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Objective Scleromyxedema (SMX) is a rare systemic sclerosis mimic that often responds to intravenous immunoglobulin (IVIG) therapy, yet the resulting clinical and biochemical changes have not been well characterized. To better understand the pathogenesis of the disease and the efficacy of IVIG, we sought to explore whether IVIG would introduce a measurable biologic effect corresponding with clinical improvement. Methods Fifteen patients with SMX were recruited for the study. Clinical information and peripheral blood mononuclear cells for flow cytometry were obtained immediately before and again 1-2 weeks after patients received IVIG therapy. Ten patients also underwent skin biopsies for gene expression analysis both before and after IVIG therapy. Clinical data included measures of skin involvement (modification of the modified Rodnan skin thickness score [MMRSS] and percentage of body surface area) and several patient-reported outcome measures assessing patients' skin. Results Posttreatment, the average MMRSS score decreased from mean ± SD 13.6 ± 2.6 to 10.3 ± 1.9; P = 0.003. There were also significant improvements in skin flexibility (mean ± SD 5.4 ± 0.8 to 3.2 ± 0.7; P = 0.003) and softening (mean ± SD 4.9 ± 0.9 to 2.6 ± 0.6; P = 0.022). Baseline levels of Tc17 cells (CD8+CCR6+CXCR3+CCR4-) correlated with the extent of skin involvement as measured by MMRSS pretreatment (r = 0.69, P = 0.012) and decreased after IVIG therapy (mean ± SD 3.4% ± 3.2% to 1.3% ± 1.7%; P = 0.008). Posttreatment analysis of RNA in skin tissue revealed a decrease in gene expression of transforming growth factor β (TGFβ) cytokines as well as several interferon-inducible proteins. Conclusion This open-label study further supports the evidence that patients with SMX respond both objectively and subjectively to IVIG therapy. Biologic studies suggest a role for T lymphocytes in the pathogenesis of the disease and reveal the potential significance of TGFβ and interferon pathways.

Resistin-like molecule α (RELMα) is highly upregulated in the lungs of mice subjected to hypoxia. It is secreted from pulmonary epithelium and causes potent mitogenic, angiogenic, and vasoconstrictive effects in the lung vasculature. By using bone marrow transplantation in mice, we previously showed that RELMα is able to increase the number of bone marrow-derived cells in lung tissue, especially in the remodeling pulmonary vasculature. The current study investigated the effect of RELMα on progenitor stem cell content in mouse lung. Hypoxia, while stimulating RELMα expression, caused an increase in the number of Sca1(+)/CD45(-) progenitor cells in lungs of wild-type mice, but not in lungs of RELMα knockout mice. An in vitro study with cultured mesenchymal stem cells (MSCs) showed that RELMα induced a robust proliferative response that was dependent on Phosphatidylinositol 3-kinase/Akt and Erk activation. RELMα treatment of MSCs caused upregulation of a large number of genes involved in cell cycle, mitosis, organelle, and cytoskeleton biogenesis, and DNA metabolism. MSCs cultured in RELMα-supplemented media were able to maintain their differentiation potential into adipogenic, osteogenic, or mesenchymal phenotypes, although adipogenic differentiation was partially inhibited. These results demonstrate that RELMα may be involved in stem cell proliferation in the lung, without affecting differentiation potential.

Chronic obstructive pulmonary disease (COPD) is a prevalent smoking-related disease for which no disease-altering therapies currently exist. As dysregulated TGF-β signaling associates with lung pathology in patients with COPD and in animal models of lung injury induced by chronic exposure to cigarette smoke (CS), we postulated that inhibiting TGF-β signaling would protect against CS-induced lung injury. We first confirmed that TGF-β signaling was induced in the lungs of mice chronically exposed to CS as well as in COPD patient samples. Importantly, key pathological features of smoking-associated lung disease in patients, e.g., alveolar injury with overt emphysema and airway epithelial hyperplasia with fibrosis, accompanied CS-induced alveolar cell apoptosis caused by enhanced TGF-β signaling in CS-exposed mice. Systemic administration of a TGF-β-specific neutralizing antibody normalized TGF-β signaling and alveolar cell death, conferring improved lung architecture and lung mechanics in CS-exposed mice. Use of losartan, an angiotensin receptor type 1 blocker used widely in the clinic and known to antagonize TGF-β signaling, also improved oxidative stress, inflammation, metalloprotease activation and elastin remodeling. These data support our hypothesis that inhibition of TGF-β signaling through angiotensin receptor blockade can attenuate CS-induced lung injury in an established murine model. More importantly, our findings provide a preclinical platform for the development of other TGF-β-targeted therapies for patients with COPD.

