Background: An emerging paradigm holds that resistance to the development of allergic diseases, including allergic rhinoconjunctivitis, relates to an intact epithelial/epidermal barrier during early childhood. Conceivably, the immunologic and genomic footprint of this resistance is preserved in nonatopic, nonallergic adults and is unmasked during exposure to an aeroallergen.Objective: The aim of this study was to obtain direct support of the epithelial/epidermal barrier model for allergic rhinoconjunctivitis.Methods: Twenty-three adults allergic to house dust mites (HDMs) (M+) and 15 nonsensitive, nonallergic (M-) participants completed 3-hour exposures to aerosolized HDM (Dermatophagoides pteronyssinus) powder on 4 consecutive days in an allergen challenge chamber. We analyzed: (1) peripheral blood leukocyte levels and immune responses; and (2) RNA sequencing-derived expression profiles of nasal cells, before and after HDM exposure.Results: On HDM challenge: (1) onlyM+ persons developed allergic rhinoconjunctivitis symptoms; and (2) peripheral blood leukocyte levels/responses and gene expression patterns in nasal cells were largely concordant between M+ and M- participants; gross differences in these parameters were not observed at baseline (pre-exposure). Two key differences were observed. First, peripheral blood CD4(+) and CD8(+) T-cell activation levels initially decreased in M- participants versus increased in M+ participants. Second, in M-compared with M+ participants, genes that promoted epidermal/epithelial barrier function (eg, filament- aggregating protein [filaggrin]) versus inflammation (eg, chemokines) and innate immunity (interferon) were upregulated versus muted, respectively.Conclusion: An imprint of resistance to HDM challenge in nonatopic, nonallergic adults was muted T-cell activation in the peripheral blood and inflammatory response in the nasal compartment, coupled with upregulation of genes that promote epidermal/epithelial cell barrier function.

