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Understanding how asthma biomarkers relate to gene expression signatures could help identify drivers of pathogenesis.This post hoc exploratory analysis of the phase II tralokinumab trial MESOS (ClinicalTrials.gov identifier NCT02449473) aimed to profile baseline airway inflammation in patients with moderate-to-severe asthma.The T2 and T17 gene expression signatures, 3-gene mean and 5-gene mean, were calculated through transcriptomic analysis of baseline bronchial brushing samples. Clustering analysis using these signatures identified 3 distinct inflammatory subgroups: T2Fractional exhaled nitric oxide (Feno) levels were highest for T2Feno level may be useful to differentiate patients with T2 or T17 gene expression. Elevated Feno levels are associated with high CST1 expression.

Background Eosinophilic inflammation has been implicated in the pathogenesis, severity, and treatment responsiveness of chronic rhinosinusitis with nasal polyps (CRSwNP). Objective We sought to assess the efficacy and safety of benralizumab-mediated eosinophil depletion for treating CRSwNP. Methods The phase 3 OSTRO study enrolled patients with severe CRSwNP who were symptomatic despite treatment with intranasal corticosteroids and who had a history of systemic corticosteroid (SCS) use and/or surgery for nasal polyps (NP). Patients were randomized 1:1 to treatment with benralizumab 30 mg or placebo every 4 weeks for the first 3 doses and every 8 weeks thereafter. Coprimary end points were change from baseline to week 40 in NP score (NPS) and patient-reported mean nasal blockage score reported once every 2 weeks. Results The study population comprised 413 randomized patients (207 in the benralizumab group and 206 in the placebo group). Benralizumab significantly improved NPS and nasal blockage score compared to placebo at week 40 (P ≤ .005). Improvements in Sinonasal Outcome Test 22 score at week 40, time to first NP surgery and/or SCS use for NP, and time to first NP surgery were not statistically significant between treatment groups. Nominal significance was obtained for improvement in difficulty in sense of smell score at week 40 (P = .003). Subgroup analyses suggested influences of comorbid asthma, number of NP surgeries, sex, body mass index, and baseline blood eosinophil count on treatment effects. Benralizumab was safe and well tolerated. Conclusion Benralizumab, when added to standard-of-care therapy, reduced NPS, decreased nasal blockage, and reduced difficulty with sense of smell compared to placebo in patients with CRSwNP. Trial registration: ClinicalTrials.gov NCT03401229

Periostin and dipeptidyl peptidase-4 (DPP-4) are proteins induced by type 2 cytokines interleukin (IL)-4 and IL-13 and show increased expression in asthma and diseases with type 2 inflammation, including atopic dermatitis and chronic rhinosinusitis. Both proteins can also be induced by other stimuli, such as profibrotic factors, which may confound their specificity as biomarkers of IL-13 pathway activation and type 2-driven disease. DPP-4 is important in glucose metabolism; therefore, serum concentrations may be confounded by the presence of concomitant metabolic disease. This review evaluates the potential of these biomarkers for anti-IL-13-directed therapy in asthma and diseases with type 2 inflammation.

Background & Aims The incidence of nonalcoholic steatohepatitis (NASH) is increasing. The pathophysiological mechanisms of NASH and the sequence of events leading to hepatic fibrosis are incompletely understood. The aim of this study was to gain insight into the dynamics of key molecular processes involved in NASH and to rank early markers for hepatic fibrosis. Methods A time-course study in low-density lipoprotein–receptor knockout. Leiden mice on a high-fat diet was performed to identify the temporal dynamics of key processes contributing to NASH and fibrosis. An integrative systems biology approach was used to elucidate candidate markers linked to the active fibrosis process by combining transcriptomics, dynamic proteomics, and histopathology. The translational value of these findings were confirmed using human NASH data sets. Results High-fat-diet feeding resulted in obesity, hyperlipidemia, insulin resistance, and NASH with fibrosis in a time-dependent manner. Temporal dynamics of key molecular processes involved in the development of NASH were identified, including lipid metabolism, inflammation, oxidative stress, and fibrosis. A data-integrative approach enabled identification of the active fibrotic process preceding histopathologic detection using a novel molecular fibrosis signature. Human studies were used to identify overlap of genes and processes and to perform a network biology-based prioritization to rank top candidate markers representing the early manifestation of fibrosis. Conclusions An early predictive molecular signature was identified that marked the active profibrotic process before histopathologic fibrosis becomes manifest. Early detection of the onset of NASH and fibrosis enables identification of novel blood-based biomarkers to stratify patients at risk, development of new therapeutics, and help shorten (pre)clinical experimental time frames., Graphical abstract

