Characterization of Circulating Tumor DNA for Genetic Assessment of solid Tumors.

Personalized cancer therapy requires characterization of the current status of an individual's cancer, necessitating invasive tumor tissue biopsies at diagnosis, during treatment and at progression. Serial acquisition of solid tumor biopsies during treatment to characterize mutations related to acquired resistance may not be medically feasible. Circulating tumor DNA (ctDNA) in plasma offers a possible noninvasive "real time" tool for tumor characterization, providing accessible genetic biomarkers for cancer diagnosis, prognosis, and response to therapy.

Global analysis of nuclear receptor expression and dysregulation in human osteoarthritic articular cartilage: reduced LXR signaling contributes to catabolic metabolism typical of osteoarthritis.

OBJECTIVE: Compare the expression and regulation of nuclear receptors (NRs) in osteoarthritic and normal human articular cartilage. METHOD: The transcriptional levels of 48 NRs and additional related proteins were measured in mRNA from human articular cartilage from subjects with osteoarthritis (OA) and compared to samples from subjects without OA, using microarrays, individual quantitative reverse transcriptase polymerase chain reaction assays, and a custom human NR TaqMan Low Density Array (TLDA). The functional effect of liver X receptor (LXR) activity in cartilage was studied by measuring proteoglycan (PG) synthesis and degradation in articular cartilage explant cultures following treatment with the synthetic LXR agonist T0901317. RESULTS: Thirty-one of 48 NRs analyzed by TLDA were found to be measurably expressed in human articular cartilage; 23 of these 31 NRs showed significantly altered expression in OA vs unaffected cartilage. Among these, LXRalpha and LXRbeta, and their heterodimeric partners retinoid X receptor (RXR)alpha and RXRbeta were all expressed at significantly lower levels in OA cartilage, as were LXR target genes ABCG1 and apolipoproteins D and E. Addition of LXR agonist to human OA articular chondrocytes and to cartilage explant cultures resulted in activation of LXR-mediated transcription and significant reduction of both basal and interleukin (IL)-1-mediated PG degradation. CONCLUSIONS: Articular cartilage expresses a substantial number of NRs, and a large proportion of the expressed NRs are dysregulated in OA. In particular, LXR signaling in OA articular cartilage is impaired, and stimulation of LXR transcriptional activity can counteract the catabolic effects of IL-1. We conclude that LXR agonism may be a possible therapeutic option for OA.

In vitro allergen challenge of peripheral blood induces differential gene expression in mononuclear cells of asthmatic patients: inhibition of cytosolic phospholipase A2alpha overcomes the asthma-associated response.

BACKGROUND: Existing treatments for asthma are not effective in all patients and disease exacerbations are common, highlighting the need for increased understanding of disease mechanisms and novel treatment strategies. The leukotriene pathway including the enzyme responsible for arachidonic acid release from cellular phospholipids, cPLA(2)alpha, is a major contributor to asthmatic responses and an attractive target in asthma therapies. OBJECTIVE: The study reported here investigates (a) the differential effects of in vitro exposure of peripheral blood mononuclear cells (PBMCs) to allergen between asthma and healthy subjects, and (b) the contribution of cPLA(2)alpha to these differences in gene expression. METHODS: In vitro responses of asthma (N=26) and healthy (N=11) subject PBMC samples to allergen stimulation in the presence and absence of cPLA(2)alpha inhibition or 5-lipoxygenase inhibition were compared at the gene expression level using oligonucleotide arrays and at the protein level using ELISA. RESULTS: Subject samples within both asthma and healthy groups showed allergen-dependent cytokine production and allergen-dependent gene expression changes, although transcriptional profiling identified 153 genes that were modulated significantly differently by allergen between asthma and healthy subjects. Among these were genes previously associated with asthma, but the majority (about 80%) have not previously been associated with asthma. CONCLUSIONS: Transcriptional profiling elucidated novel gene expression differences between the asthmatic and healthy subject samples. Although 5-lipoxygenase inhibition did not significantly affect allergen-modulated gene expression, the inhibition of cPLA(2)alpha activity affected many of the allergen-dependent, asthma-associated gene expression changes.