The selectin antagonist known as recombinant P-selectin glycoprotein ligand IgG (rPSGL-Ig) blocks leukocyte adhesion and protects against transplantation ischemia reperfusion injury (IRI) in animal models. This randomized (1:1) single-center double-blind 47-patient phase 2 study with 6-month follow-up assessed rPSGL-Ig's safety and impact on early graft function at 1 mg/kg systemic dose with pretransplant allograft ex vivo treatment in deceased-donor liver transplant recipients. Safety was assessed in all patients, whereas efficacy was assessed in a prospectively defined per-protocol patient set (PP) by peak serum transaminase (TA) and bilirubin values, and normalization thereof. In PP patients, the incidence of poor early graft function (defined as peak TA >2500 U/L or bilirubin >10 mg/dL), average peak liver enzymes and bilirubin, normalization thereof and duration of primary and total hospitalization trended consistently lower in the rPSGL-Ig group compared to placebo. In patients with donor risk index above study-average, normalization of aspartate aminotransferase was significantly improved in the rPSGL-Ig group (p < 0.03). rPSGL-Ig treatment blunted postreperfusion induction versus placebo of IRI biomarker IP-10 (p < 0.1) and augmented cytoprotective IL-10 (p < 0.05). This is the first clinical trial of an adhesion molecule antagonist to demonstrate a beneficial effect on liver transplantation IRI and supported by therapeutic modulation of two hepatic IRI biomarkers.

To evaluate the impact of two different luteal phase support protocols on gene expression of extracellular matrix (ECM) proteins and adhesion molecules in the human endometrium.Eighty-four ECM protein and adhesion molecule genes were analyzed using array-based reverse-transcription polymerase chain reaction.Academic hospital.Nine oocyte donors.Endometrial biopsies were obtained on the day of oocyte retrieval (group I) and 3-5 days later (group II) after randomization into 3 groups. Group IIa had no luteal phase support, group IIb had luteal support with micronized progesterone, and group IIc had luteal support with progesterone plus 17β-estradiol.Gene expression profiles in relation to different types of luteal phase support protocols.Compared with the day of retrieval (group I), 24 genes were significantly up-regulated (4 in group IIa, 14 in group IIb, 22 in group IIc) and 14 genes were down-regulated (5 in group IIa, 2 in group IIb, 10 in group IIc) on day 3-5 (P.05). In the luteal support group, up- regulation occurred predominantly in genes encoding for matrix metallopeptidases (MMP10, MMP3, MMP9), integrins (ITGA3, ITGA5, ITGB3, ITGB4), and laminin (LAMB3). In contrast, the most significant suppression was documented in genes encoding for catenin-D2, collagen-11A1, and the laminins (LAMA1 and LAMA3). Significant changes between groups IIb and IIc were also observed in 9 genes.Luteal phase support following controlled ovarian stimulation has a profound impact on the ECM pathway targeted genes.

Summary Background Sensitization to cockroach allergen is one of the strongest predictors of asthma morbidity, especially among African Americans. Objective Our aims were to determine the genomic basis of cockroach sensitization and the specific response to cockroach antigen. Methods We investigated the Th1/Th2 cytokine profile of co-cultured plasmacytoid dendritic cells (pDCs) and CD4+ T cells and the ‘transcript signature’ of the immune response to cockroach antigen using high-throughput expression profiling of co-cultured cells. Results We observed significantly elevated levels of IL-13, IL-10, and TNF-α, but undetectable levels of IL-12p70 and IFN-α, when cultures were exposed to crude cockroach antigen. A significant difference was observed for IL-13 between cockroach-allergic and non-allergic individuals (P=0.039). Microarray analyses demonstrated a greater response at 48 h compared with 4 h, with 50 genes being uniquely expressed in cockroach antigen-treated cells, including CD14, S100A8, CCL8, and IFI44L. The increased CD14 expression was further observed in purified pDCs, human monocytic THP-1 cells, and the supernatant of co-cultured pDCs and CD4+ T cells on exposure to cockroach extract. Furthermore, the most differential expression of CD14 between cockroach allergy and non-cockroach allergy was only observed among individuals with the CC ‘high-risk’ genotype of the CD14−260C/T. Ingenuity Pathways Analysis analyses suggested the IFN signalling as the most significant canonical pathway. Conclusion Our results suggest that these differentially expressed genes, particularly CD14, and genes in the IFN signalling pathway may be important candidates for further investigation of their role in the immune response to cockroach allergen. Cite this as: P. Gao, D. N. Grigoryev, N. M. Rafaels, D. Mu, J. M. Wright, C. Cheadle, A. Togias, T. H. Beaty, R. A. Mathias, J. T. Schroeder and K. C. Barnes, Clinical & Experimental Allergy, 2010 (40) 1353–1364.

