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1.  Adenomatous polyps are driven by microbe-instigated focal inflammation and are controlled by IL-10 producing T-cells 
Cancer research  2013;73(19):5905-5913.
Interleukin-10 (IL-10) is elevated in cancer and is thought to contribute to immune tolerance and tumor growth. Defying these expectations, the adoptive transfer of IL-10 expressing T-cells to mice with polyposis attenuates microbial-induced inflammation and suppresses polyposis. To gain better insights into how IL-10 impacts polyposis, we genetically ablated IL-10 in T-cells in APCΔ468 mice and compared the effects of treatment with broad-spectrum antibiotics. We found that T-cells and Tregs were a major cellular source of IL-10 in both the healthy and polyp-bearing colon. Notably, T-cell-specific ablation of IL-10 produced pathologies that were identical to mice with a systemic deficiency in IL-10, in both cases increasing the numbers and growth of colon polyps. Eosinophils were found to densely infiltrate colon polyps, which were enriched similarly for microbiota associated previously with colon cancer. In mice receiving broad-spectrum antibiotics, we observed reductions in microbiota, inflammation, and polyposis. Together our findings establish that colon polyposis is driven by high densities of microbes that accumulate within polyps and trigger local inflammatory responses. Inflammation, local microbe densities, and polyp growth are suppressed by IL-10 derived specifically from T-cells and Tregs.
doi:10.1158/0008-5472.CAN-13-1511
PMCID: PMC4322779  PMID: 23955389
interleukin-10; polyposis; microbiota; inflammation; colon
2.  Calcium carbonate does not affect imatinib pharmacokinetics in healthy volunteers 
Cancer chemotherapy and pharmacology  2013;73(1):10.1007/s00280-013-2337-0.
Purpose
Imatinib mesylate (Gleevec®/Glivec®), has revolutionized the treatment of chronic myeloid leukemias (CML) and gastrointestinal stromal tumors (GIST), and there is evidence for an exposure response relationship. Calcium carbonate is increasingly used as a calcium supplement and in the setting of gastric upset associated with imatinib therapy. Calcium carbonate could conceivably elevate gastric pH and complex imatinib, thereby influencing imatinib absorption and exposure. We aimed to evaluate whether use of calcium carbonate has a significant effect on imatinib pharmacokinetics.
Methods
Eleven healthy subjects were enrolled in a 2-period, open-label, single-institution, randomized cross-over, fixed-schedule study. In one period, each subject received 400 mg of imatinib p.o.. In the other period, 4000 mg calcium carbonate (Tums Ultra®) was administered p.o. 15 min before 400 mg of imatinib. Plasma concentrations of imatinib and its active N-desmethyl metabolite CGP74588 were assayed by LC-MS; data were analyzed non-compartmentally, and compared after log transformation.
Results
Calcium carbonate administration did not significantly affect the imatinib area under the plasma concentration versus time curve (AUC) (41.2 μg/mL•h alone versus 40.8 μg/mL•h with calcium carbonate, P=0.99), maximum plasma concentration (Cmax) (2.35 μg/mL alone versus 2.39 μg/mL with calcium carbonate, P=0.89).
Conclusions
Our results indicate that the use of calcium carbonate does not significantly affect imatinib pharmacokinetics.
doi:10.1007/s00280-013-2337-0
PMCID: PMC3880632  PMID: 24170263
imatinib; calcium; CML; GIST; interaction
3.  Local hypersensitivity reaction in transgenic mice with squamous epithelial IL-5 overexpression provides a novel model of eosinophilic oesophagitis 
Gut  2012;63(1):10.1136/gutjnl-2012-303631.
Objective
Eosinophilic oesophagitis (EoE) is a chronic inflammatory condition of the oesophagus with limited treatment options. No previous transgenic model has specifically targeted the oesophageal mucosa to induce oesophageal eosinophilia.
Design
We developed a mouse model that closely resembles EoE by utilising oxazolone haptenation in mice with transgenic overexpression of an eosinophil poietic and survival factor (interleukin (IL)-5) in resident squamous oesophageal epithelia.
Results
Overexpression of IL-5 in the healthy oesophagus was achieved in transgenic mice (L2-IL5) using the squamous epithelial promoter Epstein–Barr virus ED-L2. Oxazolone-challenged L2-IL5 mice developed dose-dependent pan-oesophageal eosinophilia, including eosinophil microabscess formation and degranulation as well as basal cell hyperplasia. Moreover, oesophagi expressed increased IL-13 and the eosinophil agonist chemokine eotaxin-1. Treatment of these mice with corticosteroids significantly reduced eosinophilia and epithelial inflammation.
