophils from eoCRE/V7 mice showed deficient degranulation responses in vitro, and responses continued to be decreased following ex vivo intratracheal adoptive transfer of eoCRE/V7 eosinophils into IL-5/hE2/EPX -/- mice. Consistent with diminished degranulation responses, reduced airway hyperresponsiveness was observed in ovalbumin-sensitized and challenged eoCRE/V7 mice following methacholine inhalation. Therefore, VAMP-7 mediates eosinophil degranulation both in vitro and ex vivo, and this event augments airway hyperresponsiveness.

In vivo imaging of epithelial wound healing in the cnidarian Clytia hemisphaerica demonstrates early evolution of purse string and cell crawling closure mechanisms.

BACKGROUND: All animals have mechanisms for healing damage to the epithelial sheets that cover the body and line internal cavities. Epithelial wounds heal either by cells crawling over the wound gap, by contraction of a super-cellular actin cable ("purse string") that surrounds the wound, or some combination of the two mechanisms. Both cell crawling and purse string closure of epithelial wounds are widely observed across vertebrates and invertebrates, suggesting early evolution of these mechanisms. Cnidarians evolved ~600 million years ago and are considered a sister group to the Bilateria. They have been much studied for their tremendous regenerative potential, but epithelial wound healing has not been characterized in detail. Conserved elements of wound healing in bilaterians and cnidarians would suggest an evolutionary origin in a common ancestor. Here we test this idea by characterizing epithelial wound healing in live medusae of Clytia hemisphaerica. RESULTS: We identified cell crawling and purse string-mediated mechanisms of healing in Clytia epithelium that appear highly analogous of those seen in higher animals, suggesting that these mechanisms may have emerged in a common ancestor. Interestingly, we found that epithelial wound healing in Clytia is 75 to >600 times faster than in cultured cells or embryos of other animals previously studied, suggesting that Clytia may provide valuable clues about optimized healing efficiency. Finally, in Clytia, we show that damage to the basement membrane in a wound gap causes a rapid shift between the cell crawling and purse string mechanisms for wound closure. This is consistent with work in other systems showing that cells marginal to a wound choose between a super-cellular actin cable or lamellipodia formation to close wounds, and suggests a mechanism underlying this decision. CONCLUSIONS: 1. Cell crawling and purse string mechanisms of epithelial wound healing likely evolved before the divergence of Cnidaria from the bilaterian lineage ~ 600mya 2. In Clytia, the choice between cell crawling and purse string mechanisms of wound healing depends on interactions between the epithelial cells and the basement membrane.

Enzymatic lipid oxidation by eosinophils propagates coagulation, hemostasis, and thrombotic disease.

Blood coagulation is essential for physiological hemostasis but simultaneously contributes to thrombotic disease. However, molecular and cellular events controlling initiation and propagation of coagulation are still incompletely understood. In this study, we demonstrate an unexpected role of eosinophils during plasmatic coagulation, hemostasis, and thrombosis. Using a large-scale epidemiological approach, we identified eosinophil cationic protein as an independent and predictive risk factor for thrombotic events in humans. Concurrent experiments showed that eosinophils contributed to intravascular thrombosis by exhibiting a strong endogenous thrombin-generation capacity that relied on the enzymatic generation and active provision of a procoagulant phospholipid surface enriched in 12/15-lipoxygenase-derived hydroxyeicosatetraenoic acid-phosphatidylethanolamines. Our findings reveal a previously unrecognized role of eosinophils and enzymatic lipid oxidation as regulatory elements that facilitate both hemostasis and thrombosis in response to vascular injury, thus identifying promising new targets for the treatment of thrombotic disease.

Frontline Science: Eosinophil-deficient MBP-1 and EPX double-knockout mice link pulmonary remodeling and airway dysfunction with type 2 inflammation.