The proceedings of a National Institutes of Health workshop and a meeting of an Advisory Committee of the US Food and Drug Administration,1U.S. Food and Drug Administration Center for Biologics Evaluation and Research: Allergenic Products Advisory Committee, May 12, 2011. Available at: http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/BloodVaccinesandOtherBiologics/AllergenicProductsAdvisoryCommittee/UCM258587.pdf. Accessed May 28, 2014.Google Scholar, 2Togias A. Asthma, Allergy, and Inflammation Branch, Division of Allergy, Immunology and Transplantation, NIAID/NIHEnvironmental exposure units: clinical trial design for validation. National Institute for Allergy and Infectious Diseases/National Institutes of Health, Bethesda2010: 1-12Google Scholar as well as related commentaries,3Bernstein J.A. Correlation between a pollen challenge chamber and a natural allergen exposure study design for eliciting ocular and nasal symptoms: early evidence supporting a paradigm shift in drug investigation?.J Allergy Clin Immunol. 2012; 130: 128-129Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 4Devillier P. Le Gall M. Horak F. The allergen challenge chamber: a valuable tool for optimizing the clinical development of pollen immunotherapy.Allergy. 2011; 66: 163-169Crossref PubMed Scopus (48) Google Scholar highlight the utility of an allergen challenge chamber (ACC) for conducting clinical trials for allergic rhinoconjunctivitis (AR). Mitigation of factors that might confound the design, analysis, and interpretation of these trials requires a systematic comparison of symptoms present in the natural setting versus those elicited after exposure to aeroallergens in an ACC. In prior studies we conducted out-of-season challenges with 3 separate pollens in patients with seasonal allergy.5Jacobs R.L. Harper N. He W. Andrews C.P. Rather C.G. Ramirez D.A. et al.Responses to ragweed pollen in a pollen challenge chamber versus seasonal exposure identify allergic rhinoconjunctivitis endotypes.J Allergy Clin Immunol. 2012; 130: 122-127.e8Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 6Jacobs R.L. Harper N. He W. Andrews C.P. Rather C.G. Ramirez D.A. et al.Effect of confounding cofactors on responses to pollens during natural season versus pollen challenge chamber exposure.J Allergy Clin Immunol. 2014; 133 (e1-7): 1340-1346Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar Although there was high correlation in symptom scores in the natural pollination seasons and ACC, competing environmental influences (eg, mold) in the natural setting blunted the responsiveness to these pollens, whereas this confounder was not present in the ACC.5Jacobs R.L. Harper N. He W. Andrews C.P. Rather C.G. Ramirez D.A. et al.Responses to ragweed pollen in a pollen challenge chamber versus seasonal exposure identify allergic rhinoconjunctivitis endotypes.J Allergy Clin Immunol. 2012; 130: 122-127.e8Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 6Jacobs R.L. Harper N. He W. Andrews C.P. Rather C.G. Ramirez D.A. et al.Effect of confounding cofactors on responses to pollens during natural season versus pollen challenge chamber exposure.J Allergy Clin Immunol. 2014; 133 (e1-7): 1340-1346Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar However, most patients with allergy are polysensitized to outdoor (eg, pollens) and indoor (eg, house dust mite [HDM]) aeroallergens. HDM exposure is common, associated with perennial allergy, and a significant risk factor for AR and asthma. Here we compared the symptom dynamics in the natural setting versus the ACC in HDM-sensitive (M+) and nonsensitive (M−) participants meeting the inclusion/exclusion criteria shown in Table E1 in this article's Online Repository at www.jacionline.org. M− subjects lacked both a history of AR and skin prick test (SPT) wheal reactivity (≥5 mm) to 17 allergens, including HDM (see Table E2 in this article's Online Repository at www.jacionline.org). The study comprised 4 study phases: a 4-day run-in phase followed by 2 ACC exposure phases (ACC-I and ACC-II) with an intervening 38-day observation phase (Fig 1, A). This design allowed for evaluation of the reproducibility of symptom responses and factors that could potentially confound clinical trials in the ACC, including mediators of nocebo effects,7Bingel U. Avoiding nocebo effects to optimize treatment outcome.JAMA. 2014; 312: 693-694Crossref PubMed Scopus (141) Google Scholar as discussed in the Methods section in this article's Online Repository at www.jacionline.org. The study was conducted in the early fall, when only weed pollens were detected in San Antonio, Texas (Fig 1, A and B).6Jacobs R.L. Harper N. He W. Andrews C.P. Rather C.G. Ramirez D.A. et al.Effect of confounding cofactors on responses to pollens during natural season versus pollen challenge chamber exposure.J Allergy Clin Immunol. 2014; 133 (e1-7): 1340-1346Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar To mitigate this confounding factor, we selected M+ participants with negative SPT responses for weed pollens (see Table E2). All participants were allergy drug free throughout the study (see Table E3 in this article's Online Repository at www.jacionline.org). Each ACC phase comprised exposure for 3 hours on 4 consecutive days to a purified mite body powder of Dermatophagoides pteronyssinus (Fig 1, A and B, and see the Methods section in this article's Online Repository). The end point we targeted was an increase in instantaneous total symptom scores (iTSSs) of at least 6 units in 50% of participants from baseline levels. To achieve this goal, the ACC was calibrated to deliver 70 to 110 ng/m3 HDM Der p 1 antigen, as measured by means of ELISA (see Table E4 and the Methods section in this article's Online Repository at www.jacionline.org). iTSSs in the ACC and reflective total symptom scores (rTSSs) in the natural setting were recorded by using a 5-point Likert scale (see Table E5 in this article's Online Repository at www.jacionline.org). Of the 40 participants meeting inclusion criteria, 35 (21 M+ and 14 M− participants) completed all 4 study phases, and of these, 13 M+ participants and 1 M− participant had detectable (≥0.35 kU/L) serum specific IgE (ssIgE) to D pteronyssinus. This dichotomy between SPT reactivity but undetectable ssIgE has been reported for many allergens (see the Discussion section in this article's Online Repository at www.jacionline.org).8de Vos G. Skin testing versus serum-specific IgE testing: which is better for diagnosing aeroallergen sensitization and predicting clinical allergy?.Curr Allergy Asthma Rep. 2014; 14: 430Crossref PubMed Scopus (50) Google Scholar There were no differences in the key demographic characteristics between M+ and M− participants (see Table E6 in this article's Online Repository at www.jacionline.org). Five participants withdrew from the study for nonmedical reasons. M− participants had minimal symptoms in the ACC (mean iTSS, <1; Fig 1, B). In contrast, M+ participants experienced a mean increase of 3 units in the iTSS within 30 minutes of HDM challenge, and iTSSs reached a plateau after approximately 120 minutes (Fig 1, B). There was a high degree of concordance in symptom responses in M+ participants in the ACC recorded by using the Likert and visual analog scales (see Fig E1 in this article's Online Repository at www.jacionline.org). Eleven episodes of bronchospasm occurred in 5 M+ participants, a rate consistent with prior findings.9Horak F. Toth J. Marks B. Stubner U.P. Berger U.E. Jager S. et al.Efficacy and safety relative to placebo of an oral formulation of cetirizine and sustained-release pseudoephedrine in the management of nasal congestion.Allergy. 1998; 53: 849-856Crossref PubMed Scopus (43) Google Scholar These participants exhibited slightly higher total symptom scores (TSSs; see Fig E2 in this article's Online Repository at www.jacionline.org), had greater than 15% improvement in FEV1.0 after treatment with nebulized albuterol, and returned to the ACC without additional exacerbations (see the Discussion section in this article's Online Repository). These findings suggest that M+ participants with mild intermittent asthma can be safely evaluated within ACCs. The concordance in symptom responses during and between ACC-I and ACC-II was high (Fig 1, C, and see Table E7 in this article's Online Repository at www.jacionline.org). In contrast, the correlations between rTSSs recorded in the run-in versus observation phases or between rTSSs versus iTSSs were much lower (see Fig E3 in this article's Online Repository at www.jacionline.org). During ACC-I and ACC-II, an increase in iTSSs of 6 or greater from baseline (pre-exposure) was experienced in greater than 55% of participants (Fig 1, D). While less than 10% of participants had iTSSs of 15 or greater at baseline, 67% and 57% of M+ participants achieved iTSSs of 15 or greater in ACC-I and ACC-II, respectively (Fig 1, E). Substantial data indicate that ssIgE levels to allergens might serve as biomarkers for symptom severity.10Ciprandi G. Tosca M.A. Silvestri M. The practical role of serum allergen-specific IgE as potential biomarker for predicting responder to allergen immunotherapy.Expert Rev Clin Immunol. 2014; 10: 321-324Crossref PubMed Scopus (11) Google Scholar Accordingly, TSSs were greater in M+ participants with a detectable ssIgE level for D pteronyssinus in the ACC (Fig 2, A and B). The failure to detect such an association in the natural setting (Fig 2, A and B) might relate to variable HDM levels measured in dust from mattresses in the participants' homes (see Fig E4 and the Methods section in this article's Online Repository at www.jacionline.org). Pollen SPT reactivity (P+) stratified TSSs, with M+P+ participants having higher rTSSs and iTSSs when compared with those of M+P− participants (Fig 2, C). Levels of T-cell activation were greater in M+P+ participants compared with those seen in M+P− participants before and during ACCs (Fig 2, D, and see Fig E5 and the Methods section in this article's Online Repository at www.jacionline.org). Notably, T-cell activation has been associated with symptom responses during allergy.11Majori M. Piccoli M.L. Melej R. Pileggi V. Pesci A. Lymphocyte activation markers in peripheral blood before and after natural exposure to allergen in asthmatic patients.Respiration. 1997; 64: 45-49Crossref PubMed Scopus (7) Google Scholar Thus exposure to pollens in the months preceding the ACC exposures might have rendered M+P+ participants constitutively "primed," serving as a basis for the higher T-cell activation and symptoms in the natural and ACC settings (Fig 2, C and D). In the ACC phases the effects of pollen sensitization and ssIgE status were additive, with M+IgE+P+ participants manifesting maximal responsiveness after HDM exposure (Fig 2, E). The trigger for the constitutive priming could be winter and spring tree pollens because all M+P+ participants were reactive based on SPT reactivity to tree pollens (see Table E2). Moreover, the extended tree pollination season, which terminated a few months before the start of the ACC exposures, is typically associated with intense symptoms.6Jacobs R.L. Harper N. He W. Andrews C.P. Rather C.G. Ramirez D.A. et al.Effect of confounding cofactors on responses to pollens during natural season versus pollen challenge chamber exposure.J Allergy Clin Immunol. 2014; 133 (e1-7): 1340-1346Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar At the 2 transition points from the natural settings to the ACC (run-in → ACC-I and observation phase → ACC-II), there was a decrease in TSSs (Fig 1, Fig 2, A). This decrease was greater in those with more symptoms (ie, higher in M+P+ participants than in M+P− participants [Fig 2, F] and higher in M+IgE+ participants than in M+IgE− participants [data not shown]). This decrease might relate to (1) differences in how TSSs were recorded in the natural versus ACC settings (reflective vs instantaneous scoring, respectively); (2) learned responses secondary to increased emphasis/education by research staff in the ACC on how to accurately record TSSs; and (3) the controlled environment in the ACC (see the Discussion in this article's Online Repository). Inspection of the overall TSS trajectory indicated that after initiation of ACC exposures, there was a downward shift in the TSS (Fig 1, B). The baseline (pre-ACC) iTSS recorded before commencing challenge 1 was higher than the baseline iTSS recorded before challenges 2 and 3, and the baseline iTSS before challenges 4 through 8 were similar (Fig 1, B). Furthermore, the rTSS in the observation phase was lower than the rTSS in the run-in phase (Fig 1, B). This downward shift in baseline iTSS would give the mistaken impression that responsiveness was greater in challenges 2 and 3 and lower thereafter, when in fact the responsiveness in exposures 1 and 4 through 8 was similar (Fig 2, G). These downward shifts in TSSs did not differ by pollen SPT or ssIgE status (data not shown), and we surmise this might relate to a combination of factors: learned responses (secondary to education in the ACC) and partial clinical tolerance akin to what has been observed after repetitive exposure to allergens, including HDM (see the Discussion section in this article's Online Repository).12Woodfolk J.A. High-dose allergen exposure leads to tolerance.Clin Rev Allergy Immunol. 2005; 28: 43-58Crossref PubMed Google Scholar, 13Liu L.Y. Swenson C.A. Kelly E.A. Kita H. Jarjour N.N. Busse W.W. Comparison of the effects of repetitive low-dose and single-dose antigen challenge on airway inflammation.J Allergy Clin Immunol. 2003; 111: 818-825Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar In this study exposure to HDM concentrations used in the ACC was associated with reliable and reproducible elicitation of symptoms. SPT reactivity to pollen, which was associated with increased inflammatory status, and ssIgE levels to HDM were biological markers that correlated with symptom responses in the ACC. These 2 biomarkers stratified M+ participants as higher versus lower responders in the ACC, whereas this stratification is obscured in the natural settings. Therefore we suggest that out-of-pollination season challenges with HDM in the ACC might help mitigate the confounding of factors present in the natural setting: variable or low exposure to HDM, lack of association of ssIgE levels with rTSSs, and effects of competing environmental influences in a primed polysensitized subject. We also suggest that the other factors that could potentially confound clinical trials in an ACC (and natural setting) are nocebo7Bingel U. Avoiding nocebo effects to optimize treatment outcome.JAMA. 2014; 312: 693-694Crossref PubMed Scopus (141) Google Scholar and placebo14Enck P. Bingel U. Schedlowski M. Rief W. The placebo response in medicine: minimize, maximize or personalize?.Nat Rev Drug Discov. 2013; 12: 191-204Crossref PubMed Scopus (476) Google Scholar effects (expectations, learning process, and participant-physician communication). Confounding could occur by (1) misattribution of positive therapy effects to decrease symptom scores related to more precise symptom scoring (because of learned behavior) and/or partial clinical tolerance (because of repetitive exposure) and (2) imbalance in the proportion of high versus low responders in the treatment versus placebo arms. An example that highlights the potential for this imbalance is the observation that the effectiveness of anti-IgE therapy for asthma differed by the overall sensitization status of the trial participants.15Busse W.W. Morgan W.J. Gergen P.J. Mitchell H.E. Gern J.E. Liu A.H. et al.Randomized trial of omalizumab (anti-IgE) for asthma in inner-city children.N Engl J Med. 2011; 364: 1005-1015Crossref PubMed Scopus (732) Google Scholar Therapies might be more effective in patients with greater responsiveness to allergen exposure, a trait that can be readily identified in an ACC. Thus we surmise that mindfulness of the abovementioned confounders and use of an ACC might together facilitate detection of differences in the effects of placebo versus therapy in clinical trials, especially in exploratory studies with novel therapeutic agents when both the participant numbers and therapy effect sizes might be modest. Download .docx (.23 MB) Help with docx files Online Repository Data Download .pdf (.39 MB) Help with pdf files Fig E1 Download .pdf (.16 MB) Help with pdf files Fig E2 Download .pdf (.4 MB) Help with pdf files Fig E3 Download .pdf (.37 MB) Help with pdf files Fig E4 Download .pdf (.68 MB) Help with pdf files Fig E5