Conclusions
L2-IL5 mice provide a novel experimental model that can potentially be used in preclinical testing of EoE-related therapeutics and mechanistic studies identifying pathogenetic features associated with mucosal eosinophilia.
doi:10.1136/gutjnl-2012-303631
PMCID: PMC3884674  PMID: 23161496
4.  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-β 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-β.
doi:10.1165/rcmb.2013-0009OC
PMCID: PMC3931112  PMID: 23859654
5-lipoxygenase; asthma; eosinophils; montelukast; lung
5.  Edge chipping and flexural resistance of monolithic ceramics☆ 
Objective
Test the hypothesis that monolithic ceramics can be developed with combined esthetics and superior fracture resistance to circumvent processing and performance drawbacks of traditional all-ceramic crowns and fixed-dental-prostheses consisting of a hard and strong core with an esthetic porcelain veneer. Specifically, to demonstrate that monolithic prostheses can be produced with a much reduced susceptibility to fracture.
Methods
Protocols were applied for quantifying resistance to chipping as well as resistance to flexural failure in two classes of dental ceramic, microstructurally-modified zirconias and lithium disilicate glass–ceramics. A sharp indenter was used to induce chips near the edges of flat-layer specimens, and the results compared with predictions from a critical load equation. The critical loads required to produce cementation surface failure in monolithic specimens bonded to dentin were computed from established flexural strength relations and the predictions validated with experimental data.
Results
Monolithic zirconias have superior chipping and flexural fracture resistance relative to their veneered counterparts. While they have superior esthetics, glass–ceramics exhibit lower strength but higher chip fracture resistance relative to porcelain-veneered zirconias.
Significance
The study suggests a promising future for new and improved monolithic ceramic restorations, with combined durability and acceptable esthetics.
doi:10.1016/j.dental.2013.09.004
PMCID: PMC4000448  PMID: 24139756
Edge chipping; Flexural fracture; Monolithic restorations; Zirconia-based ceramic; Glass–ceramic; Graded glass–zirconia
6.  Surfactant protein D attenuates sub-epithelial fibrosis in allergic airways disease through TGF-β 
Respiratory Research  2014;15(1):143.
Background
Surfactant protein D (SP-D) can regulate both innate and adaptive immunity. Recently, SP-D has been shown to contribute to the pathogenesis of airway allergic inflammation and bleomycin-induced pulmonary fibrosis. However, in allergic airways disease, the role of SP-D in airway remodeling remains unknown. The objective of this study was to determine the contribution of functional SP-D in regulating sub-epithelial fibrosis in a mouse chronic house dust mite model of allergic airways disease.
Methods
C57BL/6 wild-type (WT) and SP-D−/− mice (C57BL/6 background) were chronically challenged with house dust mite antigen (Dermatophagoides pteronyssinus, Dp). Studies with SP-D rescue and neutralization of TGF-β were conducted. Lung histopathology and the concentrations of collagen, growth factors, and cytokines present in the airspace and lung tissue were determined. Cultured eosinophils were stimulated by Dp in presence or absence of SP-D.
Results
Dp-challenged SP-D−/− mice demonstrate increased sub-epithelial fibrosis, collagen production, eosinophil infiltration, TGF-β1, and IL-13 production, when compared to Dp-challenged WT mice. By immunohistology, we detected an increase in TGF-β1 and IL-13 positive eosinophils in SP-D−/− mice. Purified eosinophils stimulated with Dp produced TGF-β1 and IL-13, which was prevented by co-incubation with SP-D. Additionally, treatment of Dp challenged SP-D−/− mice with exogenous SP-D was able to rescue the phenotypes observed in SP-D−/− mice and neutralization of TGF-β1 reduced sub-epithelial fibrosis in Dp-challenged SP-D−/− mice.
Conclusion
These data support a protective role for SP-D in the pathogenesis of sub-epithelial fibrosis in a mouse model of allergic inflammation through regulation of eosinophil-derived TGF-β.
Electronic supplementary material
The online version of this article (doi:10.1186/s12931-014-0143-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s12931-014-0143-9
PMCID: PMC4262976  PMID: 25472740
Surfactant protein D; Asthma; Fibrosis; Airway remodeling; Eosinophil; Transforming growth factor beta
7.  A Sensitive High Throughput ELISA for Human Eosinophil Peroxidase: A Specific Assay to Quantify Eosinophil Degranulation from Patient-derived Sources 
Journal of immunological methods  2012;384(1-2):10-20.
Quantitative high throughput assays of eosinophil-mediated activities in fluid samples from patients in a clinical setting have been limited to ELISA assessments for the presence of the prominent granule ribonucleases, ECP and EDN. However, the demonstration that these ribonucleases are expressed by leukocytes other than eosinophils, as well as cells of non-hematopoietic origin, limits the usefulness of these assays. Two novel monoclonal antibodies recognizing eosinophil peroxidase (EPX) were used to develop an eosinophil-specific and sensitive sandwich ELISA. The sensitivity of this EPX-based ELISA was shown to be similar to that of the commercially available ELISA kits for ECP and EDN. More importantly, evidence is also presented confirming that among these granule protein detection options, EPX-based ELISA is the only eosinophil-specific assay. The utility of this high throughput assay to detect released EPX was shown in ex vivo degranulation studies with isolated human eosinophils. In addition, EPX-based ELISA was used to detect and quantify eosinophil degranulation in several in vivo patient settings, including bronchoalveolar lavage fluid obtained following segmental allergen challenge of subjects with allergic asthma, induced sputum derived from respiratory subjects following hypotonic saline inhalation, and nasal lavage of chronic rhinosinusitis patients. This unique EPX-based ELISA thus provides an eosinophil-specific assay that is sensitive, reproducible, and quantitative. In addition, this assay is adaptable to high throughput formats (e.g., automated assays utilizing microtiter plates) using the diverse patient fluid samples typically available in research and clinical settings.
doi:10.1016/j.jim.2012.06.011
PMCID: PMC3432656  PMID: 22750539
EPX; eosinophilia; granule proteins; allergic inflammation
8.  Conditions for the validity of SNP-based heritability estimation 
Human genetics  2014;133(8):1011-1022.
