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Overall survival following reduced intensity conditioning (RIC) allogeneic hematopoietic cell transplantation (HCT) using alemtuzumab, fludarabine, and melphalan is favorable in patients transplanted for inborn errors of immunity (IEI), but RIC is associated with high rates of mixed chimerism (MC) and secondary graft failure (GF). We hypothesized that peri-transplant alemtuzumab levels or specific patterns of inflammation would predict these risks. We assessed samples from BMT CTN 1204 (NCT01998633) to study the impact of alemtuzumab levels and cytokine patterns on MC and impending or established secondary GF (defined as donor chimerism 0.32μg/mL (p=0.008). Impending or established secondary GF was only observed in patients with day 0 alemtuzumab levels >0.32µg/mL (p=0.08). Unexpectedly, patients with impending or established secondary GF had lower CXCL9 levels. The cumulative incidence of impending or established secondary GF in patients with a day +14 CXCL9 level ≤2394pg/mL (day +14 median) was 73.6% versus 0% in patients >2394pg/mL (p=0.002). CXCL9 levels inversely correlated with alemtuzumab levels. These findings support a relationship between alemtuzumab levels, CXCL9 levels, and sustained engraftment. These data suggest a model in which higher levels of alemtuzumab at day 0 deplete donor T-cells, inhibit the graft-versus-marrow reaction (thereby suppressing CXCL9 levels), and adversely impact sustained engraftment in the non-myeloablative HCT setting. Clinical Trial # NCT01998633.

Cytokine storm syndromes (CSS) are severe hyperinflammatory conditions characterized by excessive immune system activation leading to organ damage and death. Hemophagocytic lymphohistiocytosis (HLH), a disease often associated with inherited defects in cell-mediated cytotoxicity, serves as a prototypical CSS for which the 5-year survival is only 60%. Frontline therapy for HLH consists of the glucocorticoid dexamethasone (DEX) and the chemotherapeutic agent etoposide. Many patients, however, are refractory to this treatment or relapse after an initial response. Notably, many cytokines that are elevated in HLH activate the JAK/STAT pathway, and the JAK1/2 inhibitor ruxolitinib (RUX) has shown efficacy in murine HLH models and humans with refractory disease. We recently reported that cytokine-induced JAK/STAT signaling mediates DEX resistance in T cell acute lymphoblastic leukemia (T-ALL) cells, and that this could be effectively reversed by RUX. On the basis of these findings, we hypothesized that cytokine-mediated JAK/STAT signaling might similarly contribute to DEX resistance in HLH, and that RUX treatment would overcome this phenomenon. Using ex vivo assays, a murine model of HLH, and primary patient samples, we demonstrate that the hypercytokinemia of HLH reduces the apoptotic potential of CD8 T cells leading to relative DEX resistance. Upon exposure to RUX, this apoptotic potential is restored, thereby sensitizing CD8 T cells to DEX-induced apoptosis in vitro and significantly reducing tissue immunopathology and HLH disease manifestations in vivo. Our findings provide rationale for combining DEX and RUX to enhance the lymphotoxic effects of DEX and thus improve the outcomes for patients with HLH and related CSS.

Langerhans cell histiocytosis (LCH) is a myeloproliferative disorder that is characterized by the inflammatory lesions with pathogenic CD1a(+)CD207(+) dendritic cells (DCs). BRAFV600E and other somatic activating MAPK gene mutations have been identified in differentiating bone marrow and blood myeloid cells, but the origin of the LCH lesion CD1a(+)CD207(+) DCs and mechanisms of lesion formation remain incompletely defined. To identify candidate LCH CD1a(+)CD207(+) DC precursor populations, gene-expression profiles of LCH lesion CD1a(+)CD207(+) DCs were first compared with established gene signatures from human myeloid cell subpopulations. Interestingly, the CD1c(+) myeloid DC (mDC) gene signature was most enriched in the LCH CD1a(+)CD207(+) DC transcriptome. Additionally, the BRAFV600E allele was not only localized to CD1a(+)CD207(−) DCs and CD1a(+)CD207(+) DCs, but it was also identified in CD1c(+) mDCs in LCH lesions. Transcriptomes of CD1a(+)CD207(−) DCs were nearly indistinguishable from CD1a(+)CD207(+) DCs (both CD1a(+)CD207(low) and CD1a(+)CD207(high) subpopulations). Transcription profiles of LCH lesion CD1a(+)CD207(+) DCs and peripheral blood CD1c(+) mDCs from healthy donors were compared to identify potential LCH DC-specific biomarkers: HLA-DQB2 expression was significantly increased in LCH lesion CD1a(+)CD207(+) DCs compared with circulating CD1c(+) mDCs from healthy donors. HLA-DQB2 antigen was identified on LCH lesion CD1a(+)CD207(−) DCs and CD1a(+)CD207(+) DCs as well as on CD1c(+)(CD1a(+)CD207(−)) mDCs, but it was not identified in any other lesion myeloid subpopulations. HLA-DQB2 expression was specific to peripheral blood of patients with BRAFV600E(+) peripheral blood mononuclear cells, and HLA-DQB2(+)CD1c(+) blood cells were highly enriched for the BRAFV600E in these patients. These data support a model in which blood CD1c(+)HLA-DQB2(+) mDCs with activated ERK migrate to lesion sites where they differentiate into pathogenic CD1a(+)CD207(+) DCs.

Langerhans cell histiocytosis (LCH) is a rare inflammatory myeloid neoplasia with a broad spectrum of clinical manifestations. The activation of the MAP kinase pathway plays an integral role in its pathogenesis with genetic alterations found in the majority of cases that most frequently involve a somatic mutation of the oncogenic BRAFV600E variant. In this study we investigated the prevalence of the BRAFV600E mutation and its clinical relevance in adult Greek patients with LCH. Among 37 patients studied, the BRAFV600E mutation was identified in 12 out of 31 (38.7%), whereas in six patients (19.3%) the results were in conclusive. The presence of the mutation did not correlate with age at diagnosis, organ involvement, disease extent, response to initial treatment, development of diabetes insipidus and relapse risk. In our series the prevalence of the BRAFV600E mutation is at the lower range of the relative percentage found in children, but in line to that obtained in previous studies of adult patients with LCH that have found an up to 50% prevalence of the BRAFV600E mutation in these patients. Further studies with a larger number of adults are needed to identify the exact prevalence of mutations in the RAS-RAF-MEK-ERK pathway and their role on clinical parameters and disease outcomes. Supplemental data for this article is available online at https://doi.org/10.1080/08880018.2022.2029988 .