BACKGROUND:Modifiers of symptom severity in patients with allergic rhinoconjunctivitis (AR) are imprecisely characterized. The hygiene hypothesis implicates childhood microbial exposure as a protective factor. Cockroach sensitization (C+) might be a proxy for microbial exposure. OBJECTIVE:We sought to determine whether C+ assayed by means of skin prick tests influenced AR symptom severity in controlled and natural settings. METHODS:Total symptom scores (TSSs) were recorded by 21 participants with house dust mite allergy (M+) in the natural setting and during repeated exposures of 3 hours per day to house dust mite allergen in an allergen challenge chamber (ACC). In M+ participants the peripheral blood and nasal cells were assayed for T-cell activation and transcriptomic profiles (by using RNA sequencing), respectively. Participants allergic to mountain cedar (n = 21), oak (n = 34), and ragweed (n = 23) recorded TSSs during separate out-of-season exposures to these pollens (any pollen sensitization [P+]) in the ACC; a subset recorded TSSs in the pollination seasons. RESULTS:The hierarchy of TSSs (highest to lowest) among M+ participants tracked the following skin prick test sensitization statuses: M+P+C- > M+P+C+ > M+P-C- > M+P-C+. In nasal cells and peripheral blood the immune/inflammatory responses were rapidly resolved in M+P+C+ compared with M+P+C- participants. Among those allergic to pollen, C+ was associated with a lower TSS during pollen challenges and the pollination season. After aggregated analysis of all 4 ACC studies, C+ status was associated with a 2.8-fold greater likelihood of a lower TSS compared with C- status (odds ratio, 2.78; 95% CI, 1.18-6.67; P = .02). CONCLUSIONS:C+ status is associated with mitigation of AR symptom severity in adults with AR.

SignificanceLevels of CC chemokine receptor 5 (CCR5) on T cells are a critical factor influencing HIV/AIDS susceptibility. DNA methylation is an epigenetic feature associated with lower gene expression. Here we show that the DNA methylation status ofCCR5 cis-regulatory regions (cis-regions) correlates inversely with CCR5 levels on T cells. T-cell activation induces demethylation ofCCR5 cis-regions, upregulating CCR5 expression. Higher vs. lower sensitivity ofCCR5 cis-regions to undergoing T-cell activation-induced demethylation is associated with increased vs. decreased CCR5 levels. Polymorphisms inCCR5 cis-regions that are associated with increased vs. decreased HIV/AIDS susceptibility are also associated with increased vs. decreased sensitivity to activation-induced demethylation. Thus, interactions among T-cell activation,CCR5epigenetics, and genetics influence CCR5 levels on T cells and, by extension, HIV/AIDS susceptibility.

This study was performed to determine the operational characteristics of the Biogenics Research Chamber for elicitation of symptoms in dust mite allergic individuals. Twenty-five dust mite sensitive and 15 normal controls (32% male, mean age 40) were enrolled to undergo 4 consecutive 3 hour chamber exposures to a milled, purified mite body preparation of Dermatophagoides pteronyssinus. A 5-day run-in assessed symptoms in the natural setting. Symptoms were monitored at baseline and 30 minute intervals. Airborne samples were collected from 5 stations for 10 minutes at hourly intervals through a modified nylon filter for measurement of Der p1 by ELISA, and an Allergenco cassette sampler for microscopic evaluation of mite particles. Thirty-eight participants completed 4 consecutive chamber exposures. Mean Der p1 levels on days 1, 2, 3 and 4 in the chamber were 81, 78, 110, and 101 ng/m3, respectively. 70% of sensitive subjects had Maximum Total Nasal Symptom Scores of ≥9 (out of 16). 65% of sensitive participants had Maximum Total Symptom Scores (TSS) of ≥15 (out of 28). Three of 15 normal controls had TSS of 1, 2, and 4, and the remaining did not exhibit any symptoms. Administration of dust mite preparations containing an average of 92 ng/m3 of Der p1 levels induced symptoms in 70% of sensitive participants. The delivery and dispersal system of the Biogenics Chamber with dust mite was as efficient as previously reported for various pollens. This elicitation response will allow for testing of novel pharmacological agents.

