Highly Sensitive, Flow Cytometry-Based Measurement of Intestinal Permeability in Models of Experimental Colitis.

BACKGROUND & AIMS: Increased intestinal permeability is seen in a variety of inflammatory conditions such as enteric infections and inflammatory bowel disease. Because barrier function can provide a key biomarker of disease severity, it often is assayed in animal models. A common methodology involves gavaging mice with fluorescein isothiocyanate-conjugated dextran (FITC-D), followed by cardiac puncture to assay plasma fluorescence on a spectrophotometer. Although the FITC-D method is relatively simple, its sensitivity is limited and enables only a single measurement because the test requires killing the subject. Herein, we describe a novel flow cytometry-based method of intestinal permeability measurement based on detection of orally gavaged ovalbumin (OVA) that leaks out of the gut. Our approach uses minute blood volumes collected from the tail vein, permitting repeated testing of the same subject at multiple time points. By comparing this assay against the gold standard FITC-D method, we show the expanded utility of our OVA assay in measuring intestinal permeability. METHODS: We directly compared our OVA assay against the FITC-D assay by co-administering both probes orally to the same animals and subsequently using their respective methodologies to measure intestinal permeability by detecting probe levels in the plasma. Permeability was assessed in mice genetically deficient in intestinal mucus production or glycosylation. In addition, wild-type mice undergoing dextran sodium sulfate-induced colitis or infected by the enteric bacterial pathogen Citrobacter rodentium also were tested. RESULTS: The OVA assay showed very high efficacy in all animal models of intestinal barrier dysfunction tested. Besides identifying intestinal barrier dysfunction in mice with impaired mucin glycosylation, the assay also allowed for repeated tracking of intestinal permeability within the same animal over time, providing data that cannot be easily acquired with other currently applied methods. CONCLUSIONS: The OVA assay is a highly sensitive and effective method of measuring intestinal permeability in mouse models of barrier dysfunction and experimental colitis.

Critical Role of TSLP Receptor on CD4 T Cells for Exacerbation of Skin Inflammation.

Thymic stromal lymphopoietin (TSLP) is a key cytokine that initiates and promotes allergic inflammation both in humans and mice. It is well known that TSLP is important in initial step of inflammation by stimulating dendritic cells to promote Th2 differentiation of naive T cells. However, TSLP is abundantly produced in the late phase of inflammation, as well; therefore, we focused on the function of TSLP in chronic Th2-type inflammation. By establishing a novel (to our knowledge) chronic allergic skin inflammation mouse model with repetitive challenges of hapten after sensitization, we demonstrated that CD4 T cell-specific deletion of TSLP receptor (TSLPR) resulted in near-complete ablation of ear swelling and infiltration of CD4 T cells and eosinophils, but after second challenge. Of note, TSLPR deletion on CD4 T cells did not affect acute inflammation. As expected, transfer of Ag-sensitized wild-type CD4T cells, but not of TSLPR-deficient CD4T cells, increased skin inflammation in the model upon challenge. Furthermore, production of IL-4 from TSLPR-deficient CD4T cells in inflamed ear lesions was markedly diminished, demonstrating that TSLP-dependent IL-4 production from CD4T cells was critical for the exacerbation of skin inflammation. Similar results were obtained in Th2-type allergic skin inflammation model using MC903. Collectively, these results indicate that TSLP acts directly on CD4 T cells to elicit pathogenesis of Th2 cells, thereby having a critical role in exacerbation of skin inflammation in the chronic phase.

Animal Models of Pulmonary Fibrosis.

Pulmonary fibrosis is a debilitating disease and is often fatal. It may be the consequence of direct lung injury or the result of genetic defects and occupational, environmental, or drug-related exposures. In many cases the etiology is unknown. The pathogenesis of all forms of pulmonary fibrosis regardless of type of injury or etiology is incompletely understood. These disorders are characterized by the accumulation of extracellular matrix in the lung interstitium with a loss of lung compliance and impaired gas exchange that ultimately leads to respiratory failure. Animal models of pulmonary fibrosis have become indispensable in the improved understanding of these disorders. Multiple models have been developed each with advantages and disadvantages. In this chapter we discuss the application of two of the most commonly employed direct lung instillation models, namely, the induction of pulmonary fibrosis with bleomycin or fluorescein isothiocyanate (FITC). We provide details on design, materials, and methods and describe how these models can be best undertaken. We also discuss methods to induce fibrosis in aged mice using murine gamma-herpesvirus (γHV-68) and approaches to exacerbate bleomycin- or FITC-induced fibrosis using γHV-68.