Eosinophils and the release of cationic granule proteins have long been implicated in the development of the type 2-induced pathologies linked with respiratory inflammation. Paradoxically, the ablation of the two genes encoding the most abundant of these granule proteins, major basic protein-1 (MBP-1) and eosinophil peroxidase (EPX), results in a near collapse of eosinophilopoiesis. The specificity of this lineage ablation and the magnitude of the induced eosinopenia provide a unique opportunity to clarify the importance of eosinophils in acute and chronic inflammatory settings, as well as to identify potential mechanism(s) of action linked with pulmonary eosinophils in those settings. Specifically, we examined these issues by assessing the induced immune responses and pathologies occurring in MBP-1-/-/EPX-/- mice after 1) ovalbumin sensitization/provocation in an acute allergen-challenge protocol, and 2) crossing MBP-1-/-/EPX-/- mice with a double-transgenic model of chronic type 2 inflammation (i.e., I5/hE2). Acute allergen challenge and constitutive cytokine/chemokine expression each induced the accumulation of pulmonary eosinophils in wild-type controls that was abolished in the absence of MBP-1 and EPX (i.e., MBP-1-/-/EPX-/- mice). The expression of MBP-1 and EPX was also required for induced lung expression of IL-4/IL-13 in each setting and, in turn, the induced pulmonary remodeling events and lung dysfunction. In summary, MBP-1-/-/EPX-/- mice provide yet another definitive example of the immunoregulatory role of pulmonary eosinophils. These results highlight the utility of this unique strain of eosinophil-deficient mice as part of in vivo model studies investigating the roles of eosinophils in health and disease settings.

Lung Pathologies in a Chronic Inflammation Mouse Model Are Independent of Eosinophil Degranulation.

RATIONALE: The release of eosinophil granule proteins in the lungs of patients with asthma has been dogmatically linked with lung remodeling and airway hyperresponsiveness. However, the demonstrated inability of established mouse models to display the eosinophil degranulation occurring in human subjects has prevented a definitive in vivo test of this hypothesis. OBJECTIVES: To demonstrate in vivo causative links between induced pulmonary histopathologies/lung dysfunction and eosinophil degranulation. METHODS: A transgenic mouse model of chronic T-helper cell type 2-driven inflammation overexpressing IL-5 from T cells and human eotaxin 2 in the lung (I5/hE2) was used to test the hypothesis that chronic histopathologies and the development of airway hyperresponsiveness occur as a consequence of extensive eosinophil degranulation in the lung parenchyma. MEASUREMENT AND MAIN RESULTS: Studies targeting specific inflammatory pathways in I5/hE2 mice surprisingly showed that eosinophil-dependent immunoregulative events and not the release of individual secondary granule proteins are the central contributors to T-helper cell type 2-induced pulmonary remodeling and lung dysfunction. Specifically, our studies highlighted a significant role for eosinophil-dependent IL-13 expression. In contrast, extensive degranulation leading to the release of major basic protein-1 or eosinophil peroxidase was not causatively linked to many of the induced pulmonary histopathologies. However, these studies did define a previously unappreciated link between the release of eosinophil peroxidase (but not major basic protein-1) and observed levels of induced airway mucin. CONCLUSIONS: These data suggest that improvements observed in patients with asthma responding to therapeutic strategies ablating eosinophils may occur as a consequence of targeting immunoregulatory mechanisms and not by simply eliminating the destructive activities of these purportedly end-stage effector cells.

Eosinophil-dependent skin innervation and itching following contact toxicant exposure in mice.

BACKGROUND: Contact toxicant reactions are accompanied by localized skin inflammation and concomitant increases in site-specific itch responses. The role(s) of eosinophils in these reactions is poorly understood. However, previous studies have suggested that localized eosinophil-nerve interactions at sites of inflammation significantly alter tissue innervation. OBJECTIVE: To define a potential mechanistic link between eosinophils and neurosensory responses in the skin leading to itching. METHODS: BALB/cJ mice were exposed to different contact toxicants, identifying trimellitic anhydride (TMA) for further study on the basis of inducing a robust eosinophilia accompanied by degranulation. Subsequent studies using TMA were performed with wild type versus eosinophil-deficient PHIL mice, assessing edematous responses and remodeling events such as sensory nerve innervation of the skin and induced pathophysiological responses (ie, itching). RESULTS: Exposure to TMA, but not dinitrofluorobenzene, resulted in a robust eosinophil skin infiltrate accompanied by significant levels of degranulation. Follow-up studies using TMA with wild type versus eosinophil-deficient PHIL mice showed that the induced edematous responses and histopathology were, in part, causatively linked with the presence of eosinophils. Significantly, these data also demonstrated that eosinophil-mediated events correlated with a significant increase in substance P content of the cutaneous nerves and an accompanying increase in itching, both of which were abolished in the absence of eosinophils. CONCLUSIONS: Eosinophil-mediated events following TMA contact toxicant reactions increase skin sensory nerve substance P and, in turn, increase itching responses. Thus, eosinophil-nerve interactions provide a potential mechanistic link between eosinophil-mediated events and neurosensory responses following exposure to some contact toxicants.