The heritability of a trait (h2) is the proportion of its population variance caused by genetic differences, and estimates of this parameter are important for interpreting the results of genome-wide association studies (GWAS). In recent years, researchers have adopted a novel method for estimating a lower bound on heritability directly from GWAS data that uses realized genetic similarities between nominally unrelated individuals. The quantity estimated by this method is purported to be the contribution to heritability that could in principle be recovered from association studies employing the given panel of SNPs (hSNP2). Thus far, the validity of this approach has mostly been tested empirically. Here, we provide a mathematical explication and show that the method should remain a robust means of obtaining hSNP2 SNP under circumstances wider than those under which it has so far been derived.
doi:10.1007/s00439-014-1441-5
PMCID: PMC4159725  PMID: 24744256
9.  Eosinophil Peroxidase in Sputum Represents a Unique Biomarker of Airway Eosinophilia 
Allergy  2013;68(9):1177-1184.
Background
Sputum eosinophilia has been shown to be a predictor of asthma patient response to therapies. However, quantitative cell counts and differentials of sputum are labor intensive. The objective of this study was to validate a novel ELISA-based assay of eosinophil peroxidase (EPX) in sputum as a rapid and reliable marker of airway eosinophils.
Methods
The utility of EPX-based ELISA as an eosinophil-specific assay was achieved through comparisons with sputum eosinophil differential counts in freshly prepared and archived patient samples from a variety of clinical settings.
Results
EPX levels in sputum correlated with eosinophil percentage (rs=0.84) in asthma patients with varying degrees of airway eosinophilia. Significantly, unlike assays of other eosinophil granule proteins (e.g., ECP and EDN), which often detect the presence of these proteins even in asthma patients with neutrophilic bronchitis, EPX-based ELISA levels are not increased in this subset of asthma patients or in COPD patients lacking evidence of an airway eosinophilia. Moreover, sputum EPX was a surrogate marker of airway eosinophilia in other patient studies (e.g., allergen inhalation and treatment trials the anti-(IL-5) therapeutic Mepolizumab™). Finally, EPX levels in cyto-centrifuged prepared sputum supernatants correlated with those from rapidly prepared non-centrifuged filtrates of sputum (rs 0.94).
Conclusions
EPX-based ELISA is a valid, reliable, repeatable, and specific surrogate marker of eosinophils and/or eosinophil degranulation in the sputum of respiratory patients.
doi:10.1111/all.12206
PMCID: PMC3788081  PMID: 23931643
Sputum Eosinophils; EPX; ELISA; Asthma; COPD
10.  Applying compressed sensing to genome-wide association studies 
GigaScience  2014;3:10.
Background
The aim of a genome-wide association study (GWAS) is to isolate DNA markers for variants affecting phenotypes of interest. This is constrained by the fact that the number of markers often far exceeds the number of samples. Compressed sensing (CS) is a body of theory regarding signal recovery when the number of predictor variables (i.e., genotyped markers) exceeds the sample size. Its applicability to GWAS has not been investigated.
Results
Using CS theory, we show that all markers with nonzero coefficients can be identified (selected) using an efficient algorithm, provided that they are sufficiently few in number (sparse) relative to sample size. For heritability equal to one (h 2 = 1), there is a sharp phase transition from poor performance to complete selection as the sample size is increased. For heritability below one, complete selection still occurs, but the transition is smoothed. We find for h 2 ∼ 0.5 that a sample size of approximately thirty times the number of markers with nonzero coefficients is sufficient for full selection. This boundary is only weakly dependent on the number of genotyped markers.
Conclusion
Practical measures of signal recovery are robust to linkage disequilibrium between a true causal variant and markers residing in the same genomic region. Given a limited sample size, it is possible to discover a phase transition by increasing the penalization; in this case a subset of the support may be recovered. Applying this approach to the GWAS analysis of height, we show that 70-100% of the selected markers are strongly correlated with height-associated markers identified by the GIANT Consortium.
doi:10.1186/2047-217X-3-10
PMCID: PMC4078394  PMID: 25002967
GWAS; Genomic selection; Compressed sensing; Lasso; Underdetermined system; Sparsity; Phase transition
11.  The Development of a Sensitive and Specific ELISA for Mouse Eosinophil Peroxidase: Assessment of Eosinophil Degranulation Ex Vivo and in Models of Human Disease 
Journal of Immunological Methods  2011;375(1-2):138-147.