Hemophagocytic lymphohistiocytosis (HLH) is a syndrome characterized by pathologic immune activation in which prompt recognition and initiation of immune suppression is essential for survival. Children with HLH have many overlapping clinical features with critically ill children with sepsis and systemic inflammatory response syndrome (SIRS) in whom alternative therapies are indicated. To determine whether plasma biomarkers could differentiate HLH from other inflammatory conditions and to better define a core inflammatory signature of HLH, concentrations of inflammatory plasma proteins were compared in 40 patients with HLH to 47 pediatric patients with severe sepsis or SIRS. Fifteen of 135 analytes were significantly different in HLH plasma compared with SIRS/sepsis, including increased interferon-γ (IFN-γ)–regulated chemokines CXCL9, CXCL10, and CXCL11. Furthermore, a 2-analyte plasma protein classifier including CXCL9 and interleukin-6 was able to differentiate HLH from SIRS/sepsis. Gene expression in CD8+ T cells and activated monocytes from blood were also enriched for IFN-γ pathway signatures in peripheral blood cells from patients with HLH compared with SIRS/sepsis. This study identifies differential expression of inflammatory proteins as a diagnostic strategy to identify critically ill children with HLH, and comprehensive unbiased analysis of inflammatory plasma proteins and global gene expression demonstrates that IFN-γ signaling is uniquely elevated in HLH. In addition to demonstrating the ability of diagnostic criteria for HLH and sepsis or SIRS to identify groups with distinct inflammatory patterns, results from this study support the potential for prospective evaluation of inflammatory biomarkers to aid in diagnosis of and optimizing therapeutic strategies for children with distinctive hyperinflammatory syndromes.

Hodgkin lymphoma (HL) histopathology is characterized by rare malignant Reed&ndash, Sternberg cells among an inflammatory infiltrate. We hypothesized that characteristics of inflammation in pediatric HL lesions would be reflected by the levels of inflammatory cytokines or chemokines in pre-therapy plasma of children with HL. The study objectives were to better define the inflammatory pre-therapy plasma proteome and identify plasma biomarkers associated with extent of disease and clinical outcomes in pediatric HL. Pre-therapy plasma samples were obtained from pediatric subjects with newly diagnosed HL and healthy pediatric controls. Plasma concentrations of 135 cytokines/chemokines were measured with the Luminex platform. Associations between protein concentration and disease characteristics were determined using multivariate permutation tests with false discovery control. Fifty-six subjects with HL (mean age: 13 years, range 3&ndash, 18) and 47 controls were analyzed. The cytokine/chemokine profiles of subjects with HL were distinct from controls, and unique cytokines/chemokines were associated with high-risk disease (IL-10, TNF-&alpha, IFN-&gamma, IL-8) and slow early response (CCL13, IFN-&lambda, 1, IL-8). TNFSF10 was significantly elevated among those who ultimately relapsed and was significantly associated with worse event-free survival. These biomarkers could be incorporated into biologically based risk stratification to optimize outcomes and minimize toxicities in pediatric HL.

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia characterized by granulomatous lesions containing pathological CD207+ dendritic cells (DCs) with persistent MAPK pathway activation. Standard-of-care chemotherapies are inadequate for most patients with multisystem disease, and optimal strategies for relapsed and refractory disease are not defined. The mechanisms underlying development of inflammation in LCH lesions, the role of inflammation in pathogenesis, and the potential for immunotherapy are unknown. Analysis of the immune infiltrate in LCH lesions identified the most prominent immune cells as T lymphocytes. Both CD8+ and CD4+ T cells exhibited “exhausted” phenotypes with high expression of the immune checkpoint receptors. LCH DCs showed robust expression of ligands to checkpoint receptors. Intralesional CD8+ T cells showed blunted expression of Tc1/Tc2 cytokines and impaired effector function. In contrast, intralesional regulatory T cells demonstrated intact suppressive activity. Treatment of BRAFV600ECD11c LCH mice with anti-PD-1 or MAPK inhibitor reduced lesion size, but with distinct responses. Whereas MAPK inhibitor treatment resulted in reduction of the myeloid compartment, anti-PD-1 treatment was associated with reduction in the lymphoid compartment. Notably, combined treatment with MAPK inhibitor and anti-PD-1 significantly decreased both CD8+ T cells and myeloid LCH cells in a synergistic fashion. These results are consistent with a model that MAPK hyperactivation in myeloid LCH cells drives recruitment of functionally exhausted T cells within the LCH microenvironment, and they highlight combined MAPK and checkpoint inhibition as a potential therapeutic strategy.

Introduction The BMT-CTN 1204 study for Hemophagocytic Syndromes or Selected Primary Immune Deficiencies (NCT01998633) (RICHI) was a single arm study testing safety and efficacy of reduced intensity conditioning (RIC) with alemtuzumab (1mg/kg), fludarabine (150 mg/m2) and melphalan (140 mg/m2). Survival was favorable compared to historical studies, but patients experienced high rates of mixed chimerism (MC) and ultimate secondary graft failure (GF). Mechanisms for GF are not known. Expansion of recipient T cells and interferon-gamma pathway activation have been proposed as drivers for GF. However, high peri-transplant alemtuzumab levels have been associated with higher risk of MC and eventual secondary GF, suggesting an inverse relationship between GF and immune activation in the context of RIC. In order to elucidate mechanisms of GF for patients on the RICHI study, we systematically evaluated cytokine patterns and alemtuzumab levels and their association with durable engraftment. Methods Serial blood samples were collected, processed, and stored for consenting patients at day -14 (window: day -28 to -14), day -7 (+/- 1 day), day -1 (+/- 1), day +1 (+1 to +3), day +14 (+/- 2), day +28 (+/- 2), day +42 (+/- 3), day +70 (+/- 10), and day +100 (+/- 10). Alemtuzumab levels were measured using a flow cytometric assay as previously described. Comprehensive cytokine analysis was performed for over 100 analytes using the MagPix platform. Primary GF was defined as donor chimerism Results Thirty-three patients were included in this study with HLH (n=25), CAEBV (n=3), CGD (n=2), HIGM (n=2), and IPEX (n=1). All patients received bone marrow grafts and 27 (82%) patients had unrelated donors. Twenty-one grafts were 8/8 or 6/6 HLA-matched (64%) and 12 grafts were 7/8 HLA-matched (36%). Among all patients, 1 patient (3%) developed primary GF, 22 (67%) developed mixed chimerism (MC), and 11 patients (33%) developed secondary GF. Survival with sustained engraftment without DLI or second HCT was 40.0%. We first evaluated peripheral blood levels of 100+ cytokines. Analysis revealed significant differences between patients with and without GF as shown in Figure 1A. Notably, on day +14 and +28, patients with secondary GF had significantly lower CXCL9 levels than those without GF. We then estimated the cumulative incidence (CI) of secondary GF among patients with CXCL9 levels above and below the day +14 median level of 2394pg/mL. The CI of secondary GF in patients with a day +14 CXCL9 level ≤2394pg/mL was 73.6% vs 0% in patients with a level >2394pg/mL (p=0.002). The CI of secondary GF in patients with a day +28 CXCL9 level ≤2867pg/mL (day +28 median) was 64.3%, vs 0% in patients with levels >2867pg/mL (p=0.004). We then sought to correlate CXCL9 levels with alemtuzumab exposure, as high alemtuzumab levels would result in more efficient T cell depletion of donor grafts that could lead to lower CXCL9 levels. Indeed, CXCL9 levels inversely correlated with day 0 alemtuzumab levels. Patients with day 0 alemtuzumab levels >0.32µg/mL had lower CXCL9 levels compared to patients with levels ≤0.32µg/mL (Figure 1B). Finally, we examined the impact of alemtuzumab levels on MC and secondary GF. Patients with day 0 alemtuzumab levels ≤0.32µg/mL had a lower CI of MC compared to patients with levels >0.32µg/mL, 14.3% vs 90.9%, respectively (p=0.03). The CI of secondary GF was 0% in patients with day 0 alemtuzumab levels ≤0.32µg/mL compared to 54.3% in patients with levels >0.32µg/mL (p=0.08). Conclusions This study demonstrates a strong relationship between alemtuzumab levels and durable engraftment. Further, interferon gamma activity, as reflected by CXCL9, inversely correlates with peri-transplant alemtuzumab levels in this prospective cohort treated with RIC. Our findings support the paradigm that higher alemtuzumab levels drive efficient T cell depletion of the stem cell product which increases the risk of MC and secondary GF, suggesting that donor T cells promote engraftment via a graft versus hematopoiesis function. Precision alemtuzumab dosing strategies may offer an opportunity to improve outcomes for patients who undergo RIC HCT. Figure 1 Figure 1. Disclosures Pulsipher: Adaptive: Research Funding; Equillium: Membership on an entity's Board of Directors or advisory committees; Jasper Therapeutics: Honoraria. Bollard: Neximmune: Current equity holder in publicly-traded company; Catamaran Bio: Membership on an entity's Board of Directors or advisory committees; Cabaletta Bio: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Cellectis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Repertoire Immune Medicines: Current equity holder in publicly-traded company; ROCHE: Consultancy, Honoraria; SOBI: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kean: Regeneron: Research Funding; Bristol Myers Squibb: Patents & Royalties: From clinical trial data, Research Funding; Bluebird Bio: Research Funding; Gilead: Research Funding; Vertex: Consultancy; Novartis: Consultancy; EMD Serono: Consultancy. Jordan: Sobi: Consultancy. Allen: Sobi: Consultancy. OffLabel Disclosure: Alemtuzumab, humanized monoclonal antibody against CD52, used as part of allogeneic HCT conditioning