RationaleThe effect of exposure to Mt. Cedar pollen in a pollen challenge chamber (PCC) on white blood cell (WBC) counts and cytokines is unknown.MethodsTwenty-two Mt. Cedar positive subjects (MCPS) and 11 Mt. Cedar negative subjects (MCNS) were challenged for Mt. Cedar in a PCC for 3 hours per day for 2 days. With exposure, total symptom scores (TSS) were calculated. Blood pre-challenge and following the second challenge was analyzed.ResultsIn MCPS, there was an increase in neutrophils, lymphocytes, monocytes, and eosinophils with exposure (P = 0.004, 1.50x10-11, 3.59x10-11, 1.04x10‑7). In MCNS, lymphocytes and eosinophils increased upon exposure (P = 2.05x10-6, 0.012). Comparing MCPS to MCNS, the changes in MCPS for both monocytes and eosinophils were significantly larger (P = 0.048, 0.022). After exposure,13 out of the 38 cytokines analyzed (IFNa2, BAC1, MCP4, I.309, CTACK, IFNg, IL10, IL-12p70, IL-1b, IL-2, IL-4, IL-5, IL-7) had significant increases in MCPS while only 3 (IL-10, Eotaxin-2, TRAIL) had significant decreases. In MCPS, the fold change of eosinophils, Eotaxin, MDC, and TRAIL after exposure were significantly associated with TSS (r2 = 0.29, 0.34, 0.39, 0.34). Although none of the cytokines had a significant change in MCNS, 32 had a concordant direction of change with MCPS after pollen exposure.ConclusionsChanges in both cell counts and cytokine levels characteristic of a Th2 response were observed in MCPS. MCNS had minimal symptoms, but also experienced concordant changes in cell counts and cytokine levels, although at lower levels than MCPS. RationaleThe effect of exposure to Mt. Cedar pollen in a pollen challenge chamber (PCC) on white blood cell (WBC) counts and cytokines is unknown. The effect of exposure to Mt. Cedar pollen in a pollen challenge chamber (PCC) on white blood cell (WBC) counts and cytokines is unknown. MethodsTwenty-two Mt. Cedar positive subjects (MCPS) and 11 Mt. Cedar negative subjects (MCNS) were challenged for Mt. Cedar in a PCC for 3 hours per day for 2 days. With exposure, total symptom scores (TSS) were calculated. Blood pre-challenge and following the second challenge was analyzed. Twenty-two Mt. Cedar positive subjects (MCPS) and 11 Mt. Cedar negative subjects (MCNS) were challenged for Mt. Cedar in a PCC for 3 hours per day for 2 days. With exposure, total symptom scores (TSS) were calculated. Blood pre-challenge and following the second challenge was analyzed. ResultsIn MCPS, there was an increase in neutrophils, lymphocytes, monocytes, and eosinophils with exposure (P = 0.004, 1.50x10-11, 3.59x10-11, 1.04x10‑7). In MCNS, lymphocytes and eosinophils increased upon exposure (P = 2.05x10-6, 0.012). Comparing MCPS to MCNS, the changes in MCPS for both monocytes and eosinophils were significantly larger (P = 0.048, 0.022). After exposure,13 out of the 38 cytokines analyzed (IFNa2, BAC1, MCP4, I.309, CTACK, IFNg, IL10, IL-12p70, IL-1b, IL-2, IL-4, IL-5, IL-7) had significant increases in MCPS while only 3 (IL-10, Eotaxin-2, TRAIL) had significant decreases. In MCPS, the fold change of eosinophils, Eotaxin, MDC, and TRAIL after exposure were significantly associated with TSS (r2 = 0.29, 0.34, 0.39, 0.34). Although none of the cytokines had a significant change in MCNS, 32 had a concordant direction of change with MCPS after pollen exposure. In MCPS, there was an increase in neutrophils, lymphocytes, monocytes, and eosinophils with exposure (P = 0.004, 1.50x10-11, 3.59x10-11, 1.04x10‑7). In MCNS, lymphocytes and eosinophils increased upon exposure (P = 2.05x10-6, 0.012). Comparing MCPS to MCNS, the changes in MCPS for both monocytes and eosinophils were significantly larger (P = 0.048, 0.022). After exposure,13 out of the 38 cytokines analyzed (IFNa2, BAC1, MCP4, I.309, CTACK, IFNg, IL10, IL-12p70, IL-1b, IL-2, IL-4, IL-5, IL-7) had significant increases in MCPS while only 3 (IL-10, Eotaxin-2, TRAIL) had significant decreases. In MCPS, the fold change of eosinophils, Eotaxin, MDC, and TRAIL after exposure were significantly associated with TSS (r2 = 0.29, 0.34, 0.39, 0.34). Although none of the cytokines had a significant change in MCNS, 32 had a concordant direction of change with MCPS after pollen exposure. ConclusionsChanges in both cell counts and cytokine levels characteristic of a Th2 response were observed in MCPS. MCNS had minimal symptoms, but also experienced concordant changes in cell counts and cytokine levels, although at lower levels than MCPS. Changes in both cell counts and cytokine levels characteristic of a Th2 response were observed in MCPS. MCNS had minimal symptoms, but also experienced concordant changes in cell counts and cytokine levels, although at lower levels than MCPS.

Background Chemokines and their receptors play a role in the innate immune response as well as in the disruption of the balance between pro-inflammatory Th17 cells and regulatory T cells (Treg), underlying the pathogenesis of coronary vasculitis in Kawasaki disease (KD). Results Here we show that genetic inactivation of chemokine receptor (CCR)-2 is protective against the induction of aortic and coronary vasculitis following injection of Candida albicans water-soluble cell wall extracts (CAWS). Mechanistically, both T and B cells were required for the induction of vasculitis, a role that was directly modulated by CCR2. CAWS administration promoted mobilization of CCR2-dependent inflammatory monocytes (iMo) from the bone marrow (BM) to the periphery as well as production of IL-6. IL-6 was likely to contribute to the depletion of Treg and expansion of Th17 cells in CAWS-injected Ccr2 +/+ mice, processes that were ameliorated following the genetic inactivation of CCR2. Conclusion Collectively, our findings provide novel insights into the role of CCR2 in the pathogenesis of vasculitis as seen in KD and highlight novel therapeutic targets, specifically for individuals resistant to first-line treatments.

Objective: Dendritic cells (DCs) have long been recognized as potential therapeutic targets of rheumatoid arthritis (RA). Increasing evidence has showed that DCs are capable of suppressing autoimmunity by expanding FoxP3(+) regulatory T cells (T g), which in turn exert immunosuppression by increasing TGF beta-1. In the SKG mice, activated DC prime autoreactive T cells causing autoantibody production and an inflammatory arthritic response. Recently, we reported that CC-chemokine receptor-2 deficient (Ccr2(-/-)) mice had impaired DCs migration and reduced CD8 alpha(+) DCs in the C57B1/6J mice strain and that these mice were more susceptible to collagen antibody-induced arthritis (CAIA), compared to wild type mice. To examine the mechanism by which DCs contribute to the increased susceptibility of arthritis in Ccr2(-/-)mice, we tested the hypothesis that CD8 alpha(+) DCs are protective (tolerogenic) against autoimmune arthritis by examining the role of CD8 alpha(+) DCs in Ccr2(-/-) and SKG mice.Methods: To examine the mechanism by which DCs defects lead to the development of arthritis, we used two murine models of experimental arthritis: collagen-induced arthritis (CIA) in DBA1/J mice and zymosan-induced arthritis in SKG mice. DBA1/J mice received recombinant fms-like tyrosine kinase 3 ligand (Flt3L) injections to expand endogenous DCs populations or adoptive transfers of CD8 alpha(+) DCs.Results: Flt3L-mediated expansion of endogenous CD8 alpha(+) DCs resulted in heightened susceptibility of CIA. In contrast, supplementation with exogenous CD8 alpha(+) DCs ameliorated arthritis in Ccr2(-/-) mice and enhanced TGF beta 1 production by T cells. Furthermore, SKG mice with genetic inactivation of CCR2 did not affect the numbers of DCs nor improve the arthritis phenotype.Conclusion: CD8 alpha(+) DCs were tolerogenic to the development of arthritis. CD8 alpha(+) DCs deficiency heightened the sensitivity to arthritis in Ccr2(-/-) mice. Ccr2 deficiency did not alter the arthritic phenotype in SKG mice suggesting the arthritis in Ccr2(-/-) mice was T cell-independent. Published by Elsevier GmbH.