Mouse models of eosinophilic disorders are often part of preclinical studies investigating the underlying biological mechanisms of disease pathology. The presence of extracellular eosinophil granule proteins in affected tissues is a well established and specific marker of eosinophil activation in both patients and mouse models of human disease. Unfortunately, assessments of granule proteins in the mouse have been limited by the availability of specific antibodies and a reliance on assays of released enzymatic activities that are often neither sensitive nor eosinophil specific. The ability to immunologically detect and quantify the presence of a mouse eosinophil granule protein in biological fluids and/or tissue extracts was achieved by the generation of monoclonal antibodies specific for eosinophil peroxidase (EPX). This strategy identified unique pairs of antibodies with high avidity to the target protein and led to the development of a unique sandwich ELISA for the detection of EPX. Full factorial design was used to develop this ELISA, generating an assay that is eosinophil-specific and nearly 10 times more sensitive than traditional OPD-based detection methods of peroxidase activity. The added sensitivity afforded by this novel assay was used to detect and quantify eosinophil degranulation in several setting, including bronchoalveolar fluid from OVA sensitized/challenged mice (an animal model of asthma), serum samples derived from peripheral blood recovered from the tail vasculature, and from purified mouse eosinophils stimulated ex vivo with platelet activating factor (PAF) and PAF + ionomycin. This ability to assess mouse eosinophil degranulation represents a specific, sensitive, and reproducible assay that fulfills a critical need in studies of eosinophil-associated pathologies in mice.
doi:10.1016/j.jim.2011.10.002
PMCID: PMC3375321  PMID: 22019643
EPX; eosinophilia; granule proteins; allergic inflammation
12.  Mast cells are required for full expression of allergen/SEB-induced skin inflammation 
The Journal of investigative dermatology  2013;133(12):10.1038/jid.2013.250.
Atopic dermatitis is a chronic pruritic inflammatory skin disease. We recently described an animal model in which repeated epicutaneous applications of a house dust mite extract and staphylococcal enterotoxin B induced eczematous skin lesions. In this study we showed that global gene expression patterns are very similar between human atopic dermatitis skin and allergen/staphylococcal enterotoxin B-induced mouse skin lesions, particularly in expression of genes related to epidermal growth/differentiation, skin-barrier, lipid/energy metabolism, immune response, or extracellular matrix. In this model, mast cells and T cells, but not B cells or eosinophils, were shown to be required for the full expression of dermatitis, as revealed by reduced skin inflammation and reduced serum IgE levels in mice lacking mast cells or T cells (TCRβ−/− or Rag1−/−). The clinical severity of dermatitis correlated with the numbers of mast cells, but not eosinophils. Consistent with the idea that Th2 cells play a predominant role in allergic diseases, the receptor for the Th2-promoting cytokine thymic stromal lymphopoietin and the high-affinity IgE receptor, FcεRI, were required to attain maximal clinical scores. Therefore, this clinically relevant model provides mechanistic insights into the pathogenic mechanism of human atopic dermatitis.
doi:10.1038/jid.2013.250
PMCID: PMC3830701  PMID: 23752044
atopic dermatitis; T cell; mast cell; house dust mite; staphylococcal enterotoxin B; FcεRI; TSLP; GM-CSF
13.  Human vs. Mouse Eosinophils: “That which we call an eosinophil, by any other name would stain as red” 
The respective life histories of humans 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 physiological 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 and/or their effector functions. 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 the experimental details, comparing and contrasting eosinophils and eosinophil effector functions in humans vs. mice. In particular, our review will provide a summation and an easy to use reference guide to important studies demonstrating that while 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.
doi:10.1016/j.jaci.2012.07.025
PMCID: PMC3496419  PMID: 22935586
eosinophils; mouse; human; rodent; primate; hematology
14.  Interleukin 5 Is Protective during Sepsis in an Eosinophil-Independent Manner 
Rationale: The immune response in sepsis is characterized by overt immune dysfunction. Studies indicate immunostimulation represents a viable therapy for patients. One study suggests a potentially protective role for interleukin 5 (IL-5) in sepsis; however, the loss of eosinophils in this disease presents a paradox.
Objectives: To assess the protective and eosinophil-independent effects of IL-5 in sepsis.
Methods: We assessed the effects of IL-5 administration on survival, bacterial burden, and cytokine production after polymicrobial sepsis. In addition, we examined the effects on macrophage phagocytosis and survival using fluorescence microscopy and flow cytometry.
Measurements and Main Results: Loss of IL-5 increased mortality and tissue damage in the lung, IL-6 and IL-10 production, and bacterial burden during sepsis. Therapeutic administration of IL-5 improved mortality in sepsis. Interestingly, IL-5 administration resulted in neutrophil recruitment in vivo. IL-5 overexpression in the absence of eosinophils resulted in decreased mortality from sepsis and increased circulating neutrophils and monocytes, suggesting their importance in the protective effects of IL-5. Furthermore, novel data demonstrate IL-5 receptor expression on neutrophils and monocytes in sepsis. IL-5 augmented cytokine secretion, activation, phagocytosis, and survival of macrophages. Importantly, macrophage depletion before the onset of sepsis eliminated IL-5–mediated protection. The protective effects of IL-5 were confirmed in humans, where IL-5 levels were elevated in patients with sepsis. Moreover, neutrophils and monocytes from patients expressed the IL-5 receptor.