To the editor: Langerhans cell histiocytosis (LCH) is a hematologic disorder that presents with a wide spectrum of symptoms, ranging from focal lesions to potentially lethal multiorgan disease, affecting 4 to 8 per million children per year[1][1] and 1 to 2 per million adults per year.[2][2]

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia characterized by granulomatous lesions containing pathological CD207+ dendritic cells (DCs) with persistent mitogen-activated protein kinase (MAPK) pathway activation. Standard of care chemotherapy strategies are inadequate or overly toxic for the majority of patients with multisystem disease. The mechanisms underlying development of inflammation in LCH lesions are poorly understood, and potential for immunotherapy has not been determined. Analysis of the LCH lesion identified the most prominent immune cells as T lymphocytes, second only to pathologic CD207+/CD1a+ DCs (LCH DC). Both CD8+ and CD4+ T cells exhibited 'exhausted' phenotypes with high expression of the immune checkpoint inhibitor receptors. LCH DC cells also showed robust expression of ligands to checkpoint inhibitor receptors. Lesion CD8+ T cells exhibited blunted expression of Th1 cytokines with impaired effector function. In contrast, regulatory T cells (Tregs) isolated from LCH lesions demonstrated intact suppressive activity. Treatment of BRAFV600ECD11c LCH mice with anti-PD-1, anti-TIM-3 and MEKi decreased the lesion size: MEKi treatment resulted in reduction of the myeloid compartment; anti-PD-1 and anti-TIM-3 were associated with reduction in the lymphoid compartment. However, combined treatment with MEKi and either anti-PD-1 or anti-TIM-3 significantly decreased both CD8+ T cell and myeloid LCH cells in a synergistic fashion. These results thus indicate MAPK hyperactivation in myeloid LCH cells drives recruitment and activation of functionally exhausted T cells within the LCH microenvironment. While the results from this study did not demonstrate a significant impact of checkpoint inhibition alone on tumor burden in experimental mice, combined MAPK and checkpoint inhibition may be a potentially effective therapeutic strategy for patients with LCH. Disclosures Bollard: self: Patents & Royalties: patent on HIV T cells; NexImmune: Equity Ownership; Torque: Equity Ownership; Cellectis: Membership on an entity's Board of Directors or advisory committees; Caballetta: Equity Ownership; Mana Therapeutics: Equity Ownership.

Introduction: Pathogenic Hodgkin Reed-Sternberg (HRS) cells constitute approximately 1% of Hodgkin lymphoma (HL) tumor cells. Studies characterizing genomic lesions and gene expression of HRS gene cells have been limited due to technical challenges of studying these rare cells, and the majority of existing data has focused on adult HL. We therefore developed a multi-parameter flow sorting strategy to isolate viable cells from pediatric HL tumors and to define the transcriptomes of HRS cells and infiltrating lymphocytes in order to inform underlying mechanisms of HL pathogenesis and also create an opportunity to identify cell-specific biomarkers to predict disease risk and response to therapy. Methods : Flow cytometry was used to sort HRS cells, CD4+ T cells, CD8+ T cells, and CD20+/30+B cells from pediatric subjects' HL lesions and control tonsils. Purity was confirmed by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC). Affymetrix GeneChip HTA 2.0 was used to assess the gene expression profiles (GEPs) for 16 HRS primary tumor cell samples, 14 HL CD4+ and CD8+ T cell samples, 6 control tonsillar CD20+, CD30+, CD4+, and CD8+ cell samples, and 6 HL cell lines. Unsupervised hierarchical clustering and principal component analysis (PCA) were used to determine relatedness, and Cibersort was performed to confirm the phenotype of the sorted cell types. GEPs of HRS, HL CD4+, and HL CD8+ cells were compared to respective controls using a univariate t-test. Significance was determined using a multivariate permutation test with the confidence level of FDR assessment at 80 percent and the maximum allowed proportion of false-positive proteins at 0.1. Gene set enrichment analysis (GSEA) and ingenuity pathway analysis (IPA) were performed to analyze DEGs. Results: Effectiveness of the sorting strategy of HRS cells was confirmed by quantitative RT-PCR and IHC that demonstrated significant enrichment of CD30expression and CD30+ cells in the sorted HRS cell fraction. GEP comparisons were performed for 13 HL samples with matched HRS/CD4+/CD8+ cells: HRS vs. control tonsil CD20+/CD30+ (1934 and 3846 DEGs, respectively), HL CD4+ vs. control CD4+ (635 DEGs), HL CD8+ vs. control CD8+ (2 DEGs). We carried out a transcriptomic analysis of HRS cells, and a set of multifunctional genes were more than 2-fold downregulated (P < .001), involved in telomere maintenance and packaging (TERF2, RFC3, DNA2 and a group of HIST1) when compared to healthy lymph node CD30+ cells. A set of genes related to cytokine/chemokine dysregulation was also upregulated in HRS cells, including IL6, CCL18, and CXCL9. IPA and GSEA of specific HRS genes were also performed and demonstrated pathways associated with HL pathogenesis, including NFĸB activation and T cell exhaustion. Over-expression of genes associated with T cell pathways was demonstrated in HRS cells. While this may be a result of T cell rosetting and contamination, it may also reflect innate T cell signature within HRS cells, as HRS cells clustered separately from T cells in both unsupervised hierarchical clustering and PCA. Cibersort analysis of HRS cells revealed a heterogeneous phenotype that may reflect aberrant differentiation. In comparing clinical characteristics within HRS cells, TCEAL1 was elevated in slow vs. rapid early responders and 3 DEGs were identified when comparing EBV+/- samples. Within HL CD8 cells, KLF2 was elevated in EBV- samples. Conclusions: This study was the first to successfully isolate highly purified HRS cell populations from whole HL lesions in a pediatric HL cohort. Transcriptomic analysis of pediatric HRS cells identified mechanisms previously associated with HL pathogenesis, and also identified potential novel mechanisms, including telomere maintenance. Additional analyses demonstrated significant heterogeneity of HRS trasncriptomes across specimens that may reflect distinct differentiation pathways and differences in HRS-immune cell interactions. Finally, this study identified increased expression of some genes associated with EBV status and response to therapy. Future studies in an expanded cohort will validate these findings, compare pediatric and adult GEPs, and test these cell-specific biomarkers into the current risk stratification strategies of prospective clinical trials. Disclosures No relevant conflicts of interest to declare.