A population of stromal cells that retains osteogenic capacity in adult bone (adult bone stromal cells or aBSCs) exists and is under intense investigation. Mice heterozygous for a null allele of prkar1a ( Prkar1a +/− ), the primary receptor for cyclic adenosine monophosphate (cAMP) and regulator of protein kinase A (PKA) activity, developed bone lesions that were derived from cAMP-responsive osteogenic cells and resembled fibrous dysplasia (FD). Prkar1a +/− mice were crossed with mice that were heterozygous for catalytic subunit Cα ( Prkaca +/− ), the main PKA activity-mediating molecule, to generate a mouse model with double heterozygosity for prkar1a and prkaca ( Prkar1a +/− Prkaca +/− ). Unexpectedly, Prkar1a +/− Prkaca +/− mice developed a greater number of osseous lesions starting at 3 months of age that varied from the rare chondromas in the long bones and the ubiquitous osteochondrodysplasia of vertebral bodies to the occasional sarcoma in older animals. Cells from these lesions originated from an area proximal to the growth plate, expressed osteogenic cell markers, and showed higher PKA activity that was mostly type II (PKA-II) mediated by an alternate pattern of catalytic subunit expression. Gene expression profiling confirmed a preosteoblastic nature for these cells but also showed a signature that was indicative of mesenchymal-to-epithelial transition and increased Wnt signaling. These studies show that a specific subpopulation of aBSCs can be stimulated in adult bone by alternate PKA and catalytic subunit activity; abnormal proliferation of these cells leads to skeletal lesions that have similarities to human FD and bone tumors.

In animal models of acute lung injury (ALI), gene expression studies have focused on the acute phase of illness, with little emphasis on resolution. In this study, the acute phase of intratracheal lipopolysaccharide (IT LPS)-induced lung injury was similar in wild-type (WT) and recombinase-activating gene-1-deficient (Rag-1−/−) lymphocyte-deficient mice, but resolution was impaired and resolution-phase lung gene expression remained different from baseline only in Rag-1−/−mice. By focusing on groups of genes involved in similar biological processes (gene ontologies) pertinent to inflammation and the immune response, we identified 102 genes at days 4 and 10 after IT LPS with significantly different expression between WT and Rag-1−/−mice. After adoptive transfer of isolated CD4+CD25+Foxp3+ regulatory T cells (Tregs) to Rag-1−/−mice at the time of IT LPS, resolution was similar to that in WT mice. Of the 102 genes distinctly changed in either WT or Rag-1−/−mice from our 7 gene ontologies, 19 genes reverted from the Rag-1−/−to the WT pattern of expression after adoptive transfer of Tregs, implicating those 19 genes in Treg-mediated resolution of ALI.

In cancer cells, cyclic AMP–dependent protein kinase (PKA) is secreted into the conditioned medium. This PKA, designated as extracellular protein kinase A (ECPKA), is markedly up-regulated in the sera of patients with cancer. The currently available tumor markers are based on the antigen determination method and lack specificity and sensitivity. Here, we present an ECPKA autoantibody detection method for a universal biomarker that detects cancer of various cell types. We tested sera from 295 patients with cancers of various cell types, 155 normal controls, and 55 patients without cancer. The specificity and sensitivity of this autoantibody enzyme immunoassay method were compared with the conventional antigen determination method by receiver-operating characteristic plots. In the sera, the presence of autoantibody directed against ECPKA was highly correlated with cancer. High anti-ECPKA autoantibody titers (frequency, 90%; mean titer, 3.0) were found in the sera of patients with various cancers, whereas low or negative titers (frequency, 12%; mean titer, 1.0) were found in the control group. The receiver-operating characteristic plot showed that autoantibody enzyme immunoassay exhibited 90% sensitivity and 88% specificity, whereas the enzymatic assay exhibited 83% sensitivity and 80% specificity. These results show that the autoantibody method distinguished between patients with cancer and controls better than the antigen method could. Our results show that autoantibody ECPKA is a universal serum biomarker for cancers of various cell types. (Cancer Res 2006; 66(18): 8971-4)

The list of cancer markers of current interest has grown considerably, but none of the markers used in clinical work is a true tumor marker. These cancer biomarkers are based on the determination of tumor antigens. Here, we report a single method of autoantibody enzyme immunoassay (EIA) screens for a spectrum of serum tumor markers. A comparison of the autoantibody-based EIA to conventional antigen EIA kits, using receiver operating characteristic (ROC) plots, showed that the autoantibody EIA can significantly enhance the sensitivity and specificity of tumor markers. The detection of serum autoantibodies for a spectrum of serum tumor markers, as demonstrated here, suggests that most, if not all, serum cancer biomarkers produce autoantibodies. A unique autoantibody biomarker screening method, as presented here, might therefore facilitate achieving the accurate and early diagnosis of cancer.