Gene expression analysis in a murine model of allergic asthma reveals overlapping disease and therapy dependent pathways in the lung.

Accumulating evidence in animal models and human asthma support a central role for IL-13 signaling in disease pathogenesis. In order to identify asthma and therapy associated genes, global transcriptional changes were monitored in mouse lung following antigen challenge (ovalbumin (OVA)), either alone or in the presence of a soluble IL-13 antagonist. Changes in whole lung gene expression after instillation of mIL-13 were also measured both in wild type and STAT6 deficient mice. A striking overlap in the gene expression profiles induced by either OVA challenge or mIL-13 was observed, further strengthening the relationship of IL-13 signaling to asthma. Consistent with results from functional studies, a subset of the OVA-induced gene expression was significantly inhibited by a soluble IL-13 antagonist while IL-13-modulated gene expression was completely attenuated in the absence of STAT6-mediated signaling. Results from these experiments greatly expand our understanding of asthma and provide novel molecular targets for therapy and potential biomarkers of IL-13 antagonism.

Mechanistic toxicogenomic analysis of WAY-144122 administration in Sprague-Dawley rats.

Application of global gene expression analysis in the study of mechanisms of toxicity could provide a more comprehensive interpretation of the molecular basis of drug action. WAY-144122 has pharmacological activity against several targets improving insulin responsiveness and favorably altering lipid profiles. Normal rats treated with suprapharmacological doses of WAY-144122 for 28 days exhibited drug-related effects in the liver and ovary. To determine the molecular mechanism underlying these effects, we employed global gene expression profiling to measure RNA levels in these target organs obtained from WAY-144122-treated rats administered test article for 1, 3, 7, and 14 days. Genes altered in expression by WAY-144122 were functionally categorized and related to their biological activity. In the liver, WAY-144122 caused a widespread up-regulation of genes involved in lipid mobilization, peroxisomal proliferation, and fatty acid beta-oxidation. In the ovary, we observed reduced expression of genes encoding luteinizing hormone receptor, follistatin, and enzymes in the estradiol synthesis pathway. Transcriptional changes in both organs precede histopathological effects. Profiling analysis allowed us to formulate hypotheses for molecular mechanisms underlying the physiological observations. In the liver, transcriptional changes suggest that WAY-144122 induced increased metabolic activity and peroxisomal proliferation resulting in increased liver weight and hepatocellular hypertrophy. We propose decreased estradiol synthesis as the underlying mechanism for the observed follicular atrophy in the ovary. Importantly, in this study, we have identified potential molecular mechanisms of drug effect in expression profiles before observation of physiological changes.

Pharmacogenomic analysis of rhIL-11 treatment in the HLA-B27 rat model of inflammatory bowel disease.

Recombinant human interleukin-11 (rhIL-11) reduces the clinical signs and histological lesions of inflammatory bowel disease (IBD) in transgenic rats expressing the human major histocompatability complex (MHC) class I allele, HLA-B27. To elucidate the pharmacogenomic effects of rhIL-11 in this model, we examined the global gene expression pattern in inflamed colonic tissue before and following rhIL-11 treatment using oligonucleotide microarrays. In total, 175 disease-related genes were identified. Increased expression of genes involved in antigen presentation, cell death and inflammation, and decreased expression of metabolic genes was associated with disease. A total of 27 disease-related genes returned to normal expression levels following rhIL-11 treatment including the MHC class II gene RT1-DMbeta. rhIL-11 induced the expression of four intestinal epithelial growth factors. These gene expression patterns indicate that treatment of inflammatory bowel disease with rhIL-11 affects class II antigen processing and colonic epithelial cell proliferation and metabolism.

Protective effect of rhIL-11 in a murine model of acetaminophen-induced hepatotoxicity.