Background: In HIV infection, lymphoid tissue is disrupted by fibrosis. Angiotensin converting enzyme inhibitors have anti-fibrotic properties. We completed a pilot study to assess whether the addition of lisinopril to antiretroviral therapy (ART) reverses fibrosis of gut tissue, and whether this leads to reduction of HIV RNA and DNA levels. Methods: Thirty HIV-infected individuals on ART were randomized to lisinopril at 20mg daily or matching placebo for 24 weeks. All participants underwent rectal biopsies prior to starting the study drug and at 22 weeks, and there were regular blood draws. The primary end point was the change in HIV RNA and DNA levels in rectal tissue. Secondary outcomes included the change in 1) HIV levels in blood; 2) Gag-specific T-cell responses; 3) levels of T-cell activation; and 4) collagen deposition. Results: The addition of lisinopril did not have a significant effect on the levels of HIV RNA or DNA in gut tissue or blood, Gag-specific responses, or levels of T-cell activation. Lisinopril also did not have a significant impact on lymphoid fibrosis in the rectum, as assessed by quantitative histology or heavy water labeling. Conclusions: Treatment with lisinopril for 24 weeks in HIV-infected adults did not have an effect on lymphoid fibrosis, immune activation, or gut tissue viral reservoirs. Further study is needed to see if other anti-fibrotic agents may be useful in reversing lymphoid fibrosis and reducing HIV levels. Clinical Trials Registration: NCT01535235

Excess collagen synthesis (fibrogenesis) in the liver plays a causal role in the progression of nonalcoholic fatty liver disease (NAFLD). Methods are needed to identify patients with more rapidly progressing disease and to demonstrate early response to treatment. We describe here a novel method to quantify hepatic fibrogenesis flux rates both directly in liver tissue and non-invasively in blood. Twenty-one patients with suspected NAFLD ingested heavy water (2H2O, 50 ml aliquots) 2-3 times daily for 3 to 5 weeks prior to a clinically indicated liver-biopsy. Liver collagen fractional synthesis rate (FSR) and plasma lumican FSR were measured based on 2H labeling using tandem mass spectrometry. Patients were classified by histology for fibrosis stage (F0-F4) and as having non-alcoholic fatty liver (NAFL) or non-alcoholic steatohepatitis (NASH). Magnetic resonance elastography measurements of liver stiffness were also performed. Hepatic collagen FSR in NAFLD increased with advancing disease stage (e.g. higher in NASH than NAFL, positive correlation with fibrosis score and liver stiffness) and correlated with hemoglobin A1C. In addition, plasma lumican FSR demonstrated a significant correlation with hepatic collagen FSR. Conclusion: Utilizing a well-characterized cohort of patients with biopsy-proven NAFLD, this study demonstrates that hepatic scar in NASH is actively remodeled even in advanced fibrosis, a disease that is generally regarded as static and slowly progressive. Moreover, hepatic collagen FSR correlates with established risks for fibrotic disease progression in NASH and plasma lumican FSR correlates with hepatic collagen FSR, suggesting applications as direct or surrogate markers, respectively, of hepatic fibrogenesis in humans. This article is protected by copyright. All rights reserved.