Due to brain tissue heterogeneity, the molecular genetic profile of any neurotransmitter-specific neuronal subtype is unknown. The purpose of this study was to purify a population of dopamine neurons, construct a cDNA library, and generate an initial gene expression profile and a microarray representative of dopamine neuron transcripts. Ventral mesencephalic dopamine neurons were purified by fluorescent-activated cell sorting from embryonic day 13.5 transgenic mice harboring a 4.5-kb rat tyrosine hydroxylase promoter– lacZ fusion. Nine-hundred sixty dopamine neuron cDNA clones were sequenced and arrayed for use in studies of gene expression changes during methamphetamine neurotoxicity. A neurotoxic dose of methamphetamine produced a greater than twofold up-regulation of the mitochondrial cytochrome c oxidase polypeptide I transcript from adult mouse substantia nigra at 12 h posttreatment. This is the first work to describe a gene expression profile for a neuronal subtype and to identify gene expression changes during methamphetamine neurotoxicity.

Using cDNA microarrays we have investigated gene expression patterns in brain regions of patients with schizophrenia. A cDNA neuroarray, comprised of genes related to brain function, was used to screen pools of samples from the cerebellum and prefrontal cortex from a matched set of subjects, and middle temporal gyrus, from a separate subject cohort. Samples of cerebellum and prefrontal cortex from neuroleptic naive patients were also included. Genes that passed a 3% reproducibility criterion for differential expression in independent experiments included 21 genes for drug-treated patients and 5 genes for drug-naive patients. Of these 26 genes, 10 genes were increased and 16 were decreased. Many of the differentially expressed genes were related to synaptic signaling and proteolytic functions. A smaller number of these genes were also differentially expressed in the middle temporal gyrus. The five genes that were differentially expressed in two brain regions from separate cohorts are: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide; sialyltransferase; proteasome subunit, alpha type 1; ubiquitin carboxyl-terminal esterase L1; and solute carrier family 10, member 1. Identification of patterns of changes in gene expression may lead to a better understanding of the pathophysiology of schizophrenia disorders.

SummaryWe have constructed a fusion phage epitope library in the filamentous bacteriophage fuse5. The library was made by inserting a degenerate oligonucleotide which encodes 15 variable amino acids into the NH2-terminal region of the phage gene III protein. This library, containing over 107different epitope bearing phage, has been used in an attempt to identify inhibitors of the von Willebrand factor (vWF)-platelet Glycoprotein lb interaction. The library was screened with a monoclonal antibody (RG46) that recognizes the GPIb binding domain of vWF (amino acids 445-733). A total of 30 clones falling into 8 classes have been identified that react with the RG46 antibody. Isolates from all 8 classes are positive by immunoblot analysis. The amino acid sequence of the gene III fusion protein from positive clones showed a strong homology to the known RG46 epitope. Peptides identified from the screen were synthesized and used to demonstrate that some of the synthetic peptides exhibited inhibitory activity towards ristocetin induced binding of vWF to the GPIb receptor. Thus, we have demonstrated that screening a fusion phage epitope library with a monoclonal antibody that inhibits vWF binding to the GPIb receptor can be a useful tool not only for mapping antibody recognizing determinants, but also can serve as a source for identifying novel peptides that are antagonists for vWF binding to the platelet GPIb receptor.

A phage display library was constructed in the filamentous bacteriophage fuse5. The library was made by inserting a degenerate oligonucleotide which encodes 15 variable amino acids into the NH2-terminal region of the phage gene III protein. This library, containing over 10(7) different phage, was screened with a glutathione S-transferase (GST) fusion protein containing the Src homology 3 (Src SH3) domain and a protein kinase A phosphorylation site (GST/PKA/Src SH3). A family of proline-rich sequences was isolated following four cycles of enrichment and amplification. Phage containing these sequences were shown to specifically bind to the GST/PKA/Src SH3 protein but not to GST/PKA only. A comparison of the inferred amino acid sequence of the different phage clones revealed a consensus sequence, RPLPXXP, which conforms to a Src SH3 domain binding motif identified independently during an affinity screen of a lambda-lox mouse embryo cDNA library using a 32P-labeled Src SH3 protein fragment as the probe (Y. Ivashchenko, manuscript in preparation). Peptides based upon the 7-amino acid SH3 binding domain core motif displayed strong binding to both the Src and to the Fyn SH3 domains, but failed to bind to the SH3 domain of p21 Ras-GTPase-activating protein (Ras-GAP) and other proteins. We anticipate that further screening of the phage display library will be a useful tool for the rapid identification of additional SH3 domain binding sequences and will also help to establish the essential core motifs that define the specificity of interactions among the diverse proteins containing SH3 domains and those containing SH3 binding motifs.