Acetaminophen intoxication results in hepatotoxicity associated with increased serum concentrations of hepatocellular leakage enzymes such as aspartate aminotransferase, lactate dehydrogenase, and alanine aminotransferase, centrilobular degeneration and necrosis, and activation of Kupffer cells. Recombinant human Interleukin-11 (rhIL-11) downregulates the production of proinflammatory mediators from activated macrophages and has direct effects on hepatocyte gene expression. Based on these biological activities of rhIL-11, the effect of pretreatment with rhIL-11 in a murine model of acetaminophen-induced hepatotoxicity was examined. Administration of 500 microg/kg acetaminophen to B6C3F1 mice resulted in progressive hepatotoxicity as demonstrated by elevated serum concentrations of hepatocellular leakage enzymes and TNFalpha and histopathology. Pretreatment with 250 or 500 microg/kg of subcutaneously administered rhIL-11 2 hours before acetaminophen administration reduced serum concentrations of hepatocellular leakage enzymes and TNFalpha by 40-50%. This was associated with a statistically significant decrease in mean severity score for centrilobular hemorrhage and necrosis from grade 3 to grade 2 for rhIL-11-treated animals compared to vehicle. These results indicate that treatment with rhIL-11 has a protective effect in a model of acetaminophen-induced liver damage.

Interleukin-11 modulates Th1/Th2 cytokine production from activated CD4+ T cells.

Recombinant human interleukin-11 (rHuIL-11) is a pleiotropic cytokine with effects on multiple cell types. rHuIL-11 reduces activated macrophage activity and downregulates production of proinflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO). In vitro and in vivo, rHuIL-11 inhibits production of key immunostimulatory cytokines, including IL-12 and interferon-gamma (IFN-gamma). rHuIL-11 has recently demonstrated immunomodulatory activity to downregulate IFN-gamma production, increase IL-4 production, and reduce inflammatory tissue injury in a human psoriasis clinical trial. The cellular mechanisms of these effects are not fully elucidated. We demonstrate here that expression of gp130 and IL-11 receptor (IL-11R) alpha mRNA, components of the IL-11R complex, are detected in human and murine CD4(+) and CD8(+) lymphocytes, suggesting that rHuIL-11 can directly interact with T cells. In a cell culture model of murine T cell differentiation, rHuIL-11 acts to inhibit IL-2 production as well as IL-12-induced IFN-gamma production and enhances IL-4 and IL-10 production. rHuIL-11 had no effect on T cell proliferation. The ability of rHuIL-11 to modulate cytokine production from activated CD4(+) T cells provides a mechanism through which rHuIL-11 may ameliorate such inflammatory diseases as psoriasis.

Molecular classification of psoriasis disease-associated genes through pharmacogenomic expression profiling.

Psoriasis is recognized as the most common T cell-mediated inflammatory disease in humans. Genetic linkage to as many as six distinct disease loci has been established but the molecular etiology and genetics remain unknown. To begin to identify psoriasis disease-related genes and construct in vivo pathways of the inflammatory process, a genome-wide expression screen of multiple psoriasis patients was undertaken. A comprehensive list of 159 genes that define psoriasis in molecular terms was generated; numerous genes in this set mapped to six different disease-associated loci. To further interpret the functional role of this gene set in the disease process, a longitudinal pharmacogenomic study was initiated to understand how expression levels of these transcripts are altered following patient treatment with therapeutic agents that antagonize calcineurin or NF-KB pathways. Transcript levels for a subset of these 159 genes changed significantly in those patients who responded to therapy and many of the changes preceded clinical improvement. The disease-related gene map provides new insights into the pathogenesis of psoriasis, wound healing and cellular-immune reactions occurring in human skin as well as other T cell-mediated autoimmune diseases. In addition, it provides a set of candidate genes that may serve as novel therapeutic intervention points as well as surrogate and predictive markers of treatment outcome.