Pulmonary arterial remodeling characterized by increased vascular smooth muscle density is a common lesion seen in pulmonary arterial hypertension (PAH), a deadly condition. Clinical correlation studies have suggested an immune pathogenesis of pulmonary arterial remodeling, but experimental proof has been lacking. We show that immunization and prolonged intermittent challenge via the airways with either of two different soluble antigens induced severe muscularization in small- to medium-sized pulmonary arteries. Depletion of CD4+ T cells, antigen-specific T helper type 2 (Th2) response, or the pathogenic Th2 cytokine interleukin 13 significantly ameliorated pulmonary arterial muscularization. The severity of pulmonary arterial muscularization was associated with increased numbers of epithelial cells and macrophages that expressed a smooth muscle cell mitogen, resistin-like molecule α, but surprisingly, there was no correlation with pulmonary hypertension. Our data are the first to provide experimental proof that the adaptive immune response to a soluble antigen is sufficient to cause severe pulmonary arterial muscularization, and support the clinical observations in pediatric patients and in companion animals that muscularization represents one of several injurious events to the pulmonary artery that may collectively contribute to PAH.

Using a single vector targeting strategy, we have generated mice with a combined deficiency of interleukin (IL)-4 and IL-13 to clarify their roles in T helper type 2 (Th2) cell responses. Using immunological challenges normally characterized by a Th2-like response, we have compared the responses of the double-deficient mice with those generated by wild-type, IL-4–deficient, and IL-13–deficient mice. Using a pulmonary granuloma model, induced with Schistosoma mansoni eggs, we demonstrate that although eosinophil infiltration, immunoglobulin E, and IL-5 production are reduced in the IL-4–deficient mice and IL-13–deficient mice, they are abolished only in the combined absence of both cytokines. Furthermore, IL-4/13–deficient animals are severely impaired in their ability to expel the gastrointestinal nematode Nippostrongylus brasiliensis. Unexpectedly, N. brasiliensis–infected IL-4/13–deficient mice developed elevated IL-5 and eosinophilia, indicating that compensatory mechanisms exist for the expression of IL-5, although serum IgE remained undetectable. IL-4/13–deficient mice default to a Th1-like phenotype characterized by the expression of interferon γ and the production of IgG2a and IgG2b. We conclude that IL-4 and IL-13 cooperate to initiate rapid Th2 cell–driven responses, and that although their functions overlap, they perform additive roles.

We report that Th2 cell cultures generated using T cells or splenocytes from IL-13-deficient mice produce significantly reduced levels of IL-4, IL-5, and IL-10 compared with wild-type. In contrast, IL-4 and IL-5 production by mast cells stimulated in vitro with PMA, ionomycin, or IgE cross-linking are unaffected. In vitro Th2 cell differentiation cannot be rescued by the addition of exogenous factors, but in vivo antigen challenge and administration of IL-13 can increase Th2-like cytokine responses as can infection with the parasitic nematode Nippostrongylus brasiliensis. IL-13-deficient mice also have lower basal levels of serum IgE and biased antigen-specific immunoglobulin responses. Thus, IL-13 is an important regulator of Th2 commitment and may therefore play a central role in atopy and infectious diseases.