Langerhans cell histiocytosis (LCH) is characterized by inflammatory lesions containing pathologic CD207+ dendritic cells with constitutively activated ERK. Mutually exclusive somatic mutations in MAPK pathway genes have been identified in ∼75% of LCH cases, including recurrent BRAF-V600E and MAP2K1 mutations. To elucidate mechanisms of ERK activation in the remaining 25% of patients, we performed whole-exome sequencing (WES, n = 6), targeted BRAF sequencing (n = 19), and/or whole-transcriptome sequencing (RNA-seq, n = 6) on 24 LCH patient samples lacking BRAF-V600E or MAP2K1 mutations. WES and BRAF sequencing identified in-frame BRAF deletions in the β3-αC loop in 6 lesions. RNA-seq revealed one case with an in-frame FAM73A-BRAF fusion lacking the BRAF autoinhibitory regulatory domain but retaining an intact kinase domain. High levels of phospho-ERK were detected in vitro in cells overexpressing either BRAF fusion or deletion constructs and ex vivo in CD207+ cells from lesions. ERK activation was resistant to BRAF-V600E inhibition, but responsive to both a second-generation BRAF inhibitor and a MEK inhibitor. These results support an emerging model of universal ERK-activating genetic alterations driving pathogenesis in LCH. A personalized approach in which patient-specific alterations are identified may be necessary to maximize benefit from targeted therapies for patients with LCH.

Purpose: Tumor necrosis factor alpha (TNF-alpha) is produced in Langerhans cell histiocytosis (LCH) lesions and is elevated in active high-risk vs low-risk LCH vs control patient blood, where TNF-alpha has been reported at 12 pg/ml in juvenile rheumatoid arthritis (JRA). Anti-TNF therapeutic etanercept is used in TNF-mediated diseases, such as JRA, and was successfully used in one case of LCH. We therefore conducted phase II study for etanercept therapeutic efficacy for refractory or relapsed LCH patients. Methods: Luminex platform was used to assess patient blood TNF-alpha level. This phase II study was approved by the Baylor College of Medicine IRB with research support from Amgen/Immunex. Eligibility included LCH patients with progressive disease post-initial treatment and ≥1 salvage therapy. Etanercept was injected 0.4mg/kg subcutaneously twice/week for 12 weeks. Therapeutic response were evaluated at 4 and 8 weeks. Results: TNF-alpha is elevated in blood LCH high-risk (average 45pg/ml) vs low-risk (average 13pg/ml) vs control (average 13pg/ml). Five LCH patients (1.6-42 years) with multi-system involvement, 2 with high-risk disease were enrolled. A median of 5 doses of etanercept were administered (range 1-14). One high-risk patient died 18 days after the first dose from disease progression. At week 4 evaluation, one patient had stable disease and 3 progressed and all subjects progressed by week 8. Ultimately 0/5 patients had disease improvement with etanercept. Conclusion: Etanercept therapy, at JRA-effective dose, in this trial did not improve LCH disease. It is possible that this may not be an effective strategy. Alternatively, it is also possible that drug dose or distribution was suboptimal. While anti-TNF therapies, including etanercept, are effective for treatment of RA, etanercept is not effective for granulomatous diseases such as Crohn's, sarcoidosis or Wegner's disease. Additional studies exploring potential efficacy of higher dose or alternative forms of TNF-alpha inhibition in LCH may be warranted. Disclosures No relevant conflicts of interest to declare.

Reduced apoptosis of crypt stem/progenitor cells and elevated insulin and IGFs are linked to colon cancer risk. Insulin receptor substrate-1 (IRS-1) mediates the actions of insulin, IGF-I, and IGF-II, but the role of endogenous IRS-1 in crypt apoptosis and cancer is undefined. Using IRS-1(-/-), IRS-1(+/-), and IRS-1(+/+) mice, we tested the hypothesis that reduced IRS-1 expression increases apoptosis of intestinal crypt cells and protects against Apc(min/+) (Min)/beta-catenin-driven intestinal tumors. Expression of Sox9, a transcriptional target of Tcf/beta-catenin and putative biomarker of crypt stem cells, was assessed in intestine of different IRS-1 genotypes and cell lines. Irradiation-induced apoptosis was significantly increased in the crypts and crypt stem cell region of IRS-1-deficient mice. Tumor load was significantly reduced by 31.2 +/- 14.6% in IRS-1(+/-)/Min and by 64.1 +/- 7.6% in IRS-1(-/-)/Min mice, with more prominent reductions in tumor number than size. Compared with IRS-1(+/+)/Min, IRS-1(-/-)/Min mice had fewer Sox9-positive cells in intestinal crypts and reduced Sox9 mRNA in intestine. IRS-1 overexpression increased Sox9 expression in an intestinal epithelial cell line. We conclude that even small reductions in endogenous IRS-1 increase apoptosis of crypt stem or progenitor cells, protect against beta-catenin-driven intestinal tumors, and reduce Sox9, a Tcf/beta-catenin target and putative stem/progenitor cell biomarker.

Many low birth weight infants experience growth failure due to feeding intolerance resulting in reduced nutrient intake. The branched-chain amino acid, leucine, has been shown to have anabolic effe...

l-Arginine (l-Arg) is a semiessential amino acid that has altered availability in human ulcerative colitis (UC), a form of inflammatory bowel disease, and is beneficial in murine colitis induced by dextran sulfate sodium (DSS), a model with similarity to UC. We assessed the role of cationic amino acid transporter 2 (CAT2), the inducible transporter of l-Arg, in DSS colitis. Expression of CAT2 was upregulated in tissues from colitic mice and localized predominantly to colonic macrophages. CAT2-deficient (CAT2−/−) mice exposed to DSS exhibited worsening of survival, body weight loss, colon weight, and histological injury. These effects were associated with increased serum l-Arg and decreased tissue l-Arg uptake and inducible nitric oxide synthase protein expression. Clinical benefits of l-Arg supplementation in wild-type mice were lost in CAT2−/−mice. There was increased infiltration of macrophages, dendritic cells, granulocytes, and T cells in colitic CAT2−/−compared with wild-type mice. Cytokine profiling revealed increases in proinflammatory granulocyte colony-stimulating factor, macrophage inflammatory protein-1α, IL-15, and regulated and normal T cell-expressed and -secreted and a shift from an IFN-γ- to an IL-17-predominant T cell response, as well as an increase in IL-13, in tissues from colitic CAT2−/−mice. However, there were no increases in other T helper cell type 2 cytokines, nor was there a global increase in macrophage-derived proinflammatory cytokines. The increase in IL-17 derived from both CD4 and γδ T cells and was associated with colonic IL-6 expression. Thus CAT2 plays an important role in controlling inflammation and IL-17 activation in an injury model of colitis, and impaired l-Arg availability may contribute to UC pathogenesis.