Subcutaneous or oral administration of rhIL-11 modulated the contact hypersensitivity response.

Recombinant human interleukin 11 (rhIL-11) is a multifunctional cytokine with immunomodulatory activity on both T cells and macrophages. The effects of rhIL-11 in a murine model of contact hypersensitivity (CHS) response have been studied. The CHS response is a T cell-mediated response directed against chemically modified self-proteins following epidermal exposure to haptens. CHS is generated in two phases. The sensitization phase involves dermal dendritic cell recognition of haptenized proteins and antigen presentation. The effector phase involves T cell recognition and activation. In mice sensitized with oxazolone, CHS was induced by secondary challenge to the right ear and measured by ear swelling 24 h later. rhIL-11 significantly suppressed CHS as measured by ear swelling and tissue myeloperoxidase activity when injected subcutaneously for 5 days from the day of sensitization or when administered only on the day before and the day of challenge, but was not effective when administered prior to or on the day of sensitization. These results indicate that subcutaneously administered rhIL-11 may modulate the effector phase of CHS. Administration of rhIL-11 as an oral gavage prior to sensitization also reduced CHS. However oral administration of rhIL-11 after sensitization had no effect. These results suggest that orally and subcutaneously administered rhIL-11 may act through different mechanisms to affect CHS.

Defining mechanisms of action of interleukin-11 on the progression of radiation-induced oral mucositis in hamsters.

Oral ulcerative mucositis is a common toxicity associated with drug and radiation therapy for cancer. It impacts on quality of life and economic outcomes, as well as morbidity and mortality. Mucositis is often associated with dose limitations for chemotherapy or is a cause for dose interruption for radiation. The complexity of mucositis as a biological process has only been recently appreciated. It has been suggested that the condition represents a sequential interaction of oral mucosal cells and tissues, pro-inflammatory cytokines and local factors such as saliva and the oral microbiota. The recognition that the pathophysiology of mucositis is a multifactorial process was partially suggested by the observation that interleukin-11 (IL-11), a pleotropic cytokine, favorably altered the course of chemotherapy-induced mucositis in an animal model. In the current study, we evaluated a series of biologic and morphologic outcomes to determine their roles and sequence in the development of experimental radiation-induced mucositis and to evaluate the effects of IL-11 in attenuating them. Our results suggest that IL-11 favorably modulates acute radiation-induced mucositis by attenuating pro-inflammatory cytokine expression. Data are also presented which help define the pathobiological sequence of mucositis.

Dose-dependent and schedule-dependent effects of interleukin-12 on antigen-specific CD8 responses.

Interleukin-12 (IL-12) has been shown to play a central role in the innate and acquired immune responses. Its activities include enhancement of natural killer (NK) and cytotoxic T lymphocyte (CTL) activity and promotion of CD4 Th1 cell development. It has also been shown to provide potent activity as a vaccine adjuvant in generating antibody and T cell responses. We have investigated the efficacy of IL-12 protein in promoting CD8 T cell responses when it is used as an adjuvant for immunization. Studies using, as antigen, cDNA from an autologous antigen (P1A) as well as studies of responses to vaccinia virus-delivered self (gp100) and non-self (beta-galactosidase) antigens show that the dose and schedule of IL-12 administration can significantly affect adjuvant activity, leading to enhancement or suppression of antigen-specific responses.

IL-12-Dependent enhancement of CTL response to weak class I-restricted peptide immunogens requires coimmunization with T helper cell immunogens.

The effect of in vivo administration of rmIL-12 on the CTL response to immunization with a weakly immunogenic class I-restricted peptide emulsified in incomplete Freund's adjuvant was investigated. In the absence of IL-12, peptide-specific CTL responses were significantly greater following coimmunization with class I-restricted peptide and T helper cell antigens than following immunization with the class I-restricted peptide alone. IL-12-dependent enhancement of the CTL response to peptide immunization was demonstrated in the presence of, but not in the absence of, coimmunization with T helper cell antigen. These findings indicate that IL-12 enhancement of the CTL response to weak class I-restricted immunogens is T helper cell dependent. Treatment with rmIL-12 also enhanced the CTL response to immunization with cDNA encoding both CTL and T helper cell epitopes. These findings are relevant to the design of vaccines containing tumor-associated class I-restricted peptides currently being tested as an immunotherapy for cancer patients.