Cys-leukotrienes promote fibrosis in a mouse model of eosinophil-mediated respiratory inflammation.

Leukotrienes (i.e., products of the 5-lipoxygenase pathway) are thought to be contributors to lung pathologies. Moreover, eosinophils have been linked with pulmonary leukotriene activities both as potential sources of these mediators and as responding effector cells. The objective of the present study was to define the role(s) of leukotrienes in the lung pathologies accompanying eosinophil-associated chronic respiratory inflammation. A transgenic mouse model of chronic T helper (Th) 2-driven inflammation expressing IL-5 from T cells and human eotaxin-2 locally in the lung (I5/hE2) was used to define potential in vivo relationships among eosinophils, leukotrienes, and chronic Th2-polarized pulmonary inflammation. Airway levels of cys-leukotrienes and leukotriene B4 (LTB4) are both significantly elevated in I5/hE2 mice. The eosinophil-mediated airway hyperresponsiveness (AHR) characteristic of these mice was abolished in the absence of leukotrienes (i.e., 5-lipoxygenase-deficient I5/hE2). More importantly, the loss of leukotrienes led to an unexpectedly significant decrease in collagen deposition (i.e., pulmonary fibrosis) that accompanied elevated levels of IL-4/-13 and TGF-ß in the lungs of I5/hE2 mice. Further studies using mice deficient for the LTB4 receptor (BLT-1(-/-)/I5/hE2) and I5/hE2 animals administered a cys-leukotriene receptor antagonist (montelukast) demonstrated that the AHR and the enhanced pulmonary fibrosis characteristic of the I5/hE2 model were uniquely cys-leukotriene-mediated events. These data demonstrate that, similar to allergen challenge models of wild-type mice, cys-leukotrienes underlie AHR in this transgenic model of severe pulmonary Th2 inflammation. These data also suggest that an underappreciated link exists among eosinophils, cys-leukotriene-mediated events, and fibrotic remodeling associated with elevated levels of IL-4/-13 and TGF-ß.

Human versus mouse eosinophils: "that which we call an eosinophil, by any other name would stain as red".

The respective life histories of human subjects and mice are well defined and describe a unique story of evolutionary conservation extending from sequence identity within the genome to the underpinnings of biochemical, cellular, and physiologic pathways. As a consequence, the hematopoietic lineages of both species are invariantly maintained, each with identifiable eosinophils. This canonical presence nonetheless does not preclude disparities between human and mouse eosinophils, their effector functions, or both. Indeed, many books and reviews dogmatically highlight differences, providing a rationale to discount the use of mouse models of human eosinophilic diseases. We suggest that this perspective is parochial and ignores the wealth of available studies and the consensus of the literature that overwhelming similarities (and not differences) exist between human and mouse eosinophils. The goal of this review is to summarize this literature and in some cases provide experimental details comparing and contrasting eosinophils and eosinophil effector functions in human subjects versus mice. In particular, our review will provide a summation and an easy-to-use reference guide to important studies demonstrating that although differences exist, more often than not, their consequences are unknown and do not necessarily reflect inherent disparities in eosinophil function but instead species-specific variations. The conclusion from this overview is that despite nominal differences, the vast similarities between human and mouse eosinophils provide important insights as to their roles in health and disease and, in turn, demonstrate the unique utility of mouse-based studies with an expectation of valid extrapolation to the understanding and treatment of patients.

Eosinophils regulate dendritic cells and Th2 pulmonary immune responses following allergen provocation.

Reports have recently suggested that eosinophils have the potential to modulate allergen-dependent pulmonary immune responses. The studies presented expand these reports demonstrating in the mouse that eosinophils are required for the allergen-dependent Th2 pulmonary immune responses mediated by dendritic cells (DCs) and T lymphocytes. Specifically, the recruitment of peripheral eosinophils to the pulmonary lymphatic compartment(s) was required for the accumulation of myeloid DCs in draining lymph nodes and, in turn, Ag-specific T effector cell production. These effects on DCs and Ag-specific T cells did not require MHC class II expression on eosinophils, suggesting that these granulocytes have an accessory role as opposed to direct T cell stimulation. The data also showed that eosinophils uniquely suppress the DC-mediated production of Th17 and, to smaller degree, Th1 responses. The cumulative effect of these eosinophil-dependent immune mechanisms is to promote the Th2 polarization characteristic of the pulmonary microenvironment after allergen challenge.