Background: Langerhans Cell Histiocytosis (LCH) is characterized by inflammatory lesions with pathologic CD207+ dendritic cells (DCs) with constitutively activated ERK. LCH lesions are associated with a local cytokine storm and exhibit a heterogeneous collection of inflammatory infiltrates (eosinophils, neutrophils and lymphocytes). The mechanisms behind tumor formation, both accumulation of pathologic CD207+ DCs as well as recruitment of inflammatory infiltrate, remain to be defined. Mutually exclusive somatic mutations in MAPK pathway genes have been identified in approximately 75% of LCH cases, including recurrent BRAF-V600Eand MAP2K1 mutations. However, the inflammatory infiltrate within LCH lesions is composed of cells without any BRAF mutation or ERK activation. We hypothesize that the pathologic inflammation in LCH is orchestrated by interactions between the pathologic MAPK-activated DCs and normal immune cells. Defining the nature and function of infiltrating T cells in LCH will not only provide insights into mechanisms of pathogenesis and potential therapeutic targets, but may also inform novel physiologic mechanisms of DC-mediated T cell function. Methods: Clonality was investigated by exploring T cell receptor beta (TCRB) rearrangement in LCH patients. Quantitative analysis of different immune cell populations in the tumor microenvironment in 25 LCH patients was performed using spanning tree progression of density normalized events (SPADE). Results: Our initial experiments identified clonal populations among LCH lesion-infiltrating CD3+ T cells. The complementarity-determining region 3 (CDR3) of the TCRBV07 gene demonstrated marked monoclonal expansion, suggesting a potential involvement of antigen selection in the pathogenesis of LCH. SPADE analysis of infiltrating CD3+ T cells revealed high expression of T cell immunoglobulin mucin (TIM-3), lymphocyte activated gene 3 (LAG3) and programmed cell death protein 1 (PD-1) compared to healthy controls, indicating that effector T cell exhaustion might be an important mechanism of immune dysregulation in LCH patients. In addition, in a cohort of LCH patients harboring wild-type BRAF, but not the BRAF-V600E mutation, a distinct subset of infiltrating CD3+ T cells were detected expressing both CD4 and CD8α receptors at high levels (double-positive, DP). Preliminary analysis showed high levels of IFNγ, but not TNF-α, after stimulation of these DP T cells. Conclusions: The results from this study support a model in which LCH lesion DCs with activated MAPK signaling select and recruit activated T cells with potential for clonal expansion to LCH lesions. Multiple markers of T cell exhaustion were identified on infiltrating LCH lesion T cells. While the significance of double-positive T cells in LCH is uncertain, similar cells have been reported in the target organs of several auto-immune conditions and hematological and solid malignancies, consistent with double-positive T cells in sites of immune exhaustion. The function of this double-positive T cell population remains to be defined, but we postulate that these cells may participate in tumor immune responses in LCH due to their potential of having broad tumor reactivity. While the CD207+ DCs in LCH lesions is histologically uniform across the disease spectrum, the composition of the immune microenvironment is variable. The variability of the DP T cell population in BRAF-V600E vs BRAF wild-type DCs study suggests that specific mechanisms of MAPK activation may have some distinct impact on T cell recruitment and activation. While recent discoveries have enhanced understanding of the origin and aberrant MAPK activation of the pathologic DCs, very little is known about the role of lymphocytes or the inflammatory microenvironment. Characterizing the populations, function, and interactions of the LCH lesion immune cells may provide novel therapeutic opportunities, such as checkpoint inhibitors. Further, identifying the mechanisms that underlie the "cytokine storm" of LCH lesion may inform physiologic interactions between MAPK-activated DCs and effector immune cells. Disclosures No relevant conflicts of interest to declare.

Gastric cancer is the second leading cause of cancer mortality worldwide and is projected to rise to tenth in all-cause mortality in the near term. Early detection requires improved sensitivity and specificity of endoscopic imaging with novel methods. The objective of this study was to evaluate the utility of activatable molecular probes for the detection of gastric cancer both in vivo and ex vivo in a preclinical model. Smad4 +/− mice, which develop spontaneous gastric neoplasia, were compared to normal wild-type controls. Cathepsin-activatable and matrix metalloproteinase (MMP)-activatable molecular probes were injected 24 hours and 6 hours before imaging, respectively. In vivo imaging was performed using quantitative tomographic near-infrared fluorescence (NIRF) imaging. For validation, ex vivo imaging and histologic examination were performed. Molecular imaging in vivo of Smad4 +/− gastric cancer murine models revealed intense activation of both cathepsin B and MMP probes. Ex vivo imaging and histology confirmed that the detected neoplasms were adenocarcinomas and hyperplastic lesions. This study provides proof of principle that the cathepsin- and MMP-activatable molecular probes are activated in the Smad4 +/− murine model of spontaneous gastric adenocarcinoma and can be imaged by both in vivo and ex vivo NIRF methods. The cathepsin probe also detects hyperplastic lesions.

Recent identification of intestinal epithelial stem cell (ISC) markers and development of ISC reporter mice permit visualization and isolation of regenerating ISCs after radiation to define their functional and molecular phenotypes. Previous studies in uninjured intestine of Sox9-EGFP reporter mice demonstrate that ISCs express low levels of Sox9-EGFP (Sox9-EGFP Low), whereas enteroendocrine cells (EEC) express high levels of Sox9-EGFP (Sox9-EGFP High). We hypothesized that Sox9-EGFP Low ISCs would expand after radiation, exhibit enhanced proliferative capacities, and adopt a distinct gene expression profile associated with rapid proliferation. Sox9-EGFP mice were given 14 Gy abdominal radiation and studied between days 3 and 9 postradiation. Radiation-induced changes in number, growth, and transcriptome of the different Sox9-EGFP cell populations were determined by histology, flow cytometry, in vitro culture assays, and microarray. Microarray confirmed that nonirradiated Sox9-EGFP Low cells are enriched for Lgr5 mRNA and mRNAs enriched in Lgr5-ISCs and identified additional putative ISC markers. Sox9-EGFP High cells were enriched for EEC markers, as well as Bmi1 and Hopx, which are putative markers of quiescent ISCs. Irradiation caused complete crypt loss, followed by expansion and hyperproliferation of Sox9-EGFP Low cells. From nonirradiated intestine, only Sox9-EGFP Low cells exhibited ISC characteristics of forming organoids in culture, whereas during regeneration both Sox9-EGFP Low and High cells formed organoids. Microarray demonstrated that regenerating Sox9-EGFP High cells exhibited transcriptomic changes linked to p53-signaling and ISC-like functions including DNA repair and reduced oxidative metabolism. These findings support a model in which Sox9-EGFP Low cells represent active ISCs, Sox9-EGFP High cells contain radiation-activatable cells with ISC characteristics, and both participate in crypt regeneration.