Conclusions: Taken together, these data support a novel role for IL-5 on noneosinophilic myeloid populations, and suggest treatment with IL-5 may be a viable therapy for sepsis.
doi:10.1164/rccm.201201-0134OC
PMCID: PMC3423456  PMID: 22652030
macrophages; neutrophils; innate immunity; immunotherapy
15.  Predicting response to bacillus Calmette-Guérin (BCG) in patients with carcinoma in situ of the bladder 
Urologic oncology  2013;32(1):45.e23-45.e30.
Purpose
Currently, there is no reliable tool to predict response to intravesical bacillus Calmette-Guérin (BCG). Based on the fact that BCG is a Th1-polarizing immunotherapy, we attempt to correlate the pretreatment immunologic tumor microenvironment (Th1 or Th2) with response to therapy.
Materials and methods
Bladder cancer patients with initial diagnosis of carcinoma in situ (Tis) were stratified based on their response to BCG treatment. A total of 38 patients met inclusion criteria (20 patients who responded and 18 patients who did not respond). Immunohistochemical (IHC) methods known to assess the type of immunologic microenvironment (Th1 vs. Th2) were performed on tumor tissue obtained at initial biopsy/resection: the level of tumor eosinophil infiltration and degranulation (Th2 response); the number of tumor-infiltrating GATA-3+ (Th2-polarized) lymphocytes; and the number of tumor-infiltrating T-bet+ (Th1-polarized) lymphocytes. Results obtained from these metrics were correlated with response to treatment with BCG immunotherapy.
Results
The IHC metrics of the tumor immune microenvironment prior to BCG treatment were each statistically significant predictors of responders (R) vs. nonresponders (NR). Eosinophil infiltration and degranulation was higher for R vs. NR: 1.02±0.17 vs. 0.5±0.12 (P = 0.01) and 1.1±0.15 vs. 0.56±0.15 (P = 0.04), respectively. Ratio of GATA-3+ (Th2-polarized) lymphocytes to T-bet+ (Th1-polarized) lymphocytes was higher for R vs. NR: 4.85±0.94 vs. 0.98±0.19 (P<0.001). The 3 markers were combined to create a Th2 signature biomarker, which was a statistically significant (P<0.0001) predictor of R vs. NR. All IHC markers demonstrated that a preexisting Th1 immunologic environment within the tumor was predictive of BCG failure.
Conclusion
The Th1 vs. Th2 polarization of bladder tumor immune microenvironment prior to treatment with BCG represents a prognostic metric of response to therapy. If a patient has a preexisting Th1 immunologic response within the tumor, there is no value in using a therapy intended to create a Th1 immunologic response. An algorithm integrating 3 IHC methods provided a sensitive and specific technique that may become a useful tool for pathologists and urologists to predict response to BCG in patients with carcinoma in situ of the bladder.
doi:10.1016/j.urolonc.2013.06.008
PMCID: PMC3953142  PMID: 24055426
Eosinophils; Tumor immune microenvironment; GATA-3; T-bet; Degranulation; Bladder cancer
16.  Eosinophils Are Important for Protection, Immunoregulation and Pathology during Infection with Nematode Microfilariae 
PLoS Pathogens  2014;10(3):e1003988.
Eosinophil responses typify both allergic and parasitic helminth disease. In helminthic disease, the role of eosinophils can be both protective in immune responses and destructive in pathological responses. To investigate whether eosinophils are involved in both protection and pathology during filarial nematode infection, we explored the role of eosinophils and their granule proteins, eosinophil peroxidase (EPO) and major basic protein-1 (MBP-1), during infection with Brugia malayi microfilariae. Using eosinophil-deficient mice (PHIL), we further clarify the role of eosinophils in clearance of microfilariae during primary, but not challenge infection in vivo. Deletion of EPO or MBP-1 alone was insufficient to abrogate parasite clearance suggesting that either these molecules are redundant or eosinophils act indirectly in parasite clearance via augmentation of other protective responses. Absence of eosinophils increased mast cell recruitment, but not other cell types, into the broncho-alveolar lavage fluid during challenge infection. In addition absence of eosinophils or EPO alone, augmented parasite-induced IgE responses, as measured by ELISA, demonstrating that eosinophils are involved in regulation of IgE. Whole body plethysmography indicated that nematode-induced changes in airway physiology were reduced in challenge infection in the absence of eosinophils and also during primary infection in the absence of EPO alone. However lack of eosinophils or MBP-1 actually increased goblet cell mucus production. We did not find any major differences in cytokine responses in the absence of eosinophils, EPO or MBP-1. These results reveal that eosinophils actively participate in regulation of IgE and goblet cell mucus production via granule secretion during nematode-induced pathology and highlight their importance both as effector cells, as damage-inducing cells and as supervisory cells that shape both innate and adaptive immunity.