Background Biomarkers facilitate early detection of disease and measurement of therapeutic efficacy, both at clinical and experimental levels. Recent advances in analytics and disease models allow comprehensive screening for biomarkers in complex diseases, such as asthma, that was previously not feasible. Objective Using murine and nonhuman primate (NHP) models of asthma, identify biomarkers associated with early and chronic stages of asthma and responses to steroid treatment. Methods The total protein content from thymic stromal lymphopoietin transgenic (TSLP Tg) mouse BAL fluid was ascertained by shotgun proteomics analysis. A subset of these potential markers was further analyzed in BAL fluid, BAL cell mRNA, and lung tissue mRNA during the stages of asthma and following corticosteroid treatment. Validation was conducted in murine and NHP models of allergic asthma. Results Over 40 proteins were increased in the BAL fluid of TSLP Tg mice that were also detected by qRT-PCR in lung tissue and BAL cells, as well as in OVA-sensitive mice and house dust mite-sensitive NHP. Previously undescribed as asthma biomarkers, KLK1, Reg3γ, ITLN2, and LTF were modulated in asthmatic mice, and Clca3, Chi3l4 (YM2), and Ear11 were the first lung biomarkers to increase during disease and the last biomarkers to decline in response to therapy. In contrast, GP-39, LCN2, sICAM-1, YM1, Epx, Mmp12, and Klk1 were good indicators of early therapeutic intervention. In NHP, AMCase, sICAM-1, CLCA1, and GP-39 were reduced upon treatment with corticosteroids. Conclusions and clinical relevance These results significantly advance our understanding of the biomarkers present in various tissue compartments in animal models of asthma, including those induced early during asthma and modulated with therapeutic intervention, and show that BAL cells (or their surrogate, induced sputum cells) are a viable choice for biomarker examination.

Background Interleukin (IL)-19 has been reported to enhance chronic inflammatory diseases such as asthma but the in vivo mechanism is incompletely understood. Because IL-19 is produced by and regulates cells of the monocyte lineage, our studies focused on in vivo responses of CD11c positive (CD11c+) alveolar macrophages and lung dendritic cells. Methodology/Principal Findings IL-19-deficient (IL-19-/-) mice were studied at baseline (naive) and following intranasal challenge with microbial products, or recombinant cytokines. Naive IL-19-/- mixed background mice had a decreased percentage of CD11c+ cells in the bronchoalveolar-lavage (BAL) due to the deficiency in IL-19 and a trait inherited from the 129-mouse strain. BAL CD11c+ cells from fully backcrossed IL-19-/- BALB/c or C57BL/6 mice expressed significantly less Major Histocompatibility Complex class II (MHCII) in response to intranasal administration of lipopolysaccharide, Aspergillus antigen, or IL-13, a pro-allergic cytokine. Neurogenic-locus-notch-homolog-protein-2 (Notch2) expression by lung monocytes, the precursors of BAL CD11c+ cells, was dysregulated: extracellular Notch2 was significantly decreased, transmembrane/intracellular Notch2 was significantly increased in IL-19-/- mice relative to wild type. Instillation of recombinant IL-19 increased extracellular Notch2 expression and dendritic cells cultured from bone marrow cells in the presence of IL-19 showed upregulated extracellular Notch2. The CD205 positive subset among the CD11c+ cells was 3-5-fold decreased in the airways and lungs of naive IL-19-/- mice relative to wild type. Airway inflammation and histological changes in the lungs were ameliorated in IL-19-/- mice challenged with Aspergillus antigen that induces T lymphocyte-dependent allergic inflammation but not in IL-19-/- mice challenged with lipopolysaccharide or IL-13. Conclusions/Significance Because MHCII is the molecular platform that displays peptides to T lymphocytes and Notch2 determines cell fate decisions, our studies suggest that endogenous IL-19 is a constituent of the regulome that controls both processes in vivo.

To the editor: In the July 29, 2010, issue of Blood , Pillay et al presented a novel study on the kinetics of neutrophils in humans.[1][1] Their study used deuterium in heavy water to label proliferating cells in the marrow. Using a first-order model, the authors found that the neutrophil