Post-myocardial infarction (MI), chemokine homing of inflammatory cells into the injured left ventricle (LV) regulates ventricular remodeling, in part by stimulating the extracellular matrix response. The CC chemokine receptor 5 (CCR5) is a key chemokine receptor expressed on macrophages, and CCR5 ligands are highly upregulated post-MI. We hypothesized that deletion of CCR5 would attenuate adverse remodeling by decreasing inflammatory cell recruitment. Accordingly, we examined LV function, macrophage recruitment and activation, and collagen content in wild-type (WT, n = 25) and CCR5 null (n = 33) mice at 7 days post-MI. Both groups had similar infarct sizes (44 ± 2% in WT and 42 ± 2% in CCR5 null; P = 0.37). However, the LV remodeling index (end diastolic volume/LV mass) increased to a larger extent in CCR5 null (1.28 ± 0.08 μl/mg for CCR5 null and 1.02 ± 0.06 μl/mg for WT; P < 0.05). Although numbers of infiltrated macrophages were similar in WT and CCR5 null mice, CCR5-deficient macrophages isolated from the infarct zone displayed >50% decrease in gene expression levels of proinflammatory activation markers (interleukin-1β, interleukin-6, and tumor necrosis factor-α), as well as anti-inflammatory activation markers (arginase 1, CD163, mannose receptor, and transforming growth factor-β1) compared with WT (all P < 0.05). Concomitant with the reduced macrophage activation, heat shock protein-47 and collagen type I precursor levels in the infarct region decreased in the CCR5 null (1.2 ± 0.3 units in the CCR5 null and 2.3 ± 0.4 units in the WT; P < 0.05), while collagen fragments increased (88.3 ± 5.9 units in the CCR5 null and 32.7 ± 8.5 units in the WT; P < 0.05). We conclude that CCR5 deletion impairs LV remodeling by hindering macrophage activation, which stimulates an imbalance in collagen metabolism and increases the remodeling index.

The Standard measures of experimental arthritis fail to detect, visualize, and quantify early inflammation and disease activity. Here, we describe the use of an injectable MMP-activated fluorescence agent for in vivo quantification of acute inflammation produced by collagen-antibody-induced arthritis (CAIA) in CC chemokine receptor-2 (Ccr2−/−) null mice. Although Ccr2−/− DBA1/J mice were highly susceptible to and rapidly developed CAIA, the standard clinical assessment of fore or hind paw thicknesses was unable to detect significant acute inflammatory changes (days 3–10). Remarkably, noninvasive, in situ, MMP-activatable fluorescent imaging of Ccr2−/− DBA1/J mice with CAIA displayed acute joint pathology in advance of clinically measurable acute inflammation (days 5, 7, and 10). These results were confirmed by the histology of ankle joints, which showed significant inflammation, bone loss, and synovial hyperplasia, compared to control mice at postimmunization day 5. The MMP-mediated fluorescence technique holds tremendous implications for quantifiable examination of arthritis disease activity of acute joint inflammation.

Post-myocardial infarction (MI), chemokine homing of inflammatory cells into injured myocardium regulates ventricular remodeling, in part by stimulating the extracellular matrix (ECM) response. The...

The CC Chemokine Receptor 5 (CCR5) is a key chemokine receptor expressed on macrophages, and CCR5 ligands are highly upregulated post‐MI. However, the role of CCR5 in post‐MI remodeling of the left ventricle (LV) has not been investigated. Accordingly, we examined LV function, plasma inflammatory markers, macrophage infiltration, matrix metalloproteinase‐9 (MMP‐9), and collagen content in wild‐type mice (WT, n=25) and CCR5 null mice (Null, n=33) at 7 days post‐MI. While MI sizes were similar between the groups (44±2% in WT and 42±2% in Null), the LV remodeling index was higher in the CCR5 null mice. The higher LV remodeling index was associated with attenuated plasma IL‐6, IFN‐γ and TNFα levels and lower LV macrophage numbers (7±1% of total infarct area compared to 11±1% in WT; p<0.05). In addition, the concomitant increases of MMP‐9 and crosslinked collagen in LV infarcts were not observed in the CCR5 null mice. These results indicated that CCR5 deletion imparted a net negative effect post‐MI by decreasing macrophage numbers to impair the inflammatory response, reduce collagen content, and increase the remodeling index. NIH (R01 HL‐75360), AHA (0855119F) to M.L.L., AHA (09POST2150178) to R.Z and VA Merit to S.S.A.

We postulated that CCR2-driven activation of the transcription factor NF-kappaB plays a critical role in dendritic cell (DC) maturation (e.g., migration, costimulation, and IL-12p70 production), necessary for the generation of protective immune responses against the intracellular pathogen Leishmania major. Supporting this notion, we found that CCR2, its ligand CCL2, and NF-kappaB were required for CCL19 production and adequate Langerhans cell (LC) migration both ex vivo and in vivo. Furthermore, a role for CCR2 in upregulating costimulatory molecules was indicated by the reduced expression of CD80, CD86, and CD40 in Ccr2(-/-) bone marrow-derived dendritic cells (BMDCs) compared with wild-type (WT) BMDCs. Four lines of evidence suggested that CCR2 plays a critical role in the induction of protective immunity against L. major by regulating IL-12p70 production and migration of DC populations such as LCs. First, compared with WT, Ccr2(-/-) lymph node cells, splenocytes, BMDCs, and LCs produced lower levels of IL-12p70 following stimulation with LPS/IFN-gamma or L. major. Second, a reduced number of LCs carried L. major from the skin to the draining lymph nodes in Ccr2(-/-) mice compared with WT mice. Third, early treatment with exogenous IL-12 reversed the susceptibility to L. major infection in Ccr2(-/-) mice. Finally, disruption of IL-12p70 in radioresistant cells, such as LCs, but not in BMDCs resulted in the inability to mount a fully protective immune response in bone marrow chimeric mice. Collectively, our data point to an important role for CCR2-driven activation of NF-kappaB in the regulation of DC/LC maturation processes that regulate protective immunity against intracellular pathogens.

The inflammatory response following a myocardial infarction (MI) regulates multiple components of the wound healing process. In particular, macrophages regulate wound debridement, scar formation, and neovascularization. The CC Chemokine Receptor 5 (CCR5) is a key chemokine receptor expressed on macrophages, and CCR5 ligands are highly upregulated post‐MI. However, the roles of CCR5 post‐MI have not been investigated. Accordingly, we examined macrophage infiltration and collagen concentration in the left ventricle (LV) and plasma inflammatory markers in wild‐type mice (wt, n=25) and CCR5 null mice (null, n=33). The groups had similar infarct sizes (44±2% in wt and 42±2% in null, p= n.s.). Macrophage infiltration was lower in the null (7±1% of total infarct area compared to 11±1% in wt; p<0.05). The collagen insoluble to soluble ratio was 36.7% higher in the null (p<0.05), suggesting reduced collagen synthesis. In addition, plasma levels of IL‐6, IFN‐ ? and TNFá increased in wt, but not in null mice, post‐MI (p<0.05). We conclude that CCR5 deletion has a net negative effect on remodeling post‐MI, as evidenced by a decreased inflammatory response and collagen synthesis. NIH (R01 HL‐75360) and American Heart Association grant (0855119F) to M.L.L. and T32 (HL07446) to R.Z.