Author Summary
Eosinophil recruitment is a classic characteristic of both allergic and parasitic helminth diseases. Elucidation of the role of eosinophils in these diseases is of pivotal importance for understanding the mechanisms of protection and the development of pathology. In the last few years, the part played by eosinophils in helminth-defence has been dissected using in vivo models and their importance in protection has been shown to be highly specific to the host-parasite combination. This study dissects the role of eosinophils during infection with the human lymphatic filarial parasite, Brugia malayi, which causes the major neglected tropical disease, lymphatic filariasis. In particular, we study the role of the eosinophil as a double–edged sword in generating both protection and pathology. We definitively confirm the importance of eosinophils in protection against B. malayi microfilariae and show that protection is not mediated by release of the eosinophil granule proteins, major basic protein or eosinophil peroxidase alone. Overall, we reveal that during an infection with B. malayi microfilariae, eosinophils are critical for primary protective responses. However, eosinophils contribute to nematode-induced lung dysfunction, while additionally, eosinophil granules are important negative regulators of parasite-induced lung inflammatory and some adaptive immune responses.
doi:10.1371/journal.ppat.1003988
PMCID: PMC3953434  PMID: 24626328
17.  Eosinophil deficiency compromises parasite survival in chronic nematode infection 
Immune responses elicited by parasitic worms share many features with those of chronic allergy. Eosinophils contribute to the inflammation that occurs in both types of disease and helminths can be damaged or killed by toxic products released by eosinophils in vitro. Such observations inform the widely held view that eosinophils protect the host against parasitic worms. The mouse is a natural host for Trichinella spiralis, a worm that establishes chronic infection in skeletal muscle. We tested the influence of eosinophils on T. spiralis infection in two mouse strains in which the eosinophil lineage is ablated. Eosinophils were prominent in infiltrates surrounding infected muscle cells of wild-type mice; however, in the absence of eosinophils T. spiralis muscle larvae died in large numbers. Parasite death correlated with enhanced IFN-γ and decreased IL-4 production. Larval survival improved when mice were treated with inhibitors of inducible nitric oxide synthase, implicating the nitric oxide pathway in parasite clearance. Thus, the long-standing paradigm of eosinophil toxicity in nematode infection requires reevaluation, as our results suggest that eosinophils may influence the immune response in a manner that would sustain chronic infection and insure worm survival in the host population. Such a mechanism may be deployed by other parasitic worms that depend upon chronic infection for survival.
PMCID: PMC3923382  PMID: 19155506
Eosinophils; T cells; parasitic infections
18.  The Construction of Transgenic and Gene Knockout/Knockin Mouse Models of Human Disease 
Transgenic research  2011;21(2):327-349.
The genetic and physiological similarities between mice and humans have focused considerable attention on rodents as potential models of human health and disease. Together with the wealth of resources, knowledge, and technologies surrounding the mouse as a model system, these similarities have propelled this species to the forefront of biomedical research. The advent of genomic manipulation has quickly led to the creation and use of genetically engineered mice as powerful tools for cutting edge studies of human disease research, including the discovery, refinement, and utility of many currently available therapeutic regimes. In particular, the creation of genetically modified mice as models of human disease has remarkably changed our ability to understand the molecular mechanisms and cellular pathways underlying disease states. Moreover, the mouse models resulting from gene transfer technologies have been important components correlating an individual’s gene expression profile to the development of disease pathologies. The objective of this review is to provide physician-scientists with an expansive historical and logistical overview of the creation of mouse models of human disease through gene transfer technologies. Our expectation is that this will facilitate on-going disease research studies and may initiate new areas of translational research leading to enhanced patient care.
doi:10.1007/s11248-011-9537-3
PMCID: PMC3516403  PMID: 21800101
Animal Model; Biomedical Research; Genetic Engineering; Murine; Rodent
19.  Update on clinical and immunological features of eosinophilic gastrointestinal diseases 
Purpose of review
Eosinophilic gastrointestinal diseases (EGIDs) are an increasingly common heterogeneous group of intestinal diseases. The purpose of this review is to present the latest developments in the care of patients with EGIDs and to summarize a growing literature defining the clinical features and mechanistic elements of eosinophils and their complex relationships with the gastrointestinal tract.
Recent findings
Recent studies continue to define what constitutes ‘normal’ and ‘abnormal’ numbers of eosinophils in the different sections of the gastrointestinal tract. Symptom complexes of EGIDs appear to be related primarily to the mucosal, as opposed to the muscular or serosal, forms of EGIDs. Dissection of the mucosal microenvironment is uncovering a complex array of cells, other than eosinophils, that likely contribute to the inflammatory response associated with EGIDs. Mechanistic studies have identified genetic perturbations (eotaxin-3, thymic stromal lymphopoietin, IL-13, and filaggrin) that may also contribute to the development of the most often encountered and well studied EGID, eosinophilic esophagitis.