BACKGROUND: In patients with chronic lymphocytic leukemia (CLL), the Bruton tyrosine kinase(BTK) inhibitor ibrutinib induces a rapid reduction in lymphadenopathy, accompanied by a transient lymphocytosis. Continuation of therapy leads to a reduction in peripheral blood disease burden with achievement of objective responses in a majority of patients over time. Inhibition of B cell receptor (BCR) and chemokine receptor signaling are considered the mechanisms for redistribution lymphocytosis and CLL cell death. However, these mechanisms of action have not been formally documented directly in patients. To address this, before initiating therapy with ibrutinib, we labeled the DNA of proliferating CLL cells with deuterium (2H) by asking patients to drink deuterated water (2H2O) and then determined the effects of ibrutinib on leukemia cell kinetics (proliferation and death rates) and mobilization of cells from lymphoid tissues (trafficking) after ibrutinib administration. METHODS: 30 previously untreated CLL/SLL patients were enrolled between December 2012 and June 2013 at MD Anderson Cancer Center. Patients required treatment per iwCLL guidelines, had adequate hematopoietic (platelets > 50,000/μL, ANC > 750/μL) and organ functions. Patients drank 50 mL of 70% 2H2O 3 times a day for 5 days, followed by 60 mL daily for a total of 4 weeks (“labeling phase”). After a 6-12 week “washout phase”, patients started once-daily ibrutinib 420 mg continuously on 28 day cycles. Study objectives were to determine the impact on CLL cell proliferation and death rates, before and after ibrutinib, and on CLL cell trafficking after ibrutinib. PATIENT CHARACTERISTICS: Median age of the 30 patients was 64 years (range 48–78) with 19 males. 16 patients exhibited early stage (Rai stage 0-2), and 14 advanced stage (Rai stage 3-4) disease. 17 patients expressed unmutated IGHVs, 11 patients had mutated IGHVs, and 2 had inconclusive IGHV results. 8 were CD38+ and 15 ZAP-70 + by immunohistochemistry. FISH cytogenetics revealed 11 patients with del13q, 6 with trisomy 12, 3 with del17p or TP53 mutation, 4 with del11q, and 6 without abnormalities. Median β2 microglobulin level was 2.8 mg/L (1.6 - 7.4). RESPONSE TO THERAPY: At a median follow up of 13 months, 28 of 30 patients continued on therapy without disease progression. All patients were evaluable for response assessment per 2008 IWCLL guidelines: 28 (93%) achieved partial remission, 1 (3%) complete remission, and 1 (3%) had stable disease, yielding an ORR of 97%. Two patients came off study at 184 and 480 days, respectively. CLL CELL KINETICS AND TRAFFICKING: The average CLL cell birth rate determined before ibrutinib therapy was 0.42% per day (range 0.32 – 1.42%). After initiating ibrutinib, there was a rapid increase in absolute lymphocyte count in the blood in 26 of 30 patients; this contained previously labeled CLL cells rather than newly-divided unlabeled cells. Over time, the proportion of circulating CLL cells that were labeled did not decrease appreciably despite a concomitant fall in circulating lymphocytes, indicating that newly divided cells were not entering the circulation and strongly suggesting that ibrutinib had a major inhibitory effect on leukemia cell proliferation. In 3 of the 4 patients in whom there was a rapid influx of unlabeled cells with the onset of ibrutinib treatment, maintenance of circulating labeled cells concomitant with a significant fall in circulating lymphocytes suggested proliferation of CLL cells was inhibited with extended treatment. Prior to ibrutinib therapy, the measured proliferation rate of CLL cells was 0.42 % per day. Because after ibrutinib therapy both cell proliferation and exiting from the blood were markedly reduced, the elimination rate should equal the death rate. Notably, with treatment the elimination rate of CLL cells was much faster than before ibrutinib, revealing a “true” death rate of 1.48 % of the clone per day. CONCLUSIONS: Using 2H-labelling of dividing leukemic cells, we have shown for the first time directly in CLL patients that ibrutinib significantly inhibits leukemia cell proliferation, leads to an efflux of CLL cells from tissues into the blood, and promotes a remarkably high death rate of CLL cells in an apparently indirect manner, likely due to the interruption of survival signals from the BCR and other receptors that are engaged in lymphoid tissues. Disclosures Burger: Pharmacyclics, Inc.: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees. Li:KineMed, Inc.: Employment, Equity Ownership. O'Brien:Pharmacyclics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Hellerstein:KineMed Inc: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Turner:KineMed, Inc.: Employment, Equity Ownership. Emson:KineMed, Inc.: Employment, Equity Ownership. Chiorazzi:Pharmacyclics, Inc.: Research Funding; KineMed: Equity Ownership.