Atherosclerosis is a progressive disease characterized by the accumulation of lipids and fibrous elements in the arteries and is a leading cause of heart disease and stroke in developed and developing countries.1 Animal models have become increasingly important tools for addressing key mechanistic and therapeutic questions that cannot be answered from human studies of atherosclerosis. However, the small-scale vascular structures in genetically engineered mice require labor-intensive histomorphometric techniques to quantify lesions. Recently, a new technique has emerged to image ex vivo blocks of soft tissue by staining tissue with metal solutions, then scanning with a microscopic computed tomography (microCT) instrument (Figure I in the online-only Data Supplement).2 This technique was originally applied to the study of the developing heart in embryos3 and fetuses (Figure II in the online-only Data Supplement) but can also be applied to the en bloc imaging of the heart, great vessels, and lesions thereof. By this method, tissues are left intact, but one can employ image analysis to create “virtual” histological …

HomeCirculationVol. 120, No. 9Microscopic Computed Tomography–Based Virtual Histology for Visualization and Morphometry of Atherosclerosis in Diabetic Apolipoprotein E Mutant Mice Free AccessReview ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissionsDownload Articles + Supplements ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toSupplementary MaterialsFree AccessReview ArticlePDF/EPUBMicroscopic Computed Tomography–Based Virtual Histology for Visualization and Morphometry of Atherosclerosis in Diabetic Apolipoprotein E Mutant Mice Hernan G. Martinez, MD, Suresh I. Prajapati, MSc, Carlos A. Estrada, MD, Fabio Jimenez, BSc, Marlon P. Quinones, MD, Isabel Wu, BSc, Ali Bahadur, MSc, Allen Sanderson, PhD, Christopher R. Johnson, PhD, Minsub Shim, PhD, Charles Keller, MD and Seema S. Ahuja, MD Hernan G. MartinezHernan G. Martinez From the Departments of Medicine (H.G.M., C.A.E., F.J., M.P.Q., S.S.A.), Pediatrics (C.K.), and Cellular and Structural Biology (C.K.) and Greehey Children's Cancer Research Institute (S.I.P., I.W., A.B., C.K.), University of Texas Health Science Center, San Antonio; Scientific Computing and Imaging Institute, University of Utah, Salt Lake City (A.S., C.R.J.); National Institute of Environmental Health Science, Research Triangle Park, NC (M.S.); and South Texas Veterans Health Care System, Audie L. Murphy Division, San Antonio (S.S.A.). , Suresh I. PrajapatiSuresh I. Prajapati From the Departments of Medicine (H.G.M., C.A.E., F.J., M.P.Q., S.S.A.), Pediatrics (C.K.), and Cellular and Structural Biology (C.K.) and Greehey Children's Cancer Research Institute (S.I.P., I.W., A.B., C.K.), University of Texas Health Science Center, San Antonio; Scientific Computing and Imaging Institute, University of Utah, Salt Lake City (A.S., C.R.J.); National Institute of Environmental Health Science, Research Triangle Park, NC (M.S.); and South Texas Veterans Health Care System, Audie L. Murphy Division, San Antonio (S.S.A.). , Carlos A. EstradaCarlos A. Estrada From the Departments of Medicine (H.G.M., C.A.E., F.J., M.P.Q., S.S.A.), Pediatrics (C.K.), and Cellular and Structural Biology (C.K.) and Greehey Children's Cancer Research Institute (S.I.P., I.W., A.B., C.K.), University of Texas Health Science Center, San Antonio; Scientific Computing and Imaging Institute, University of Utah, Salt Lake City (A.S., C.R.J.); National Institute of Environmental Health Science, Research Triangle Park, NC (M.S.); and South Texas Veterans Health Care System, Audie L. Murphy Division, San Antonio (S.S.A.). , Fabio JimenezFabio Jimenez From the Departments of Medicine (H.G.M., C.A.E., F.J., M.P.Q., S.S.A.), Pediatrics (C.K.), and Cellular and Structural Biology (C.K.) and Greehey Children's Cancer Research Institute (S.I.P., I.W., A.B., C.K.), University of Texas Health Science Center, San Antonio; Scientific Computing and Imaging Institute, University of Utah, Salt Lake City (A.S., C.R.J.); National Institute of Environmental Health Science, Research Triangle Park, NC (M.S.); and South Texas Veterans Health Care System, Audie L. Murphy Division, San Antonio (S.S.A.). , Marlon P. QuinonesMarlon P. Quinones From the Departments of Medicine (H.G.M., C.A.E., F.J., M.P.Q., S.S.A.), Pediatrics (C.K.), and Cellular and Structural Biology (C.K.) and Greehey Children's Cancer Research Institute (S.I.P., I.W., A.B., C.K.), University of Texas Health Science Center, San Antonio; Scientific Computing and Imaging Institute, University of Utah, Salt Lake City (A.S., C.R.J.); National Institute of Environmental Health Science, Research Triangle Park, NC (M.S.); and South Texas Veterans Health Care System, Audie L. Murphy Division, San Antonio (S.S.A.). , Isabel WuIsabel Wu From the Departments of Medicine (H.G.M., C.A.E., F.J., M.P.Q., S.S.A.), Pediatrics (C.K.), and Cellular and Structural Biology (C.K.) and Greehey Children's Cancer Research Institute (S.I.P., I.W., A.B., C.K.), University of Texas Health Science Center, San Antonio; Scientific Computing and Imaging Institute, University of Utah, Salt Lake City (A.S., C.R.J.); National Institute of Environmental Health Science, Research Triangle Park, NC (M.S.); and South Texas Veterans Health Care System, Audie L. Murphy Division, San Antonio (S.S.A.). , Ali BahadurAli Bahadur From the Departments of Medicine (H.G.M., C.A.E., F.J., M.P.Q., S.S.A.), Pediatrics (C.K.), and Cellular and Structural Biology (C.K.) and Greehey Children's Cancer Research Institute (S.I.P., I.W., A.B., C.K.), University of Texas Health Science Center, San Antonio; Scientific Computing and Imaging Institute, University of Utah, Salt Lake City (A.S., C.R.J.); National Institute of Environmental Health Science, Research Triangle Park, NC (M.S.); and South Texas Veterans Health Care System, Audie L. Murphy Division, San Antonio (S.S.A.). , Allen SandersonAllen Sanderson From the Departments of Medicine (H.G.M., C.A.E., F.J., M.P.Q., S.S.A.), Pediatrics (C.K.), and Cellular and Structural Biology (C.K.) and Greehey Children's Cancer Research Institute (S.I.P., I.W., A.B., C.K.), University of Texas Health Science Center, San Antonio; Scientific Computing and Imaging Institute, University of Utah, Salt Lake City (A.S., C.R.J.); National Institute of Environmental Health Science, Research Triangle Park, NC (M.S.); and South Texas Veterans Health Care System, Audie L. Murphy Division, San Antonio (S.S.A.). , Christopher R. JohnsonChristopher R. Johnson From the Departments of Medicine (H.G.M., C.A.E., F.J., M.P.Q., S.S.A.), Pediatrics (C.K.), and Cellular and Structural Biology (C.K.) and Greehey Children's Cancer Research Institute (S.I.P., I.W., A.B., C.K.), University of Texas Health Science Center, San Antonio; Scientific Computing and Imaging Institute, University of Utah, Salt Lake City (A.S., C.R.J.); National Institute of Environmental Health Science, Research Triangle Park, NC (M.S.); and South Texas Veterans Health Care System, Audie L. Murphy Division, San Antonio (S.S.A.). , Minsub ShimMinsub Shim From the Departments of Medicine (H.G.M., C.A.E., F.J., M.P.Q., S.S.A.), Pediatrics (C.K.), and Cellular and Structural Biology (C.K.) and Greehey Children's Cancer Research Institute (S.I.P., I.W., A.B., C.K.), University of Texas Health Science Center, San Antonio; Scientific Computing and Imaging Institute, University of Utah, Salt Lake City (A.S., C.R.J.); National Institute of Environmental Health Science, Research Triangle Park, NC (M.S.); and South Texas Veterans Health Care System, Audie L. Murphy Division, San Antonio (S.S.A.). , Charles KellerCharles Keller From the Departments of Medicine (H.G.M., C.A.E., F.J., M.P.Q., S.S.A.), Pediatrics (C.K.), and Cellular and Structural Biology (C.K.) and Greehey Children's Cancer Research Institute (S.I.P., I.W., A.B., C.K.), University of Texas Health Science Center, San Antonio; Scientific Computing and Imaging Institute, University of Utah, Salt Lake City (A.S., C.R.J.); National Institute of Environmental Health Science, Research Triangle Park, NC (M.S.); and South Texas Veterans Health Care System, Audie L. Murphy Division, San Antonio (S.S.A.). and Seema S. AhujaSeema S. Ahuja From the Departments of Medicine (H.G.M., C.A.E., F.J., M.P.Q., S.S.A.), Pediatrics (C.K.), and Cellular and Structural Biology (C.K.) and Greehey Children's Cancer Research Institute (S.I.P., I.W., A.B., C.K.), University of Texas Health Science Center, San Antonio; Scientific Computing and Imaging Institute, University of Utah, Salt Lake City (A.S., C.R.J.); National Institute of Environmental Health Science, Research Triangle Park, NC (M.S.); and South Texas Veterans Health Care System, Audie L. Murphy Division, San Antonio (S.S.A.). Originally published1 Sep 2009https://doi.org/10.1161/CIRCULATIONAHA.108.829531Circulation. 