Summary
Clinicians should remain aware of EGIDs as a diagnostic possibility for patients with common gastrointestinal symptoms. Additional research is needed to determine mechanistic processes leading to dysfunction associated with eosinophilic gastrointestinal inflammation.
doi:10.1097/MOG.0b013e32834b314c
PMCID: PMC3915867  PMID: 21897226
eosinophilic esophagitis; eotaxin; filaggrin; mucosa; thymic stromal lymphopoetin
20.  Proton Pump Inhibitor Impact on Esophageal Eosinophilia 
Objective
Differentiation between the common etiologies of dense esophageal eosinophilia such as gastroesophageal reflux disease (GERD) and eosinophilic esophagitis (EoE), can be difficult. We hypothesized that histologic features may provide diagnostic clues concerning the etiology of esophageal eosinophilia.
Methods
We performed a retrospective chart review of 204 children with the diagnosis of esophagitis characterized by ≥ 15 eos/ HPF in at least one biopsy. We then restricted our analysis to subjects who had received at least 8 weeks of only proton pump inhibitors (PPIs) followed by endoscopy and who had a clinicopathologic response to this treatment. Symptoms, endoscopic findings, and pathologic descriptions were reviewed and an eosinophil peroxidase (EPX) index was determined to assess for degranulation/eosinophil activation.
Results
Of the 204 identified charts, 7 subjects identified met the inclusion criteria. Five of these 7 patients showed a clinicopathologic response to PPIs after their follow up endoscopy, (mean peak eosinophil count- 92 vs 5 eos/ HPF, and EPX index-39.2 vs 14.6, pre- and post-treatment respectively). Two patients experienced initial resolution of symptoms and esophageal eosinophilia with PPI therapy however; within 17–23 months redeveloped symptoms and esophageal eosinophilia while on PPI therapy at the time of a third endoscopy (mean peak eosinophil count- 40 vs 11 vs 36 eos / HPF, and EPX index- 44 vs 21 vs 36.5, pre-, post- and post-treatment respectively). No clinicopathologic features or degranulation patterns differentiated subjects with GERD / PPI responsive esophageal eosinophilia (PPIREE) from those who had transient response to PPI treatment.
Conclusions
No clinicopathologic features differentiated subjects who responded to PPI treatment. PPI treatment can be helpful to exclude GERD and PPIREE but long-term follow up is critical in the management of esophagitis.
doi:10.1097/MPG.0b013e3182716b7a
PMCID: PMC3552376  PMID: 23325438
Histopathology; Eosinophil; Gastroesophageal reflux disease; eosinophil peroxidase
21.  Eosinophils regulate dendritic cells and Th2 pulmonary immune responses following allergen provocation 1 
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 (DC) 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, antigen-specific T effector cell production. These effects on DCs and antigen-specific T cells did not require MHC 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 following allergen challenge.
doi:10.4049/jimmunol.1102299
PMCID: PMC3375323  PMID: 22048766
22.  Why Is It Hard to Find Genes that are Associated with Social Science Traits? Theoretical and Empirical Considerations 
American journal of public health  2013;103(0 1):S152-S166.
Though most behavioral traits are moderately to highly heritable, the genes that influence them are elusive: many published genetic associations fail to replicate. With physical traits like eye color and skin pigmentation, in contrast, several genes with large effects have been discovered and replicated. We draw on R.A. Fisher’s geometric model of adaptation to explain why traits of interest to behavioral scientists may have a genetic architecture featuring hundreds or thousands of alleles with tiny individual effects, rather than a few with large effects, and why such an architecture makes it difficult to find robust associations between traits and genes. In the absence of strong directional selection on a trait, alleles with large effect sizes will probably remain rare, and such a lack of strong directional selection is likely to characterize most traits currently of interest in social science. We evaluate these predictions via a genome-wide association study (GWAS) that carefully measured over 100 physical and behavioral traits with a sample size typical of candidate gene studies. While we replicated several known genetic associations with physical traits, we found only two associations with behavioral traits that met the nominal genome-wide significance threshold. We use the theory and findings to discuss (1) the challenges for social science genomics, particularly the likelihood that genes are connected to behavioral variation by lengthy, nonlinear, interactive causal chains; (2) the prospects for dealing with these challenges; and (3) the inherent tradeoff between two ways of meeting these challenges: increasing sample size and improving phenotype measurement.
doi:10.2105/AJPH.2013.301327
PMCID: PMC3778125  PMID: 23927501
23.  A Novel Model of TH2-polarized Chronic Ileitis: The SAMP1 Mouse 
Inflammatory bowel diseases  2010;16(5):743-752.
Background & Aims
SAMP1/Yit mice develop spontaneous, segmental, transmural ileitis recapitulating many features of Crohn’s disease (CD). The ileitic phenotype may have arisen during crosses of SAMP1 mice selected for the presence of skin lesions. We hereby describe that the original SAMP1 strain similarly develops ileitis. Our aim was to characterize the histopathological and immunological features of this model and assess its responsiveness to standard IBD therapy.
Methods
The time course of histopathological features of ileitis was assessed. Immune compartments were characterized by flow cytometry. Ileal cytokine profiles and transcription factors were determined by real-time RT-PCR. Finally, response to corticosteroid therapy and its effect on immune compartments and cellularity was evaluated.