Leishmania major infection is useful as an experimental model to define factors responsible for the development and maintenance of Th cell immune responses. Studies using inbred mouse strains have identified that the Th1 response characteristic of C57BL/6 mice results in healing, whereas BALB/c mice fail to control the infection due to the generation of an inappropriate Th2 response. We now demonstrate that IL-13 is a key factor in determining susceptibility to L. major infection. Overexpression of IL-13 in transgenic mice makes the normally resistant C57BL/6 mouse strain susceptible to L. major infection even in the absence of IL-4 expression. This susceptibility correlates with a suppression of IL-12 and IFN-γ expression. Furthermore, using BALB/c mice deficient in the expression of IL-4, IL-13, or both IL-13 and IL-4, we demonstrate that IL-13-deficient mice are resistant to infection and that there is an additive effect of deleting both IL-4 and IL-13.

Recent studies using interleukin (IL)-4–deficient animals have highlighted the existence of IL-4–independent immunoglobulin (Ig)E induction. We have established transgenic mice expressing IL-13 from a transgene comprising a genomic fragment containing the IL-13 gene and the human CD2 locus control region. The transgenes were expressed in lymphoid tissues and induced by T cell activators, suggesting regulation by elements of the IL-13 promoter. IL-13 transgenic lines expressed 10–100-fold higher levels of serum IgE than their littermate controls, but had normal levels of other serum Ig isotypes. Elevated IgE levels were also detected in sera from IL-4–deficient mice carrying IL-13 transgenes, indicating that IL-4 is not required for IL-13–induced IgE expression in the mouse. Expression of IL-13 also perturbed the development of thymocytes. Although thymocyte development was normal up to 4 wk of age, thymocyte number decreased dramatically thereafter, reaching 10% of normal by 10 wk, and despite normal size and appearance, histological examination demonstrated that transgenic thymi contained only small foci of thymocytes. The reduction in thymocyte number was due mainly to a depletion of CD4+CD8+ thymocytes, and did not affect significantly the composition of peripheral T cell populations. These data indicate that expression of IL-13 transgenes in vivo can regulate IgE production in the mouse, and that IL-13 may also influence thymocyte development.

Abstract 184 Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of mature B cells in peripheral blood (PB), bone marrow (BM) and lymph node (LN). Recent studies identified the LN as an important site of tumor cell activation and proliferation (Herishanu et al 2011). Using in vivo labeling of “newly-born” cells with deuterated water (2H2O; heavy water), the proliferation rate of CLL cells was estimated to range between 0.1 to >1% of the clone per day (Messmer et al 2005). Furthermore, a CXCR4dimCD5bright population of CLL cells in the PB contained more deuterium (2H)-labeled DNA and hence “recently-born cells than the CXCR4brightCD5dim population (Calissano et al 2011). Possible explanations for this observation include that CXCR4dimCD5bright cells proliferate more rapidly in the PB or that these cells are recent emigrants from tissues where proliferation occurs. As prior studies were performed on PB cells, the growth rates and characterization of the proliferative fraction of CLL cells in the LN remain unknown. Here we used 2H-labelling to directly compare cellular growth rates in PB, BM, and LN. Patients drank 2H2O for 28 days; on day 13 an excisional LN biopsy and a BM aspirate were obtained. PB samples were obtained at baseline and on days 13 and 28. CLL cells were isolated using positive selection, or sorted based on reciprocal differences in CXCR4 and CD5 density for isolation of “proliferative” (CXCR4dimCD5bright), “intermediate” (CXCR4intCD5int), and “resting” (CXCR4brightCD5dim) fractions of CLL clones. 2H incorporation into the DNA of newly divided cells was measured by mass spectrometry. Raw values were normalized to the 2H2O content in total body water. Cellular growth rates were calculated by dividing the fraction of 2H-labeled cells by the number of days from the start of the labeling period. To date, samples from 6 treatment-naïve CLL patients have been analyzed. On day 13, up to 16%, 9%, and 24% of the CLL cells sampled from PB, BM, and LN, respectively were 2H-labeled. The resulting mean estimated growth rate in % of the clone per day was for PB 0.41 (0.09 – 1.13), for BM 0.41 (0.28 – 0.68), and for LN 0.83 (0.31 – 1.84). The difference in growth rates between PB and LN was statistically significant (P Taken together our data show that the proliferation rate of CLL cells is higher in the LN than in the BM and PB and suggest that some of the newborn cells exit the LN within days. A clonal subset of CXCR4dimCD5bright cells is present in both the LN and PB and might harbor the proliferative core of the disease. Disclosures: Gatmaitan: KineMed: Employment. Emson:KineMed: Employment. Chiorazzi:KineMed: Dr. Chiorazzi holds stock options in KineMed, Inc. Other.