Inflammatory bowel disease (IBD), consisting of Crohn's disease and ulcerative colitis (UC), results in substantial morbidity and is difficult to treat. New strategies for adjunct therapies are needed. One candidate is the semi-essential amino acid, L-arginine (L-Arg), a complementary medicine purported to be an enhancer of immunity and vitality in the lay media. Using dextran sulfate sodium (DSS) as a murine colonic injury and repair model with similarities to human UC, we assessed the effect of L-Arg, as DSS induced increases in colonic expression of the y(+) cationic amino acid transporter 2 (CAT2) and L-Arg uptake. L-Arg supplementation improved the clinical parameters of survival, body weight loss, and colon weight, and reduced colonic permeability and the number of myeloperoxidase-positive neutrophils in DSS colitis. Luminex-based multi-analyte profiling demonstrated that there was a marked reduction in proinflammatory cytokine and chemokine expression with L-Arg treatment. Genomic analysis by microarray demonstrated that DSS-treated mice supplemented with L-Arg clustered more closely with mice not exposed to DSS than to those receiving DSS alone, and revealed that multiple genes that were upregulated or downregulated with DSS alone exhibited normalization of expression with L-Arg supplementation. Additionally, L-Arg treatment of mice with DSS colitis resulted in increased ex vivo migration of colonic epithelial cells, suggestive of increased capacity for wound repair. Because CAT2 induction was sustained during L-Arg treatment and inducible nitric oxide (NO) synthase (iNOS) requires uptake of L-Arg for generation of NO, we tested the effect of L-Arg in iNOS(-/-) mice and found that its benefits in DSS colitis were eliminated. These preclinical studies indicate that L-Arg supplementation could be a potential therapy for IBD, and that one mechanism of action may be functional enhancement of iNOS activity.

A strong cellular cross-talk exists between the pathogen Helicobacter pylori and high-output NO production. However, how NO and H. pylori interact to signal in gastric epithelial cells and modulate the innate immune response is unknown. We show that chemical or cellular sources of NO induce the anti-inflammatory effector heme oxygenase-1 (HO-1) in gastric epithelial cells through a pathway that requires NF-κB. However, H. pylori decreases NO-induced NF-κB activation, thereby inhibiting HO-1 expression. This inhibitory effect of H. pylori results from activation of the transcription factor heat shock factor-1 by the H. pylori virulence factor CagA and by the host signaling molecules ERK1/2 and JNK. Consistent with these findings, HO-1 is downregulated in gastric epithelial cells of patients infected with cagA+ H. pylori but not in gastric epithelial cells of patients infected with cagA– H. pylori. Enhancement of HO-1 activity in infected cells or in H. pylori-infected mice inhibits chemokine generation and reduces inflammation. These data define a mechanism by which H. pylori favors its own pathogenesis by inhibiting HO-1 induction through the action of CagA.

International audience; Once acquired, Helicobacter pylori infection is lifelong due to an inadequate innate and adaptive immune response. Our previous studies indicate that interactions among the various pathways of arginine metabolism in the host are critical determinants of outcomes following infection. Cationic amino acid transporter 2 (CAT2) is essential for transport of L-arginine (L-Arg) into monocytic immune cells during H. pylori infection. Once within the cell, this amino acid is utilized by opposing pathways that lead to elaboration of either bactericidal nitric oxide (NO) produced from inducible NO synthase (iNOS), or hydrogen peroxide, which causes macrophage apoptosis, via arginase and the polyamine pathway. Because of its central role in controlling L-Arg availability in macrophages, we investigated the importance of CAT2 in vivo during H. pylori infection. CAT2(-/-) mice infected for 4 months exhibited decreased gastritis and increased levels of colonization compared to wild type mice. We observed suppression of gastric macrophage levels, macrophage expression of iNOS, dendritic cell activation, and expression of granulocyte-colony stimulating factor in CAT2(-/-) mice suggesting that CAT2 is involved in enhancing the innate immune response. In addition, cytokine expression in CAT2(-/-) mice was altered from an antimicrobial Th1 response to a Th2 response, indicating that the transporter has downstream effects on adaptive immunity as well. These findings demonstrate that CAT2 is an important regulator of the immune response during H. pylori infection.

Background: Obesity induced by high fat (HF) diet is associated with inflammation which contributes to development of insulin resistance. Most prior studies have focused on adipose tissue as the source of obesity-associated inflammation. Increasing evidence links intestinal bacteria to development of diet-induced obesity (DIO). This study tested the hypothesis that HF western diet and gut bacteria interact to promote intestinal inflammation, which contributes to the progression of obesity and insulin resistance. Methodology/Principal Findings: Conventionally raised specific-pathogen free (CONV) and germ-free (GF) mice were given HF or low fat (LF) diet for 2-16 weeks. Body weight and adiposity were measured. Intestinal inflammation was assessed by evaluation of TNF-alpha mRNA and activation of a NF-kappa B-EGFP reporter gene. In CONV but not GF mice, HF diet induced increases in body weight and adiposity. HF diet induced ileal TNF-alpha mRNA in CONV but not GF mice and this increase preceded obesity and strongly and significantly correlated with diet induced weight gain, adiposity, plasma insulin and glucose. In CONV mice HF diet also resulted in activation of NF-kappa B-EGFP in epithelial cells, immune cells and endothelial cells of small intestine. Further experiments demonstrated that fecal slurries from CONV mice fed HF diet are sufficient to activate NF-kappa B-EGFP in GF NF-kappa B-EGFP mice. Conclusions/Significance: Bacteria and HF diet interact to promote proinflammatory changes in the small intestine, which precede weight gain and obesity and show strong and significant associations with progression of obesity and development of insulin resistance. To our knowledge, this is the first evidence that intestinal inflammation is an early consequence of HF diet which may contribute to obesity and associated insulin resistance. Interventions which limit intestinal inflammation induced by HF diet and bacteria may protect against obesity and insulin resistance.

Epigenetic in vitro and in vivo studies suggest that suppressor of cytokine signaling-2 (SOCS2) may normally limit tumorigenesis in the intestine; however, this theory has not been directly tested. We hypothesized that SOCS2 deficiency promotes spontaneous intestinal tumorigenesis in Apc(Min/+) mice. Therefore, we quantified tumor number, size, and load in the small intestine and colon using SOCS2(+/+)/Apc(Min/+), SOCS2(+/-)/Apc(Min/+), and SOCS2(-/-)/Apc(Min/+) mice and assayed hematocrit as an indirect marker of disease severity. Biochemical and histological assays were used to assess mechanisms. Heterozygous and homozygous disruption of SOCS2 alleles promoted 166 and 441% increases in tumor load in the small intestine, respectively, accelerated development of colon tumors, and caused severe anemia. SOCS2 deletion promoted significant increases in intestinal insulin-like growth factor-I mRNA but did not affect plasma insulin-like growth factor-I. Western blots and immunohistochemical analysis demonstrated that tumor and nontumor intestinal tissue of SOCS2(-/-)/Apc(Min/+) mice had increased serine 727 phosphorylation of signal transducer and activator of transcription 3 compared with SOCS2(+/+)/Apc(Min/+) mice. Moreover, electromobility shift assays showed that SOCS2 deletion did not alter signal transducer and activator of transcription 3 DNA binding. However, tumors and small intestine from SOCS2(-/-)/Apc(Min/+) showed dramatic increases in activator protein-1 (AP-1) DNA binding, and SOCS2 overexpression in vitro reduced levels of AP-1. These studies indicate that SOCS2 deletion promotes the spontaneous development of intestinal tumors driven by mutations in the adenomatous polyposis coli/beta-catenin pathway and activates AP-1. Therefore, reduced expression or epigenetic silencing of SOCS2 may serve as a useful biomarker for colorectal cancer risk.