A CTL assay requiring only 150 microliter of mouse blood.

In this paper, we present a method for measuring antigen specific cytotoxic T lymphocyte (CTL) activity from individual mouse peripheral blood samples without animal sacrifice. Peripheral blood cells are stimulated in vitro with a cocktail of antigen, cytokines, costimulatory molecules and irradiated feeder cells resulting, 7 days later, in a readily detectable antigen specific signal from a well plated under limiting dilution conditions. This highly sensitive and antigen specific assay is more efficient than conventional CTL assays and thus increases the number of mice that can be tested in a single assay. Since blood samples can be assayed from an individual mouse at multiple times during the course of an in vivo study, the assay can facilitate and strengthen correlative studies on CTL responses and in vivo results.

Interleukin-11 therapy selectively downregulates type I cytokine proinflammatory pathways in psoriasis lesions.

Psoriasis is a chronic inflammatory skin disease in which epidermal hyperplasia results from skin infiltration by type I T lymphocytes and release of associated cytokines. A multifunctional cytokine, rhIL-11, modulates macrophage and type I T-lymphocyte function in cell culture and shows anti-inflammatory activity in animal models. We are testing subcutaneous delivery of rhIL-11 to patients with psoriasis in a phase 1 open-label dose-escalation clinical trial. Tissue was obtained from lesional and uninvolved skin before and during treatment with rhIL-11 and was examined by histology/immunohistochemistry and quantitative RT-PCR. Expression of over 35 genes was examined in all patients, and multiple genetic markers of psoriasis were identified. Expression of numerous proinflammatory genes was elevated in psoriatic tissue compared with nonlesional skin. Seven of 12 patients responded well to rhIL-11 treatment. Amelioration of disease by rhIL-11, as shown by reduced keratinocyte proliferation and cutaneous inflammation, was associated with decreased expression of products of disease-related genes, including K16, iNOS, IFN-gamma, IL-8, IL-12, TNF-alpha, IL-1beta, and CD8, and with increased expression of endogenous IL-11. We believe that this is the first study in humans to indicate that type I cytokines can be selectively suppressed by an exogenous immune-modifying therapy. The study highlights the utility of pharmacogenomic monitoring to track patient responsiveness and to elucidate anti-inflammatory mechanisms.

Interleukin-11 reduces T-cell-dependent experimental liver injury in mice.

Recombinant human interleukin-11 (rhIL-11) is a multifunctional cytokine that can reduce inflammation through the downregulation of multiple pro-inflammatory mediators from activated macrophages. rhIL-11 also inhibits production of several immunostimulatory cytokines such as IL-12 and interferon gamma (IFN-gamma) and has shown biological activity in multiple animal models of inflammatory disease consistent with immunomodulatory effects on macrophages and T cells. To further elucidate the anti-inflammatory activity of rhIL-11 in vivo, the effect of rhIL-11 in a model of Concanavalin A (Con-A)-induced T-cell-mediated hepatotoxicity was examined. Administration of a single dose of rhIL-11 before Con-A administration reduced centrilobular liver necrosis and enhanced survival. A dose-dependent reduction in serum levels of liver enzymes, tumor necrosis factor alpha (TNF-alpha), and IFN-gamma corresponded with this amelioration of liver damage. No significant change in infiltrating lymphocyte populations in the liver was observed following rhIL-11 treatment. Taken together, these results indicate that rhIL-11 ameliorates T-cell-mediated hepatic injury and suggests its therapeutic potential to treat inflammatory liver disease.