Interleukin 19 (IL-19) is thought to have important roles in the immune control of pathogens, and in enhancing chronic inflammatory diseases of the skin (psoriasis), lung (asthma) and intestine (colitis). However, the biological function and the networked control of IL-19 are poorly understood. Naive mice deficient in IL-19 (IL-19-/-) on a 129xBL6 background had decreased alveolar macrophages and increased eosinophils in the broncho-alveolar lavage (BAL) and decreased B cells in the spleen and bone marrow. However, naïve IL-19-/- mice backcrossed to C57BL/6 or BALB/c, and mice housed in different locations exhibited an attenuated phenotype. Therefore, genetic backcross and challenge experiments with environmental stimuli (allergen, urban particulate matter) were conducted. The studies showed that abnormal composition of the cells in the BAL fluid, spleen and bone marrow in naïve IL-19-/- 129xBL6 mice was due to the deficiency in IL-19 combined with the activity of another gene inherited from the 129 strain. IL-19-/- mice on C57BL/6 or BALB/c backgrounds that were intranasally challenged with antigen or with antigen and urban particulate matter had decreased BAL eosinophils, alveolar macrophages and B cells when compared to wild type. Together our studies indicate that endogenous IL-19 is a critical component of immune homeostasis to the airborne environment and that mouse-strain specific genes control the function of IL-19.