2009;120:821–822Atherosclerosis is a progressive disease characterized by the accumulation of lipids and fibrous elements in the arteries and is a leading cause of heart disease and stroke in developed and developing countries.1 Animal models have become increasingly important tools for addressing key mechanistic and therapeutic questions that cannot be answered from human studies of atherosclerosis. However, the small-scale vascular structures in genetically engineered mice require labor-intensive histomorphometric techniques to quantify lesions.Recently, a new technique has emerged to image ex vivo blocks of soft tissue by staining tissue with metal solutions, then scanning with a microscopic computed tomography (microCT) instrument (Figure I in the online-only Data Supplement).2 This technique was originally applied to the study of the developing heart in embryos3 and fetuses (Figure II in the online-only Data Supplement) but can also be applied to the en bloc imaging of the heart, great vessels, and lesions thereof. By this method, tissues are left intact, but one can employ image analysis to create "virtual" histological sections that allow the identification of an individual lesion in a 2-dimensional image. Furthermore, the 3-dimensional nature of microCT scans permits the volumetric assessment of multifocal atherosclerotic lesions.Shown in Figure 1 are excised hearts from an apolipoprotein E knockout mouse (ApoE null), a well-established murine model for atherosclerosis, as well as a control mouse. Both mice are on a diabetic background strain, KK-Ay, maintained on a standard diet. ApoE null mice are spontaneously hypercholesterolemic and develop fatty streaks as well as advanced lesions at aortic sites where atherosclerosis is also typically seen in humans.4 In these coronal virtual histological sections, the ventricular myocardium and atria can be appreciated (Figure 1A through 1D), as well as atherosclerotic lesions of the aortic root (Figure 1B) and ascending aorta (Figure 1D). After microCT-based virtual histology scans, the ApoE null aortic root was sectioned by traditional histology for staining of atherosclerotic lesions with Oil Red O, which stains the fatty plaque red (Figure 2A), thereby confirming the atherosclerotic lesion seen by virtual histology. With higher-resolution (1.8 μm) microCT instrument scanning, these lesions are even more readily visualized (Figure III and Movies I through III in the online-only Data Supplement). Download figureDownload PowerPointFigure 1. Coronal microCT-based virtual histology sections of wild-type (WT) mice (A and C) and ApoE null mice (B and D) (28-week-old females). Specimens were stained with a precommercial staining solution (Numira Biosciences, Salt Lake City, Utah) and scanned in a Scanco μCT40 instrument at 6 μm isometric resolution. Insets E, F, G, and H correspond to yellow boxed regions in A, B, C, and D, respectively. Insets of the aortic leaflet (F) and ascending aorta (H) demonstrate atherosclerotic plaques (bracketed by yellow lines) in the ApoE null mice. Bar=1 mm.Download figureDownload PowerPointFigure 2. Oil Red O histological stain (red) (A) and microCT-based virtual histology scan (B) of the same atherosclerotic lesion from an ApoE null mouse. The yellow dotted lines highlight the area of atherosclerotic plaque in the aortic leaflet region. Bar=250 μm.An advantage of microCT-based virtual histology is that the data are entirely digital and not subject to loss of individual sections in handling or improper plane of sectioning. Furthermore, freely available software tools exist for the visualization of natural edge boundary features of 3-dimensional tissues (Figure 3, left) as well as volume quantification of atherosclerotic lesions at multiple foci to microliter accuracy (Figure 3, right) (Seg3D, http://www.sci.utah.edu/cibc/software). Download figureDownload PowerPointFigure 3. Left and right, Renderings of the same ApoE null mouse heart from different angles, demonstrating atherosclerotic plaques (red) that were segmented by image analysis and their volumes quantified. Total volume refers to the entire volume of the red region, which comprises several regions including 1, 2, and 3 (shown by dotted boxes). Purple indicates myocardium; green, atria and vessels. Bar=1 mm.Thus, evaluation of mechanisms underlying atherosclerosis and interventions can be accelerated by this time-saving, higher-accuracy research imaging technique. In addition, because microCT-based virtual histology is inherently digital, data analysis for virtual histology is poised to take advantage of an emerging new generation of powerful software tools such as those mentioned above, which will further speed vascular disease research.The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/120/9/821/DC1.*Drs Martinez and Prajapati contributed equally to this work and†Drs Keller and Ahuja contributed equally to this work.AcknowledgmentsWe thank Itzik Goldberger and Tiffany Fong for the scans graciously provided by Xradia Inc.Sources of FundingThis research was supported by the Veterans Administration (Merit Review) and National Institutes of Health grant R01 AR 052755 to Dr Ahuja. microCT scans were performed at Numira Biosciences (Salt Lake City, Utah) through a reciprocal scanning agreement. This work was made possible in part by software from the National Institutes of Health/National Center for Research Resources Center for Integrative Biomedical Computing (5P41RR012553).DisclosuresDr Keller is cofounder of Numira Biosciences (http://www.numirabio.com), which licenses microCT-based virtual histology from the University of Texas Health Science Center at San Antonio for commercial applications. The other authors report no conflicts.FootnotesCorrespondence to Charles Keller, MD, or Seema S. Ahuja, MD, Division of Nephrology, Department of Medicine, University of Texas Health Science Center, 7703 Floyd Curl Dr, MC7870, San Antonio, TX 78229-3900. E-mail [email protected]References1 Libby P. Inflammation in atherosclerosis. Nature. 2002; 420: 868–874.CrossrefMedlineGoogle Scholar2 Langheinrich AC, Bohle RM, Greschus S, Hackstein N, Walker G, von Gerlach S, Rau WS, Holschermann H. Atherosclerotic lesions at micro CT: feasibility for analysis of coronary artery wall in autopsy specimens. Radiology. 2004; 231: 675–681.CrossrefMedlineGoogle Scholar3 Johnson JT, Hansen MS, Wu I, Healy LJ, Johnson CR, Jones GM, Capecchi MR, Keller C. Virtual histology of transgenic mouse embryos for high-throughput phenotyping. PLoS Genet. 2006; 2: e61.CrossrefMedlineGoogle Scholar4 Zhang SH, Reddick RL, Piedrahita JA, Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science. 1992; 258: 468–471.CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsCited By Choi J, Haan J and Sharma A (2021) Animal models of diabetes‐associated vascular diseases: an update on available models and experimental analysis, British Journal of Pharmacology, 10.1111/bph.15591, 179:5, (748-769), Online publication date: 1-Mar-2022. Yang Y, Li Z, Liu Q, Guo Y, Mei Y, Lyu J, Zhao M, Feng Y and Xie G (2022) Carotid arterial wall MRI of apolipoprotein e–deficient mouse at 7 T using DANTE-prepared variable-flip-angle rapid acquisition with relaxation enhancement, Magnetic Resonance Imaging, 10.1016/j.mri.2021.10.026, 86, (1-9), Online publication date: 1-Feb-2022. Willemink M, Varga-Szemes A, Schoepf U, Codari M, Nieman K, Fleischmann D and Mastrodicasa D (2021) Emerging methods for the characterization of ischemic heart disease: ultrafast Doppler angiography, micro-CT, photon-counting CT, novel MRI and PET techniques, and artificial intelligence, European Radiology Experimental, 10.1186/s41747-021-00207-3, 5:1, Online publication date: 1-Dec-2021. Stadelmann V, Boyd G, Guillot M, Bienvenu J, Glaus C, Varela A and Chen J (2021) Automatic Quantification of Atherosclerosis in Contrast-Enhanced MicroCT Scans of Mouse Aortas Ex Vivo, International Journal of Biomedical Imaging, 10.1155/2021/4998786, 2021, (1-9), Online publication date: 20-Sep-2021. HANDSCHUH S, BEISSER C, RUTHENSTEINER B and METSCHER B (2017) Microscopic dual-energy CT (microDECT): a flexible tool for multichannel ex vivo 3D imaging of biological specimens , Journal of Microscopy, 10.1111/jmi.12543, 267:1, (3-26), Online publication date: 1-Jul-2017. Robinet P and Smith J (2015) Development and Use of Mouse Models of Atherosclerosis Atherosclerosis, 10.1002/9781118828533.ch16, (197-206) Scudamore C (2013) Cardiovascular system A Practical Guide to the Histology of the Mouse, 10.1002/9781118789568.ch4, (63-73) Tilly P, Charles A, Ludwig S, Slimani F, Gross S, Meilhac O, Geny B, Stefansson K, Gurney M and Fabre J (2013) Blocking the EP3 receptor for PGE2 with DG-041 decreases thrombosis without impairing haemostatic competence, Cardiovascular Research, 10.1093/cvr/cvt276, 101:3, (482-491), Online publication date: 1-Mar-2014., Online publication date: 1-Mar-2014. Tahara R and Larsson H (2013) Quantitative analysis of microscopic X-ray computed tomography imaging: Japanese quail embryonic soft tissues with iodine staining, Journal of Anatomy, 10.1111/joa.12081, 223:3, (297-310), Online publication date: 1-Sep-2013. Kim J, Min J, Recknagel A, Riccio M and Butcher J (2010) Quantitative Three-Dimensional Analysis of Embryonic Chick Morphogenesis Via Microcomputed Tomography, The Anatomical Record: Advances in Integrative Anatomy and Evolutionary Biology, 10.1002/ar.21276, 294:1, (1-10), Online publication date: 1-Jan-2011. Prajapati S, Kilcoyne A, Samano A, Green D, McCarthy S, Blackman B, Brady M, Zarzabal L, Tatiparthy A, Sledz T, Duong T, Ohshima-Hosoyama S, Giles F, Michalek J, Rubin B and Keller C (2010) MicroCT-Based Virtual Histology Evaluation of Preclinical Medulloblastoma, Molecular Imaging and Biology, 10.1007/s11307-010-0372-3, 13:3, (493-499), Online publication date: 1-Jun-2011. Jung C, Christiansen S, Kaul M, Koziolek E, Reimer R, Heeren J, Adam G, Heine M, Ittrich H and Cheng X (2017) Quantitative and qualitative estimation of atherosclerotic plaque burden in vivo at 7T MRI using Gadospin F in comparison to en face preparation evaluated in ApoE KO mice, PLOS ONE, 10.1371/journal.pone.0180407, 12:8, (e0180407) Kirkby N, Low L, Seckl J, Walker B, Webb D, Hadoke P and Zernecke A (2011) Quantitative 3-Dimensional Imaging of Murine Neointimal and Atherosclerotic Lesions by Optical Projection Tomography, PLoS ONE, 10.1371/journal.pone.0016906, 6:2, (e16906) Lloyd D, Helmering J, Kaufman S, Turk J, Silva M, Vasquez S, Weinstein D, Johnston B, Hale C, Véniant M and Zernecke A (2011) A Volumetric Method for Quantifying Atherosclerosis in Mice by Using MicroCT: Comparison to En Face, PLoS ONE, 10.1371/journal.pone.0018800, 6:4, (e18800) September 1, 2009Vol 120, Issue 9 Advertisement Article InformationMetrics https://doi.org/10.1161/CIRCULATIONAHA.108.829531PMID: 19720948 Originally publishedSeptember 1, 2009 PDF download Advertisement SubjectsComputerized Tomography (CT)Diabetes, Type 1Genetically Altered and Transgenic ModelsImagingMetabolism