The functions of key oncogenic transcription factors independent of context have not been fully delineated despite our richer understanding of the genetic alterations in human cancers. The MYC oncogene, which produces the Myc transcription factor, is frequently altered in human cancer and is a major regulatory hub for many cancers. In this regard, we sought to unravel the primordial signature of Myc function by using high-throughput genomic approaches to identify the cell-type independent core Myc target gene signature. Using a model of human B lymphoma cells bearing inducible MYC, we identified a stringent set of direct Myc target genes via chromatin immunoprecipitation (ChIP), global nuclear run-on assay, and changes in mRNA levels. We also identified direct Myc targets in human embryonic stem cells (ESCs). We further document that a Myc core signature (MCS) set of target genes is shared in mouse and human ESCs as well as in four other human cancer cell types. Remarkably, the expression of the MCS correlates with MYC expression in a cell-type independent manner across 8,129 microarray samples, which include 312 cell and tissue types. Furthermore, the expression of the MCS is elevated in vivo in Eμ-Myc transgenic murine lymphoma cells as compared with premalignant or normal B lymphocytes. Expression of the MCS in human B cell lymphomas, acute leukemia, lung cancers or Ewing sarcomas has the highest correlation with MYC expression. Annotation of this gene signature reveals Myc's primordial function in RNA processing, ribosome biogenesis and biomass accumulation as its key roles in cancer and stem cells.

Alveolar enlargement, which is characteristic of bronchopulmonary dysplasia, congenital matrix disorders, and cigarette smoke-induced emphysema, is thought to result from enhanced inflammation and ensuing excessive matrix proteolysis. Although there is recent evidence that cell death and oxidative stress punctuate these diseases, the mechanistic link between abnormal lung extracellular matrix and alveolar enlargement is lacking. We hypothesized that the tight-skin (TSK) mouse, which harbors a spontaneous internal duplication in the microfibrillar glycoprotein fibrillin-1, might show whether matrix alterations are sufficient to promote oxidative stress and cell death, injury cascades central to the development of clinical emphysema. We observed no evidence of increased metalloprotease activation by histochemical and zymographic methods. We did find initial oxidative stress followed by increased apoptosis in the postnatal TSK lung. Both blunted antioxidant production and reduced extracellular superoxide dismutase activity were evident in the neonatal lung. High-dose antioxidant treatment with N-acetylcysteine improved airspace caliber and attenuated oxidative stress and apoptosis in neonatal and adult TSK mice. These data establish that an abnormal extracellular matrix without overt elastolysis is sufficient to confer susceptibility to postnatal normoxia, reminiscent of bronchopulmonary dysplasia. The resultant oxidative stress and apoptosis culminate in profound airspace enlargement. The TSK lung exemplifies the critical interplay between extracellular matrix, oxidative stress, and cell-death cascades that may contribute to genetic and acquired airspace enlargement.

We hypothesize that pulmonary arterial hypertension (PAH)-associated genes identified by expression profiling of peripheral blood mononuclear cells (PBMCs) from patients with idiopathic pulmonary arterial hypertension (IPAH) can also be identified in PBMCs from scleroderma patients with PAH (PAH-SSc). Gene expression profiles of PBMCs collected from IPAH (n = 9), PAH-SSc (n = 10) patients, and healthy controls (n = 5) were generated using HG_U133A_2.0 GeneChips and were processed by the RMA/GCOS_1.4/SAM_1.21 data analysis pipeline. Disease severity in consecutive patients was assessed by functional status and hemodynamic measurements. The expression profiles were analyzed using PAH severity-stratification, and identified candidate genes were validated with real-time polymerase chain reaction (PCR). Transcriptomics of PBMCs from IPAH patients was highly comparable with that of PMBCs from PAH-SSc patients. The PBMC gene expression patterns significantly correlate with right atrium pressure (RA) and cardiac index (CI), which are known predictors of survival in PAH. Array stratification by RA and CI identified 364 PAH-associated candidate genes. Gene ontology (GO) analysis revealed significant (Z score > 1.96) alterations in angiogenesis genes according to PAH severity: matrix metalloproteinase 9 (MMP9) and vascular endothelial growth factor (VEGF) were significantly upregulated in mild as compared with severe PAH and healthy controls, as confirmed by real-time PCR. These data demonstrate that PBMCs from patients with PAH-SSc carry distinct transcriptional expression. Furthermore, our findings suggest an association between angiogenesis-related gene expression and severity of PAH in PAH-SSc patients. Deciphering the role of genes involved in vascular remodeling and PAH development may reveal new treatment targets for this devastating disorder.