Results
Histological features and time course of disease were conserved, compared to those reported in SAMP1/Yit strains, with similar expansion of CD19+, CD4+ and CD8+ effector (CD44highCD62Llow), and central memory lymphocytes (CD44highCD62Lhigh). However, different from SAMP1/YitFc mice, analysis of ileal cytokine profiles revealed initial TH1 polarization followed by TH2-polarized profile accompanied by prominent eosinophilia during late disease. Lastly, corticosteroids attenuated ileitis resulting in decreased lymphocyte subsets and cellularity of compartments.
Conclusions
Here we report that the ileitic phenotype of SAMP1-related strains was already present in the original SAMP1 strain. By contrast the cytokine profile within terminal ilea of SAMP1 is distinct from the mixed TH1/TH2 profile of SAMP1/YitFc mice during late disease, as it shows predominant TH2 polarization. Dissemination of these strains may advance our understanding of CD pathogenesis, which in 60% of patients involves the terminal ileum.
doi:10.1002/ibd.21148
PMCID: PMC3786705  PMID: 19856411
24.  The causal meaning of Fisher’s average effect 
Genetics research  2013;95(0):89-109.
Summary
In order to formulate the Fundamental Theorem of Natural Selection, Fisher defined the average excess and average effect of a gene substitution. Finding these notions to be somewhat opaque, some authors have recommended reformulating Fisher’s ideas in terms of covariance and regression, which are classical concepts of statistics. We argue that Fisher intended his two averages to express a distinction between correlation and causation. On this view, the average effect is a specific weighted average of the actual phenotypic changes that result from physically changing the allelic states of homologous genes. We show that the statistical and causal conceptions of the average effect, perceived as inconsistent by Falconer, can be reconciled if certain relationships between the genotype frequencies and non-additive residuals are conserved. There are certain theory-internal considerations favouring Fisher’s original formulation in terms of causality; for example, the frequency-weighted mean of the average effects equaling zero at each locus becomes a derivable consequence rather than an arbitrary constraint. More broadly, Fisher’s distinction between correlation and causation is of critical importance to gene-trait mapping studies and the foundations of evolutionary biology.
doi:10.1017/S0016672313000074
PMCID: PMC3779663  PMID: 23938113
25.  Predictive Modeling of In Vivo Response to Gemcitabine in Pancreatic Cancer 
PLoS Computational Biology  2013;9(9):e1003231.
A clear contradiction exists between cytotoxic in-vitro studies demonstrating effectiveness of Gemcitabine to curtail pancreatic cancer and in-vivo studies failing to show Gemcitabine as an effective treatment. The outcome of chemotherapy in metastatic stages, where surgery is no longer viable, shows a 5-year survival <5%. It is apparent that in-vitro experiments, no matter how well designed, may fail to adequately represent the complex in-vivo microenvironmental and phenotypic characteristics of the cancer, including cell proliferation and apoptosis. We evaluate in-vitro cytotoxic data as an indicator of in-vivo treatment success using a mathematical model of tumor growth based on a dimensionless formulation describing tumor biology. Inputs to the model are obtained under optimal drug exposure conditions in-vitro. The model incorporates heterogeneous cell proliferation and death caused by spatial diffusion gradients of oxygen/nutrients due to inefficient vascularization and abundant stroma, and thus is able to simulate the effect of the microenvironment as a barrier to effective nutrient and drug delivery. Analysis of the mathematical model indicates the pancreatic tumors to be mostly resistant to Gemcitabine treatment in-vivo. The model results are confirmed with experiments in live mice, which indicate uninhibited tumor proliferation and metastasis with Gemcitabine treatment. By extracting mathematical model parameter values for proliferation and death from monolayer in-vitro cytotoxicity experiments with pancreatic cancer cells, and simulating the effects of spatial diffusion, we use the model to predict the drug response in-vivo, beyond what would have been expected from sole consideration of the cancer intrinsic resistance. We conclude that this integrated experimental/computational approach may enhance understanding of pancreatic cancer behavior and its response to various chemotherapies, and, further, that such an approach could predict resistance based on pharmacokinetic measurements with the goal to maximize effective treatment strategies.
Author Summary
There are few treatment options for advanced pancreatic cancer. The chemotherapeutic drug Gemcitabine is routinely used, yet 95% of patients die within 5 years of diagnosis. Surprisingly, Gemcitabine experiments with pancreatic tumor cells in the laboratory dish show that most cells will be killed by this drug. It is obvious that the dish does not adequately represent the more complex condition in real tumors. We apply mathematical modeling to simulate tumor growth to try to understand how results from the laboratory could be used to predict the treatment response in real tumors. The model simulates flow of substances such as oxygen within tumors and how this flow affects the response of cells to drug treatment. We set the inputs for the model with values obtained from the laboratory experiments. The model predicts the treatment to mostly fail in real tumors regardless of the characteristics of individual cells. We confirm these results by treating real tumors in mice, showing that our integrated experimental/computational approach may improve the understanding of pancreatic cancer behavior and response to chemotherapy, and also help to optimize treatment strategies.
doi:10.1371/journal.pcbi.1003231
PMCID: PMC3777914  PMID: 24068909

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