Members of the chemokine system, play a central role in inflammatory processes that underlie the pathogenesis of atherosclerosis and possibly, aortic valve sclerosis. Here we show that genetic inactivation of CC chemokine receptor 5 (CCR5) in the atherosclerosis-prone Apoe−/− mice (Apoe−/−Ccr5−/−) fed a normal chow or a high-fat diet (HFD) are protected against advanced atherosclerosis as well as age-associated aortic valve thickening (AAAVT)—a murine correlate of aortic valve sclerosis. Notably, human sclerotic valves contained CCR5+ cells. We confirm that Apoe−/−Ccr5−/− mice does not influence early-atherosclerotic stage. Adoptive transfer studies showed that the atheroprotective effect of CCR5 inactivation resided in the bone marrow compartment, but was not dependent on T-cells. The CCR5-null state was associated with phenotypes postulated to be atheroprotective such as reduced macrophage accumulation in the plaque, and lower circulating levels of IL-6 and MCP-5. The lack of CCR5 expression in Apoe−/− mice was also associated with higher numbers of endothelial progenitor cells (EPCs)—another postulated athero-protective factor. Compared with controls, carriers of a polymorphism in the Ccr5 gene that leads to the lack of CCR5 in the cell surface had an increased mean percentage of EPCs, but this difference did not reach statistical significance. Collectively, these findings underscore a critical role of CCR5 in age-associated cardiovascular diseases, and highlight that the effects of the chemokine system can be temporally constrained to distinct stages of these disease processes.