Background Myosin light chain kinase (MYLK) is a multifunctional protein involved in regulation of airway hyperreactivity and other activities relevant to asthma. Objective To determine the role of MYLK gene variants in asthma among African Caribbean and African American populations. Methods We performed association tests between single nucleotide polymorphisms (SNPs) in the MYLK gene and asthma susceptibility and total serum IgE concentrations in 2 independent, family-based populations of African descent. Previously we identified variants/haplotypes in MYLK that confer risk for sepsis and acute lung injury; we compared findings from our asthma populations to findings in the African American sepsis and acute lung injury groups. Results Significant associations between MYLK SNPs and asthma and total serum IgE concentrations were observed in the African Caribbean families: a promoter SNP (rs936170) in the smooth muscle form gave the strongest association ( P = .009). A haplotype including rs936170 corresponding to the actin-binding activity of the nonmuscle and smooth muscle forms was negatively associated with asthma (eg, decreased risk) in both the American ( P = .005) and Caribbean families ( P = .004), and was the same haplotype that conferred risk for severe sepsis ( P = .002). RNA expression studies on PBMCs and rs936170 suggested a significant decrease in MYLK expression among patients with asthma with this variant ( P = .025). Conclusion MYLK polymorphisms may function as a common genetic factor in clinically distinct diseases involving bronchial smooth muscle contraction and inflammation. Clinical implications Genetic variants in MYLK are significantly associated with both asthma and sepsis in populations of African ancestry.

Background The gene encoding acyloxyacyl hydroxylase (AOAH), an enzyme that hydrolyzes secondary fatty acyl chains of LPS, is localized on chromosome 7p14-p12, where evidence for linkage to total IgE (tIgE) concentrations and asthma has been previously reported. Objective We hypothesized that variants in AOAH are associated with asthma and related phenotypes. Because both AOAH and soluble CD14 respond to LPS, we tested for gene-gene interaction. Methods We investigated the association between 28 single nucleotide polymorphisms throughout the AOAH gene and asthma, concentrations of tIgE, the ratio of IL-13/IFN-γ, and soluble CD14 levels among 125 African Caribbean, multiplex asthmatic pedigrees (n = 834). Real-time PCR was used to assess whether AOAH cDNA expression differed with AOAH genotype. Results Significant effects were observed for all 4 phenotypes and AOAH markers in 3 distinct regions (promoter, introns 1-6, and the intron 12/exon 13 boundary/intron 13 region) by means of single-marker and haplotype analyses, with the strongest evidence for a 2-single-nucleotide-polymorphism haplotype and log[tIgE] ( P = .006). There was no difference in AOAH expression levels by AOAH genotype for any of the markers. Comparing genotypic distributions at both the AOAH marker rs2727831 and CD14 (−260)C>T raises the possibility of gene-gene interaction ( P = .006-.036). Conclusion Our results indicate that polymorphisms in markers within the AOAH gene are associated with risk of asthma and associated quantitative traits (IgE and cytokine levels) among asthmatic subjects and their families in Barbados, and there is an interactive effect on tIgE and asthma concentrations between an AOAH marker and the functional CD14 (−260)C>T polymorphism. Clinical implications AOAH is a novel innate immunity candidate gene associated with asthma and related phenotypes in an African ancestry population.

In mice, the recreational drug (±)3,4-methylenedioxymethamphetamine [MDMA (“ecstasy”)] produces a selective toxic effect on brain dopamine (DA) neurons. Using cDNA microarray technology in combination with an approach designed to facilitate recognition of relevant changes in gene expression, the present studies sought to identify genes potentially involved in murine MDMA-induced toxicity to DA neurons. Of 15,000 mouse cDNA fragments studied, metallothionein (Mt)-1andMt2emerged as candidate genes possibly involved in MDMA-induced toxicity to DA neurons. Northern blot analysis confirmed the microarray findings and revealed a dynamic upregulation ofMt1andMt2mRNA in the ventral midbrain within 4-12 hr after MDMA treatment. Western blot analysis showed a similar increase in MT protein levels, with peak times occurring subsequent to increases in mRNA levels.Mt1-2double knock-out mice were more vulnerable to MDMA-induced toxicity to DA neurons than corresponding wild-type mice. Stimulation of endogenous expression of MT protein with zinc acetate conferred complete protection against MDMA-induced toxicity to DA neurons, and administration of exogenous MT protein afforded partial protection. Collectively, these results indicate that MDMA-induced toxicity to DA neurons is associated with increasedMt1andMt2gene transcription and translation, possibly as part of a neuroprotective mechanism. The present findings may have therapeutic implications for neuropathological conditions involving DA neurons.