Intraoperative imaging of folate receptor alpha positive ovarian and breast cancer using the tumor specific agent EC17.

INTRODUCTION: Intraoperative fluorescence imaging of the folate-receptor alpha (FRα) could support completeness of resection in cancer surgery. Feasibility of EC17, a FRα-targeting agent that fluoresces at 500nm, was demonstrated in a limited series of ovarian cancer patients. Our objective was to evaluate EC17 in a larger group of ovarian cancer patients. In addition, we assessed the feasibility of EC17 in patients with breast cancer. METHODS: Two-to-three hours before surgery 0.1mg/kg EC17 was intravenously administered to 12 patients undergoing surgery for ovarian cancer and to 3 patients undergoing surgery for biopsy-proven FRα-positive breast cancer. The number of lesions/positive margins detected with fluorescence and concordance between fluorescence and tumor- and FRα-status was assessed in addition to safety and pharmacokinetics. RESULTS: Fluorescence imaging in ovarian cancer patients allowed detection of 57 lesions of which 44 (77%) appeared malignant on histopathology. Seven out of these 44 (16%) were not detected with inspection/palpation. Histopathology demonstrated concordance between fluorescence and FRα- and tumor status. Fluorescence imaging in breast cancer patients, allowed detection of tumor-specific fluorescence signal. At the 500nm wavelength, autofluorescence of normal breast tissue was present to such extent that it interfered with tumor identification. CONCLUSIONS: FRα is a favorable target for fluorescence-guided surgery as EC17 produced a clear fluorescent signal in ovarian and breast cancer tissue. This resulted in resection of ovarian cancer lesions that were otherwise not detected. Notwithstanding, autofluorescence caused false-positive lesions in ovarian cancer and difficulty in discriminating breast cancer-specific fluorescence from background signal. Optimization of the 500nm fluorophore, will minimize autofluorescence and further improve intraoperative tumor detection.

Cannabinoid 1 receptors in keratinocytes attenuate fluorescein isothiocyanate-induced mouse atopic-like dermatitis.

Atopic dermatitis is a chronic inflammatory disease characterized by an impaired epidermal barrier function combined with a chronic Th2-type inflammatory response and an intense pruritus. Here, we used an experimental mouse model for Th2-type contact hypersensitivity (CHS) to fluorescein isothiocyanate (FITC) to investigate the potential role of cannabinoid 1 receptors (CB1) in the pathophysiology of mouse atopic-like dermatitis. Mice lacking CB1 receptors globally (Cnr1(-/-) ) or specifically in keratinocytes (KC-Cnr1(-/-) ) as well as wild-type (WT) control mice were sensitized and challenged with FITC. We examined ear swelling responses, transepidermal water loss, Th2-type skin inflammatory responses and serum IgE levels. Both Cnr1(-/-) and KC-Cnr1(-/-) showed enhanced CHS responses to FITC and a delayed epidermal barrier repair when compared with WT mice. mRNA levels for IL-4, thymic stromal lymphopoietin (TSLP) and CCL8, as well as eosinophil activity, were significantly increased in inflamed ear tissue of FITC-challenged Cnr1(-/-) and KC-Cnr1(-/-) mice. Importantly, CB1 receptor-deficient keratinocytes secreted increased levels of TSLP, a proinflammatory mediator that drives Th2-type skin inflammation in atopic dermatitis, under basal and Th2-type inflammatory conditions. Taken together, our results demonstrate that CB1 receptors in keratinocytes help to maintain epidermal barrier homoeostasis and attenuate Th2-type allergic inflammatory responses. Based on our work, we propose that enhanced epidermal allergen penetrance cooperates with increased production of TSLP and CCL8 by epidermal keratinocytes for the induction of type 2 CD4+ T helper cells. Our results place keratinocytes at the cross-roads of outside-in and inside-out pathophysiologic mechanisms of atopic dermatitis.

The differential role of L-selectin and ICAM-1 in Th1-type and Th2-type contact hypersensitivity.