Immune response enhancement by in vivo administration of B7.2Ig, a soluble costimulatory protein.

The identification of both class I- and class II-restricted tumor-associated peptides recognized by T cells has led to the test of these peptides as immunogens in experimental immunotherapy for cancer patients. However, optimal T cell activation requires signaling both through the T cell receptor for antigen and through costimulatory pathways. B7.1 and B7.2 are powerful costimulatory molecules expressed on the surface of antigen-presenting cells. Using a mouse model, we have sought to optimize costimulatory signals during antipeptide responses by administering a soluble form of B7.2 at the time of peptide immunization. Administration of B7. 2Ig fusion protein significantly enhanced T helper cell and CTL responses. These findings suggest that soluble forms of human B7.2 protein may provide a straightforward and practical method of supplying optimal costimulation during clinical immunotherapy.

Hematopoietic, immunomodulatory and epithelial effects of interleukin-11.

Interleukin 11 (IL-11) is a pleiotropic cytokine with biological activities on many different cell types. Recombinant human IL-11 (rhIL-11) is produced by recombinant DNA technology in Escherichia coli. Both in vitro and in vivo, rhIL-11 has shown effects on multiple hematopoietic cell types. Its predominant in vivo hematopoietic activity is the stimulation of peripheral platelet counts in both normal and myelosuppressed animals. This activity is mediated through effects on both early and late progenitor cells to stimulate megakaryocyte differentiation and maturation. rhIL-11 has been approved for the treatment of chemotherapy-induced thrombocytopenia. The hematopoietic effects of rhIL-11 are most likely direct effects on progenitor cells and megakaryocytes in combination with other cytokines or growth factors. rhIL-11 also induces secretion of acute phase proteins (ferritin, haptoglobin, C-reactive protein, and fibrinogen) from the liver. The induction of heme oxidase and inhibition of several P450 oxidases have been reported from in vitro studies. In vivo, rhIL-11 treatment decreases sodium excretion by the kidney by an unknown mechanism and induces hemodilution. rhIL-11 also exhibits anti-inflammatory effects in a variety of animal models of acute and chronic inflammation, including inflammatory bowel disease, inflammatory skin disease, autoimmune joint disease, and various infection-endotoxemia syndromes. rhIL-11 has trophic effects on non-transformed intestinal epithelium under conditions of mucosal damage. The mechanism of the anti-inflammatory activity of rhIL-11 has been extensively studied. rhIL-11 directly affects macrophage and T cell effector function. rhIL-11 inhibits tumor necrosis factor-alpha (TNF alpha), interleukin 1beta (IL-1beta), interleukin 12 (IL-12), interleukin 6 (IL-6), and nitric oxide (NO) production from activated macrophages in vitro. The inhibition of cytokine production was associated with inhibition of nuclear translocation of the transcription factor, nuclear factor kappa B (NF-kappaB). The block to NF-kappaB nuclear translocation correlates with the ability of rhIL-11 to maintain or enhance production of the inhibitors of NF-kappaB, IkappaB-alpha and IkappaB-beta. In addition to effects on macrophages, rhIL-11 also reduces CD4+ T cell production of Th1 cytokines, such as IFN gamma induced by IL-12, while enhancing Th2 cytokine production. rhIL-11 also blocks IFN gamma production in vivo. The molecular effects of rhIL-11 have also been studied in a clinical trial. Molecular analysis of skin biopsies of patients with psoriasis before and during rhIL-11 treatment demonstrates a decrease in mRNA levels of TNF alpha, IFN gamma and iNOS. These activities suggest that in addition to its thrombopoietic clinical use, rhIL-11 may also be valuable in the treatment of inflammatory diseases. The clinical utility of the anti-inflammatory properties of rhIL-11 is being investigated in patients with Crohn's disease, psoriasis and rheumatoid arthritis. These diseases are believed to be initiated and maintained by activated CD4+ Th1 cells in conjunction with activated macrophages.