Global changes in miRNA expression in airway epithelium, a cell type critical in the mucosal response to microbial invasion, allergens and pollutants, have been only partially investigated. Cell stimulation with a TLR3 agonist ± cytokines was used to model disease-specific miRNA changes. PBEC (n=5) were cultured under air-liquid interface and treated with medium or polyI:C (1-10 μg/ml) ±TNFα/IFNγ (10 ng/ml each) for 24 hr. Total RNA was used for miRNA profiling (Agilent) and real-time-PCR analysis of inflammatory genes (control). PBEC supernatants were assayed using antibody arrays. Genome Ontology analysis was performed by Ingenuity pathway analysis. Expression of miR409-3p and miR624 was downregulated >30-fold compared to control following polyI:C treatment. Expression of 16 miRNAs was downregulated following polyI:C/TNFα/IFNγ treatment, with >30-fold reduction for miR617 and miR136 (calculated as Fold Change ≥ 1.5, p< 0.05 after Robust Multichip Average normalization). In cells challenged with the combined treatment, 145 genes (including 16 transcription factors) were predicted with high confidence (by 6/6 programs) as molecular targets for 6 of 16 differentially expressed miRNAs. PolyI:C plus TNFα/IFNγ strongly synergized in upregulating the mRNA and protein expression of CXCL8, GM-CSF, IL-6 and CCL2. The identification of the miRNA signature may identify new mechanisms of posttranscriptional gene control and generate new anti-inflammatory strategies.

Background: Blockade of the immunosuppressive PD-1/PD-L1 pathway has shown promising clinical results in patients with treatment-refractory solid tumors including melanoma. PD-L1, a protein displayed by many tumors, ligates the PD-1 co-inhibitory receptor on activated T cells. We previously found that IFN-γ, a potent inducer of PD-L1, was expressed by TILs in PD-L1(+) but not PD-L1(-) melanomas, creating an immunosuppressive microenvironment by a mechanism that we term “adaptive immune resistance” (Taube et al., Science Transl Med 2012). In the current study, we assessed other factors associated with PD-L1 expression in melanoma through differential gene expression analysis, in order to better understand the biology of this pathway and identify potential targets for combination immunotherapies. Methods: PD-L1(+) vs. (-) melanomas including immune cell infiltrates (n=11) were laser-capture microdissected (LCM) from formalin-fixed paraffin embedded (FFPE) specimens and analyzed by whole genome array analysis with cDNA-mediated Annealing, Selection, extension and Ligation (DASL), a novel technology that allows for gene expression analysis with partially degraded mRNAs obtained from FFPE specimens. Differentially expressed genes were submitted to the NIH functional analysis tool DAVID to search for enrichment in biologically related groups. Multiplex quantitative (q)RT-PCR was used to validate differential expression of genes of interest and to examine other candidate genes in new set of PD-L1(+) vs.(-) melanomas (n=11). Results: DASL/DAVID analysis of PD-L1(+) vs. PD-L1(-) melanomas uncovered genes of interest in several immunologically relevant pathways, including “Defense Response” (p Conclusions: These experiments identified groups of functionally related, differentially expressed immuno-regulatory genes in PD-L1(+) melanomas. These factors may coordinately create an immunosuppressive tumor microenvironment, by synergizing to enhance PD-L1 expression on tumor cells and to inhibit T cell function. Immunohistochemical and in vitro functional validation assays of candidate gene products are currently underway in order to identify new ways to overcome adaptive immune resistance in synergistic treatment combinations with PD-1/PD-L1 blockade. Citation Format: Geoffrey D. Young, Tracee L. McMiller, Haiying Xu, Shuming Chen, Alan E. Berger, Jinshui Fan, Robert A. Anders, Christopher Cheadle, Drew M. Pardoll, Suzanne L. Topalian, Janis M. Taube. Differential expression of immuno-regulatory genes associated with PD-L1 display: Implications for clinical blockade of the PD-1/PD-L1 pathway in melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 446. doi:10.1158/1538-7445.AM2013-446