Objective: Ileocaecal resection (ICR) is common in Crohn’s disease. Inflammation and fibrosis frequently recur at the site of anastomosis or in the small intestine (SI). No animal models of postsurgical inflammation and fibrosis exist. A model of ICR was developed in interleukin 10 (IL10) null and wild-type (WT) mice to test the hypothesis that ICR promotes postsurgical inflammation and fibrosis in the SI or anastomosis of genetically susceptible IL10 null, but not WT or germ-free (GF)-IL10 null mice. Methods: GF-IL10 null mice were conventionalised (CONV) and 3 weeks later randomised to ICR, transection (T) or no treatment (NoTx). Age-matched conventionally raised (CONV) WT and GF-IL10 null mice received ICR, T or NoTx. Animals were killed 28 days later. Histological scoring, real-time PCR for tumour necrosis factor α and collagen, and immunostaining for CD3 + T cells assessed inflammation and fibrosis. Results: After ICR, CONV-IL10 null, but not CONV-WT mice, developed significant inflammation and fibrosis in the SI and inflammation in anastomosis compared with NoTx or T controls. Fibrosis occurred in the anastomosis of both CONV-IL10 null and CONV-WT mice following ICR. GF-IL10 null mice developed little or no inflammation or fibrosis in the SI or anastomosis after ICR. Conclusions: ICR in CONV-IL10 null mice provides a new animal model of postsurgical inflammation and fibrosis in the SI and anastomosis. Absence of inflammation and fibrosis in the SI of CONV-WT and GF-IL10 null mice following ICR indicates that postsurgical small bowel disease occurs only in genetically susceptible IL10 null mice and is bacteria dependent.

Background Current capsule endoscopy (CE) provides minimally invasive technology for GI imaging but has limited ability to discriminate different types of polyps. Near infrared fluorescent (NIRF) probes activated by biomarkers upregulated in adenomas (eg, cathepsin B) are potentially powerful tools to distinguish premalignant or malignant lesions from benign or inflammatory lesions. Objectives To examine whether CE can be integrated with NIRF probes to detect adenomas and whether cathepsin B–activated NIRF probes are activated by benign or inflammatory lesions. Design Mouse models of adenomas, hyperplactic/lymphoid polyps, and acute or chronic intestinal inflammation were injected intravenously with a cathepsin B–activated probe (Prosense 680). Dissected intestine was imaged with CE under white or NIRF light. For NIRF excitation (680 nm), dichroic and emission (700 nm) filters were combined with CE when images were recorded. Prosense 680 samples with or without protease were used as positive and negative controls. CE-based imaging data were verified by using and independent imaging system (Xenogen IVIS system). Main Outcome Measurements Proof of principal that CE integrated with NIRF probes can detect and discriminate adenomas from other lesions. Results CE-based NIRF imaging with Prosense 680 readily visualized adenomas, including in the colitis model. NIRF signals of different intensities were detected. Prosense 680 was not activated by benign or inflammatory lesions. Limitation Optical filters external to the capsule were used. Conclusions We demonstrate proof of the principle that biochromoendoscopy—CE combined with molecular probes––provides a novel approach that differentiates adenomas from benign polyps and inflammatory lesions.

Purpose: Langerhans cell histiocytosis (LCH) is a myeloproliferative disorder with clinical manifestations ranging from single lesions to lethal multi-organ disease, and front-line therapy fails in the majority of patients. No histologic features of LCH lesions have yet been demonstrated to correlate with clinical outcomes. We hypothesize that function of differentiated CD207+, including interactions with recruited activated lymphocytes, results in plasma protein profiles that reflect disease burden and tissue-specific DC-lymphocyte interactions. Plasma proteins may therefore inform mechanisms of pathogenesis and serve to predict risk and follow disease burden in LCH patients. Methods: We evaluated inflammatory proteins in the plasma of LCH patients using the Luminex platform. We compared plasma concentrations of 121 analytes in 178 patients with LCH to 90 controls with discovery and validation sets. Comparisons were made based on disease status, age, and clinical risk categories. 152 samples collected from 78 patients at serial time points were also analyzed. Comparisons were made between diagnosis and cure, as well as multiple time points in therapy. Results: Thirty-one analytes were significantly different between all LCH and controls, including proteins mediating chemotaxis and differentiation of lymphocytes and/or dendritic cells: osteopontin, SDF1A, 6CKine, CCL19, CCL20, sIL4R, IL20, and IL23. Twenty-three analytes were significantly different when comparing adult vs pediatric LCH. When comparing LCH vs control by age groups, 5 were significantly different in adults and 23 were different in children. Eighteen were different in pediatric low risk compared to high risk patients, with the most significant differences in inflammatory proteins including sTNFRI, sTNFRII, TNFa, sIL2Ra, and IL-8. Classifier analysis was able to predict risk organ involvement with a sensitivity of 91.5% and a specificity of 87.5%. Seventeen analytes changed significantly after curative treatment. Conclusions: Distinct Plasma protein profiles exist in LCH, suggesting pathologic myeloid cells drive the inflammatory pathology of disease. Profiles are vastly different among children and adults with LCH, and differ based on burden of disease, suggesting separate inflammatory pathology. Seven analytes reliably predicted high-risk disease, and analysis of serial samples identified normalization of inflammatory proteins after cure. Disclosures Allen: Roche: Consultancy, Other: unpaid; NovImmune: Consultancy, Other: unpaid.

Introduction: Langerhans cell histiocytosis (LCH) is a disease characterized by inflammatory lesions including pathologic CD207+ dendritic cells. Clinically, LCH is highly variable ranging from single lesions to highly aggressive, disseminated disease involving multiple organs and requiring intensive chemotherapy. Recent data support a model of pathogenesis in which activating somatic mutations in MAPK pathway genes arise in myeloid DC precursors. However, little is known about genetic susceptibility to this condition. Therefore, we conducted a genome-wide association study to characterize the role of inherited genetic variants on disease risk. Methods: We utilized a case-parent trio approach, which is immune to the effects of population stratification bias. Specifically, this allows for the inclusion of individuals regardless of genetic ancestry. LCH case-parent trios (n=134) were recruited from Texas Children's Cancer Center. Genotyping was performed using the Illumina Omni-5 Quad BeadChip. Genetic ancestry was determined using the bioinformatics algorithm STRUCTURE. To inform this algorithm, a set of 12,898 autosomal ancestry informative markers specifically identified to infer population substructure was extracted from the study trios. Estimated genomic ancestral proportions were then used to classify each study participant as either of European ancestry, Amerindian ancestry, or of African ancestry. For the association analysis, we focused on the role of common variants (i.e., minor allele frequency ≥5%). The association analysis was conducted utilizing the PREMIM-EMIM algorithm, an established, multinomial log-likelihood approach for assessing case-parent trios GWAS data. This method allows for the inclusion of "incomplete" trios (e.g., mother-case duos). We applied a genome-wide statistical significance cutoff of p Results: In this GWAS, LCH cases were predominantly male (54%), and based on the genetic ancestry analysis, 60% were of European ancestry, 36% Amerindian ancestry, and 4% African ancestry. Among the 343 individuals included in the analysis, 1,672,105 SNPs autosomal SNPs were assessed and an overview of the results is displayed using a Manhattan plot. We identified five potential inherited genomic regions associated with LCH susceptibility. The strongest associations between inherited SNPs and childhood LCH were seen in SMAD6 on chromosome 15 (p-value = 2.38x10-7) and in ECE1 on chromosome 1 (p-value = 2.15x10-6). Conclusions: In this genome-wide assessment of the role of inherited genetic variation on the risk of LCH, we identified SNPs with significant effects in genes implicated in diverse pathways including embryogenesis and cellular division. Among the proteins encoded by the regions identified, SMAD6and ECE1 have both been reported to impact ERK activation, a critical feature of LCH pathogenesis. These findings support potential for inherited genetic variants to influence risk of developing LCH. Disclosures Allen: NovImmune: Consultancy, Other: unpaid; Roche: Consultancy, Other: unpaid.