Sensitization and challenge using DNFB induce contact hypersensitivity (CHS) with predominant type 1 helper (Th1) cell infiltration, whereas those using FITC generate CHS with Th2 cell infiltration. CHS results from inflammatory cell infiltration, a process that is highly regulated by the expression of multiple adhesion molecules. We attempted to determine the role of L-selectin and ICAM-1 in Th1- and Th2-type CHS induced by DNFB or FITC in mice lacking either L-selectin, ICAM-1, or both. Th1-type CHS induced by DNFB was inhibited by L-selectin and/or ICAM-1 deficiency, which was associated with reduced IFN-gamma expression. Similarly, Th2-type CHS induced by FITC was inhibited by L-selectin deficiency. However, Th2-type CHS was increased by ICAM-1 deficiency and accompanied by increased Th2 cytokine expression. Infiltration of in vitro-generated Th1 cells into the FITC-challenged skin decreased in ICAM-1-deficient mice, whereas in vitro-generated Th2 cell infiltration increased, suggesting that ICAM-1 mediates Th1 cell migration and that in the absence of ICAM-1, Th1 cell recruitment decreased, whereas relative Th2 cell migration increased. These results suggest that ICAM-1 mediates Th1 cell recruitment irrespective of DNFB or FITC and that L-selectin recruits Th1 cells in Th1-type CHS, whereas it recruits Th2 cells in Th2-type CHS.

TRPA1 and TRPV1 activation is a novel adjuvant effect mechanism in contact hypersensitivity.

We have revealed that local stimulation of sensory neurons is involved in the adjuvant effect of dibutyl phthalate (DBP) in a fluorescein isothiocyanate-induced mouse contact hypersensitivity model. Transient receptor potential (TRP) A1 and TRPV1 seemed to be candidate DBP targets. Here we directly demonstrated that DBP activates a subset of neurons in mouse dorsal root ganglia responsive to TRPA1 and TRPV1 agonists. TRPA1 and TRPV1 activation was further demonstrated using cultured cells expressing TRP channels. Among structurally different phthalate esters, there is a positive relationship between the activation of TRPA1- or TRPV1-expressing cells and the adjuvant effect.

A small molecule CRTH2 antagonist inhibits FITC-induced allergic cutaneous inflammation.

A FITC-induced allergic contact hypersensitivity model was used to investigate the role that the prostaglandin D(2) receptor-chemoattractant receptor-homologous molecule expressed on T(h)2 cells (CRTH2) plays in modulating cutaneous inflammation. Our results show that inhibition of CRTH2, achieved via administration of a potent, small molecule antagonist, Compound A (Cmpd A), effectively blocked edema formation and greatly reduced the inflammatory infiltrate and skin pathology observed in drug vehicle-treated animals. Gene expression analysis revealed that Cmpd A administration down-regulated the transcription of a wide range of pro-inflammatory mediators. This correlated with decreases in cytokine and chemokine protein levels, notably IL-4, IL-1beta, tumor necrosis factor-alpha, transforming growth factor-beta, GRO-alpha, MIP-2 and thymic stromal lymphopoietin (TSLP) in FITC-challenged ears. The administration of an anti-TSLP-neutralizing antibody was only partially effective in lowering the FITC-induced inflammatory infiltrate and cytokine production compared with the CRTH2 antagonist. Taken together, these data suggest that blockade of CRTH2 inhibits multiple pathways leading to cutaneous inflammation in this model. This suggests that CRTH2 antagonism may be a viable route for therapeutic intervention in allergic skin diseases, such as atopic dermatitis.

UVB irradiation decreases the magnitude of the Th1 response to hapten but does not increase the Th2 response.

Exposure of murine skin to low doses of ultraviolet-B (UVB) radiation before sensitization with hapten reduces the ability of antigen presenting cells (APC) in the draining lymph nodes to initiate contact hypersensitivity responses in vivo and results in the induction of hapten-specific suppressor T cells. In the present study, we tested the hypothesis that exposure of skin to UVB radiation suppresses T cell responses to hapten in vivo by altering the functions of APC, resulting in decreased stimulation of Th1 lymphocytes, which mediate contact hypersensitivity responses, and preferential activation of Th2 cells. C3H/HeN mice were exposed to either a single 2 kJ/m2 dose of UVB or to 400 J/m2 of UVB daily from FS40 sunlamps for four consecutive days and sensitized with fluorescein isothiocyanate on UV-irradiated skin. Draining lymph node cells were collected 18 h after sensitization and co-cultured with nylon wool-purified T cells from naive or fluorescein-immunized mice. Unseparated lymph node cells or sorter-purified fluorescein-bearing APC from UV-irradiated mice induced less T cell proliferation than APC from non-UV-exposed mice. Lymph node cells produced less Th1 and Th2-associated cytokines, interferon-gamma and interleukin-4, respectively, in response to APC from UV-irradiated animals compared with APC from unirradiated, fluorescein-sensitized mice. Thus, low doses of UV radiation do not result in preferential stimulation of Th2 response in lymph nodes, and results from cloned cell lines may incompletely reflect T cell responses in vivo.