The gut epithelial functions to provide a protective barrier from enteric pathogens as well as facilitate the absorption and distribution of needed nutrients. These functions are accomplished by the concerted action of a number of interacting cell types including epithelial cells, stromal elements and immune cells. We reason that cells of the immune system are an essential component for the functioning gut epithelia and are likely to play a role in the maintenance of epithelial integrity and function. We have used a genomic-based approach to understand how a novel subset of intraepithelial lymphocytes, γ/δ T cells, contributes to the functioning of the gut epithelial barrier. Using mouse strains deficient for TCR γ/δ, we examined the genes and pathways of the GI tract that are affected by the absence of the TCR γ/δ bearing subset. Our initial analysis revealed that the expression profiles of genes encoding proteins involved in maintaining epithelial integrity and tissue remodeling are regionally and selectively altered in TCR γ/δ deficient mice. In addition, we examined the affect of TCR γ/δ deficiency on the gene sets and pathways activated in the early stages of enteric infection with Salmonella enterica typhimurium. These results reveal abnormal regulation of the acute phase response implying a role for γ/δ T cells in regulating the response to injury. Collectively these studies reveal novel insights into the function of this novel immune cell subset.

Recombinant DNA techniques were used to clone and express the FV portion of MOPC315, a mouse myeloma protein with a high affinity for 2,4-dinitrophenyl (DNP). The FV fragment consists of a heterodimer of heavy and light chain variable domains (VH and VL). Two separate bacterial plasmid constructs, containing either a variable region cDNA for the light chain or a variable region synthetic gene for the heavy chain demonstrated high levels of expression (150-200 mg/L) under control of the bacteriophage T7 promoter. Recombinant chains were initially recovered as inclusion bodies and then dissolved separately in 8 M urea, combined together, and refolded by subsequent chaotrope removal. Biologically active FV was affinity purified from the chain mixture by specific binding to DNP-lysine-Sepharose. Yields of active material as high as 20% were obtained with activity confirmed by fluorescence quench analysis. The purified FV displayed a binding affinity of 4.8 +/- 0.3 x 10(-7) M which was identical to the native FV. Chimeric FVs composed of recombinant and native chain mixtures yielded similar results. Recombinant MOPC315 FV activity was also obtained using a single chain construct (sFV), in which recombinant VH and VL were linked via a (Gly4Ser)3 spacer region. Binding affinity of the sFV was shown to be the same as the recombinant and native FVs. The ease of purification and characterization of active MOPC315 as the recombinant and native FVs. The ease of purification and characterization of active MOPC315 FV makes this system useful in the study of the optimization of antibody production in bacteria.

e22048 Background: There is a well-established association between certain autoimmune diseases and the development of specific malignancies. It has been demonstrated that myositis-specific autoantigens are expressed at higher levels in tumors associated with myositis compared to normal tissue, suggesting that immune responses to antigens expressed in nascent tumors may contribute to the autoimmune process. Whether this observation is a general feature of autoantigen expression in tumor tissue, and whether the relative expression of these antigens is enriched in relation to the rest of the tumor transcriptome is currently unknown. Methods: Tumor tissue from ten lung cancer biopsies (adenocarcinoma (4), carcinoid (4), and squamous cell carcinoma (2)) and normal lung from the same patients were obtained. Total RNA was extracted and hybridized to Illumina Sentrix BeadChips. Hiearchical clustering was used to visualize the expression levels of 146 known autoantigens. Gene Set Enrichment Analysis was performed using 20 disease-specific autoantigen gene sets and 1892 gene sets from the Molecular Signatures Database. Protein levels of selected autoantigens were assessed by immunoblotting detergent tissue lysates. Results: Single-linkage hierarchical clustering analysis reveals groups of autoantigens that are differentially expressed between normal lung tissue, carcinoid tumors, and adenocarcinomas. Adenocarcinoma tumor samples were significantly enriched for myositis (nominal p-value No significant financial relationships to disclose.

Epstein-Barr virus (EBV) replicons which include the genetic element oriP and a functional gene for Epstein-Barr nuclear antigen (EBNA-1) can be maintained episomally in a variety of mammalian cell lines [Yates et al., Nature 313 (1985) 812–815]. We have assessed the application of an EBV replicon for foreign gene expression. Two cDNAs, human interferon-γ (IFN-γ) and the extracellular domain of the human epidermal growth factor receptor (EGF-R ex ), cloned in an EBV replicon, were efficiently expressed and the protein was secreted into the extracellular media. Expression in human embryonic 293 cells was approximately ten-fold higher than in CV-1 cells. The expression of the human protein is dependent upon the orientation of the IFN-γ transcriptional cassette relative to the other genetic elements within the vector.