Suppressors of cytokine signaling (SOCS) typically limit cytokine receptor signaling via the JAK-STAT pathway. Considerable evidence demonstrates that SOCS2 limits growth hormone (GH) action on body and organ growth. Biochemical evidence that SOCS2 binds to the IGF-I receptor (IGF-IR) supports the novel possibility that SOCS2 limits IGF-I action. The current study tested the hypothesis that SOCS2 normally limits basal or IGF-I-induced intestinal growth and limits IGF-IR signaling in intestinal epithelial cells. Intestinal growth was assessed in mice homozygous for SOCS2 gene deletion (SOCS2 null) and wild-type (WT) littermates at different ages and in response to infused IGF-I or vehicle or EGF and vehicle. The effects of SOCS2 on IGF-IR signaling were examined in ex vivo cultures of SOCS2 null and WT intestine and Caco-2 cells. Compared with WT, SOCS2 null mice showed significantly enhanced small intestine and colon growth, mucosal mass, and crypt cell proliferation and decreases in radiation-induced crypt apoptosis in jejunum. SOCS2 null mice showed significantly greater growth responses to IGF-I in small intestine and colon. IGF-I-stimulated activation of IGF-IR and downstream signaling intermediates were enhanced in the intestine of SOCS2 null mice and were decreased by SOCS2 overexpression in Caco-2 cells. SOCS2 bound directly to the endogenous IGF-IR in Caco-2 cells. The intestine of SOCS2 null mice also showed enhanced growth responses to infused EGF. We conclude that SOCS2 normally limits basal and IGF-I- and EGF-induced intestinal growth in vivo and has novel inhibitory effects on the IGF-IR tyrosine kinase pathway in intestinal epithelial cells.

Accurate and high-throughput technologies are needed for identification of new therapeutic targets and for optimizing therapy in inflammatory bowel disease. Our aim was to assess multi-analyte protein-based assays of cytokines/chemokines using Luminex technology. We have reported that Luminex-based profiling was useful in assessing response to L-arginine therapy in the mouse model of dextran sulfate sodium colitis. Therefore, we studied prospectively collected samples from ulcerative colitis (UC) patients and control subjects. Serum, colon biopsies, and clinical information were obtained from subjects undergoing colonoscopy for evaluation of UC or for non-UC indications. In total, 38 normal controls and 137 UC cases completed the study. Histologic disease severity and the Mayo Disease Activity Index (DAI) were assessed. Serum and colonic tissue cytokine/chemokine profiles were measured by Luminex-based multiplex testing of 42 analytes. Only eotaxin-1 and G-CSF were increased in serum of patients with histologically active UC vs. controls. While 13 cytokines/chemokines were increased in active UC vs. controls in tissues, only eotaxin-1 was increased in all levels of active disease in both serum and tissue. In tissues, eotaxin-1 correlated with the DAI and with eosinophil counts. Increased eotaxin-1 levels were confirmed by real-time PCR. Tissue eotaxin-1 levels were also increased in experimental murine colitis induced by dextran sulfate sodium, oxazolone, or Citrobacter rodentium, but not in murine Helicobacter pylori infection. Our data implicate eotaxin-1 as an etiologic factor and therapeutic target in UC, and indicate that Luminex-based assays may be useful to assess IBD pathogenesis and to select patients for anti-cytokine/chemokine therapies.

Background: L-arginine (L-Arg) is the substrate for both inducible nitric oxide synthase and arginase, which are upregulated in IBD and in mouse colitis models. L-Arg uptake by cells is mediated by cationic amino acid transporter (CAT) proteins, and CAT2 is the inducible form. We have found that L-Arg supplementation enhances restitution in wound repair models In Vitro, and improves clinical parameters of weight loss, survival, and colon weight/length, but not histology, in experimental colitis in mice induced by dextran sulfate sodium (DSS). In CAT2-/mice there is exacerbation of DSS colitis, accumulation of L-Arg in serum, and favorable effects of L-Arg treatment are lost, indicating that inducible uptake of L-Arg is involved in compensatory host responses. Our aim was to define mechanisms underlying the clinical benefit of L-Arg supplementation. Methods: Male C57BL/6 mice were given 4% DSS for 6 days followed by 4 days of 1% L-Arg (DSS/L-Arg group) or regular water (DSS group); controls were given water alone for 10 days (control group), or for 6 days followed by L-Arg for 4 days (control/L-Arg group). Colon tissue from 3 mice in each group was obtained for RNA isolation and then microarray gene expression analysis. The Affymetrix® Mouse Gene 1.0 ST array containing >16,000 protein-coding genes was used. Hierarchical clustering of global gene expression patterns was performed. Differentially expressed genes (DEGs) were selected by both a p value 2. Functional features of the DEGs were evaluated by Ingenuity Pathway Analysis. Results: Hierarchical clustering comparing expression values in the 12 samples revealed that the DSS group was markedly distinct from the control or the control/L-Arg groups. Surprisingly, the DSS/L-Arg group segregated closely with the two control groups not receiving DSS, rather than with the DSS group. There were 342 DEGs with DSS versus control, but only 59 of these genes were altered in DSS/L-Arg versus control. We specifically identified 132 genes upregulated in response to DSS that were downregulated to control levels after L-Arg supplementation. There were also 27 genes that were downregulated with DSS and returned to control levels in the DSS/L-Arg. The detailed patterns were analyzed by heatmaps, that showed marked differences with DSS that reverted to a control pattern in the DSS/L-Arg group. The pathways enriched with DEGs between DSS/L-Arg and DSS included cell-to-cell signaling (leukocyte extravasation; IL-17, IL-8, and IL-10 signaling), cell migration, and cell proliferation pathways.Conclusions:Ourmicroarray gene expression analysis indicates that L-Arg supplementation dramatically alters gene expression patterns in the DSS model, and plays a substantive role in repair, mucosal integrity, and regulation of inflammation.

molecular insights to IBD, we find 17-AAG use profoundly suppresses development of DSSinduced colitis. In another model of colitis, adoptive transfer of CD4+CD45Rbhi cells to Rag deficient mice leads to colitis at ~50 days, and compared with DMSO-treated controls, mice treated with 17-AAG did not develop colitis, gained weight(p