Abstract 60 Chronic lymphocytic leukemia (CLL) was formerly considered an accumulative disease of immunologically incompetent lymphocytes that have a slow rate of cell turnover. However, recent studies suggest that CLL cells of some patients may have an unexpectedly high birth rate, which in some subjects, is counterbalanced by a relatively high death rate. In particular, studies on the leukemia cells of patients who ingested heavy water (2H2O) revealed that some patients had leukemia cells that incorporated deuterium into DNA at rates consistent with a birth rate of 1% of the leukemia population per day. This high birth rate, when counterbalanced with comparable rates of leukemia cell turnover, allows patients to maintain a steady state that was suggestive of indolent disease. Preliminary data also suggested a relationship between disease activity and CLL cell birth. To examine the association between measured rates of leukemia-cell turnover with established surrogate markers of aggressive disease, the CLL Research Consortium (CRC) recently completed enrolment of 119 patients with early stage disease. Patients were to be untreated, Rai Stage 0, 1, or 2 and diagnosed within the previous 3 years. The leukemia cells of each of these patients were evaluated by the central tissue core of the CRC for expression of ZAP-70, CD38, and immunoglobulin heavy chain variable region gene (IGHV) mutation status. We report here a subset analysis of baseline data for 59 patients (57% male, age 58.7 ± 10.1 y). Thirty-seven percent of patients (n=22) had leukemic cells that expressed unmutated IGHV, 39% (n=23) had cells that expressed ZAP-70 (> 20% positive by cytoplasmic flow cytometery), and 19% (n=11) had CD38 positive cells (> 30% positive by multiparameter flow cytometry). Associations between the traditional prognostic markers were exceedingly strong (ZAP-70 with mutation status, p < 0.001; ZAP-70 with CD38, p = 0.013; CD38 with mutation status, p =< 0.001). Birth rate was expressed as the peak percent new cells per day calculated from 2H enrichment in DNA using isotope ratio mass spectrometry (IRMS). Data from the previous study suggested more aggressive disease activity associated with a birth rate above 0.35% per day. Birth rates in the present study ranged from 0.07 - 1.31 % per day, spanning both sides of the previously published 0.35% per day cut-off. Using the Wilcoxon rank sum test, patients whose clones expressed unmutated IGHV, were ZAP-70 positive, or were CD38 positive had a significantly higher daily leukemia cell birth rates than subjects who were IGHV mutated (median of 0.39 v 0.24, p = 0.01), ZAP-70 negative (median of 0.36 v. 0.22, p = 0.002), or CD38 negative (0.62 V. 0.24, p = 0.001). Thus, we have shown that CLL cell birth rates can be determined in a multi-centered trial in a large number of patients, and that patients can be stratified into groups with varying rates of perceived cell turnover. The cell turnover data correlate directly in multivariate analysis with established surrogate markers of aggressive disease, a poor clinical course and decreased survival with higher birth rates associating with unmutated IGHV and expression of ZAP-70 and CD38. Clinical follow-up of these patients is underway to determine if these four parameters serve as complementary prognostic markers. Nevertheless, the strong association between established surrogate markers of aggressive disease with high leukemia-cell birth rates suggests that high-rates of leukemia-cell turnover define a biology that is associated with adverse outcome. As such, these studies strengthen the notion that leukemia cell populations that have high rates of cell turnover can more rapidly accumulate secondary and tertiary genetic mutations that potentially govern the progression of this disease. Disclosures: Murphy: KineMed, Inc.: Consultancy, Equity Ownership. Emson:KineMed, Inc.: Employment. Hayes:KineMed, Inc: Consultancy. Holochwost:KineMed, Inc: Employment. Hellerstein:KineMed, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Chiorazzi:KineMed, Inc: Membership on an entity's Board of Directors or advisory committees.

Pulmonary arterial hypertension is associated with pulmonary vascular remodeling (PVRm) and its incidence is increased in many autoimmune diseases and chronic inflammatory lung diseases. Currently it is not known whether inflammatory and immune responses are cause or consequence of PVRm. In this study immunized mice were intermittently exposed to inhaled soluble antigen over a period of several weeks. The antigen exposure induced dramatic PVRm in a CD4 T cell dependent manner. PVRm was characterized by hyperplasia of smooth muscle cells. To identify pathogenic mediators, IL-4 deficient mice and wild type mice transiently given an IL-13 blocker were examined. In both cases, the mice developed significantly less PVRm relative to immunized, antigen challenged, control wild type mice. Mice given recombinant IL-13 intranasally did not develop severe vascular muscularization indicating that IL-13 and, most likely IL-4 promote PVRm indirectly. In conclusion, our data are the first to provide proof of concept that the adaptive immune response to inhaled antigen can cause PVRm. Supported by: Flight Attendant Medical Research Institute, Stony Wold Herbert Fund, American Lung Association of the City of New York

Functional redundancy is highly prevalent among the Th2 interleukins (IL)-4, IL-5, IL-9, and IL-13. To define the critical functions of these cytokines, we have generated a novel panel of compound Th2 cytokine-deficient mice (from single to quadruple cytokine knockouts). We find that these Th2 cytokines are not essential for fetal survival even during allogeneic pregnancy. Using intestinal parasite infection and a pulmonary granuloma model, we demonstrate cryptic roles for IL-4, IL-5, IL-9, and IL-13 in these responses. Significantly, although IL-5, IL-9, and IL-13 add to the speed and magnitude of the response, a threshold is reached at which IL-4 alone can activate all Th2 effector functions. These mice reveal distinct spatial, temporal, and hierarchical cytokine requirements in immune function.