In vitro primary sensitization of hapten-specific T cells by cultured human epidermal Langerhans cells--a screening predictive assay for contact sensitizers.

BACKGROUND: The need to develop predictive tests which could identify potential allergens has been recognized for many years. There is as yet no accepted in vitro method for the assessment of contact sensitizers. OBJECTIVE: We have tested the ability of a range of contact allergens to induce in vitro primary sensitization of autologous T cells. METHOD: T-cell proliferation induced by haptens using 2-day cultured human Langerhans cells as antigen-presenting cell was assessed by 3H thymidine incorporation. Antigen specific stimulation was calculated as stimulation indexes. RESULTS: Strong allergens induced in vitro a primary T-cell response in all (trinitrophenyl, TNP: 13/13) or in the majority (fluorescein isothiocyanate, FITC: 7/10) of experiments. An irritant, sodium dodecyl sulfate (SDS), failed to generate a significant T-cell proliferation in any of the experiments (0/10). We obtained a significant lymphoproliferative response to weak sensitizers only in a limited number of experiments: (coumarin: 1/12, citronellal: 0/10, hydroxycitronellal: 2/8). p-Phenylenediamine (PPDA), a prohapten and highly sensitizing chemical in vivo, generated primary sensitization in vitro in only one of six experiments, while Bandrowski's base (BB), a metabolization product of PPDA induced a significant T-cell response in all six experiments. CONCLUSION: The present in vitro model allows discrimination between two groups of substances: strong contact sensitizers (TNP, FITC, BB) on the one hand and weak sensitizers (coumarin, citronellal and hydroxycitronellal) and irritants (SDS) on the other hand. It could be used as a screening in vitro assay to eliminate strong contact allergens before further predictive animal tests have to be performed.

Comparison of sensitizing protocols for ultraviolet B-induced immunosuppression in C3H mice.

To compare previously used protocols for ultraviolet (UV)-induced suppression of contact hypersensitivity in mice, and to develop an optimized protocol for C3H mice, the effect of 3 different allergens, varying allergen concentrations in the induction or challenge phase, local and distant sites of allergen application in respect to irradiation site, 2 mouse substrains and 2 different light sources was studied. A concentration of 0.5% of oxazolone (OXA) gave a slightly better contact sensitization than a 1% concentration of trinitrochlorobenzene (TNCB). Titration experiments revealed that for both OXA and TNCB, a 1% sensitization concentration was optimal, while the optimal challenge concentration was 0.5% for OXA and 1% for TNCB. The magnitude of the resulting contact sensitization was not influenced by either the mouse substrain (C3H/HeJ or C3H/HeN) or the site of allergen application (back or belly), but application of fluorescein isothiocyanate to the ears only produced weak sensitization. A standard UVB dose of 1.3 kJ/m2 suppressed TNCB contact sensitivity to a greater extent than that of OXA. A similar degree of UV-induced suppression was obtained with a given UVB dose, irrespective of a 50-fold difference in the concomitant UVA dose. Based on our results, a proper protocol of contact sensitization for UV-induced immunosuppression in C3H mice includes sensitization with 0.5% OXA on either the mouse back or belly, ear challenge with 0.5% OXA and ear swelling reading 24 h after challenge.

Differentiation of light-dye effects in the microcirculation.

Activation of photosensitive compounds has been used in the treatment of tumors and as a technique to study various microcirculatory phenomena. This technique may be accompanied by deleterious effects which may complicate interpretations of experimental results. However, the relevant physiological mechanisms that induce toxicity and the light doses needed to produce different toxic reactions have not been well defined. In the current study, the rat cremaster muscle preparation was used with in vivo fluorescent television microscopy and subsequently with electron and light microscopy to evaluate toxic reactions of light activation of fluorescein isothiocyanate. The most sensitive photoactive reactions were macromolecular leakage and platelet activation, occurring with 120 J/cm2 activation energy. Macromolecular leakage was at least partially restricted by perivenular and pericapillary pericytes and there was no morphological damage with this light dose. Since macromolecular leakage was significantly inhibited by pretreatment with diphenhydramine or Compound 48/80, it is in part due to the release of histamine from tissue mast cells. 720 J/cm2 reduced the red blood cell column in the venules by over 50% due to platelet thrombus formation, an effect that was accentuated by pretreatment with indomethacin. This suggests an inhibitory role of prostaglandins in platelet thrombus formation. In addition, 720 J/cm2 caused endothelial and smooth muscle cell swelling and ruptures, gap formation, and leukocyte and protein accumulation in the vessel walls.