Unstable Phenotype Research Articles

Genome Wide Adaptations of Plasmodium falciparum in Response to Lumefantrine Selective Drug Pressure

The combination therapy of the Artemisinin-derivative Artemether (ART) with Lumefantrine (LM) (Coartem®) is an important malaria treatment regimen in many endemic countries. Resistance to Artemisinin has already been reported, and it is feared that LM resistance (LMR) could also evolve quickly. Therefore molecular markers which can be used to track Coartem® efficacy are urgently needed. Often, stable resistance arises from initial, unstable phenotypes that can be identified in vitro. Here we have used the Plasmodium falciparum multidrug resistant reference strain V1S to induce LMR in vitro by culturing the parasite under continuous drug pressure for 16 months. The initial IC50 (inhibitory concentration that kills 50% of the parasite population) was 24 nM. The resulting resistant strain V1SLM, obtained after culture for an estimated 166 cycles under LM pressure, grew steadily in 378 nM of LM, corresponding to 15 times the IC50 of the parental strain. However, after two weeks of culturing V1SLM in drug-free medium, the IC50 returned to that of the initial, parental strain V1S. This transient drug tolerance was associated with major changes in gene expression profiles: using the PFSANGER Affymetrix custom array, we identified 184 differentially expressed genes in V1SLM. Among those are 18 known and putative transporters including the multidrug resistance gene 1 (pfmdr1), the multidrug resistance associated protein and the V-type H+ pumping pyrophosphatase 2 (pfvp2) as well as genes associated with fatty acid metabolism. In addition we detected a clear selective advantage provided by two genomic loci in parasites grown under LM drug pressure, suggesting that all, or some of those genes contribute to development of LM tolerance – they may prove useful as molecular markers to monitor P. falciparum LM susceptibility.

A Microfluidic 3D Hepatocyte Culture System -HepaChip®- For Drug And Toxicity Investigations

PRESENTLY used in vitro test systems for drug and toxicity investigations lack the complexity of the liver and reduce test systems solely to hepatocytes or random co-cultures with non-parenchymal cells. Unfortunately, hepatocytes rapidly lose their typical morphology and the expression of metabolizing enzymes, which leads to cell phenotypes that differ from those found in the human physiology. This unstable phenotype is one of the main reasons for the failure to correctly predict drug-induced liver toxicity.

RANTES/CCL5 and Risk for Coronary Events: Results from the MONICA/KORA Augsburg Case-Cohort, Athero-Express and CARDIoGRAM Studies

BackgroundThe chemokine RANTES (regulated on activation, normal T-cell expressed and secreted)/CCL5 is involved in the pathogenesis of cardiovascular disease in mice, whereas less is known in humans. We hypothesised that its relevance for atherosclerosis should be reflected by associations between CCL5 gene variants, RANTES serum concentrations and protein levels in atherosclerotic plaques and risk for coronary events.Methods and FindingsWe conducted a case-cohort study within the population-based MONICA/KORA Augsburg studies. Baseline RANTES serum levels were measured in 363 individuals with incident coronary events and 1,908 non-cases (mean follow-up: 10.2±4.8 years). Cox proportional hazard models adjusting for age, sex, body mass index, metabolic factors and lifestyle factors revealed no significant association between RANTES and incident coronary events (HR [95% CI] for increasing RANTES tertiles 1.0, 1.03 [0.75–1.42] and 1.11 [0.81–1.54]). None of six CCL5 single nucleotide polymorphisms and no common haplotype showed significant associations with coronary events. Also in the CARDIoGRAM study (>22,000 cases, >60,000 controls), none of these CCL5 SNPs was significantly associated with coronary artery disease. In the prospective Athero-Express biobank study, RANTES plaque levels were measured in 606 atherosclerotic lesions from patients who underwent carotid endarterectomy. RANTES content in atherosclerotic plaques was positively associated with macrophage infiltration and inversely associated with plaque calcification. However, there was no significant association between RANTES content in plaques and risk for coronary events (mean follow-up 2.8±0.8 years).ConclusionsHigh RANTES plaque levels were associated with an unstable plaque phenotype. However, the absence of associations between (i) RANTES serum levels, (ii) CCL5 genotypes and (iii) RANTES content in carotid plaques and either coronary artery disease or incident coronary events in our cohorts suggests that RANTES may not be a novel coronary risk biomarker. However, the potential relevance of RANTES levels in platelet-poor plasma needs to be investigated in further studies.

Arabidopsis RGLG2, Functioning as a RING E3 Ligase, Interacts with AtERF53 and Negatively Regulates the Plant Drought Stress Response

Transcriptional activities of plants play important roles in responses to environmental stresses. ETHYLENE RESPONSE FACTOR53 (AtERF53) is a drought-induced transcription factor that belongs to the AP2/ERF superfamily and has a highly conserved AP2 domain. It can regulate drought-responsive gene expression by binding to the GCC box and/or the dehydration-responsive element in the promoter of downstream genes. Overexpression of AtERF53 driven by the cauliflower mosaic virus 35S promoter resulted in an unstable drought-tolerant phenotype in T2 transgenic Arabidopsis (Arabidopsis thaliana) plants. Using a yeast two-hybrid screen, we identified a RING domain ubiquitin E3 ligase, RGLG2, which interacts with AtERF53 in the nucleus. The copine domain of RGLG2 exhibited the strongest interacting activity. We also demonstrated that RGLG2 could move from the plasma membrane to the nucleus under stress treatment. Using an in vitro ubiquitination assay, RGLG2 and its closest sequelog, RGLG1, were shown to have E3 ligase activity and mediated AtERF53 ubiquitination for proteasome degradation. The rglg1rglg2 double mutant but not the rglg2 or rglg1 single mutant exhibited a drought-tolerant phenotype when compared with wild-type plants. AtERF53-green fluorescent proteins expressed in the rglg1rglg2 double mutants were stable. The 35S:AtERF53-green fluorescent protein/rglg1rglg2 showed enhanced AtERF53-regulated gene expression and had greater tolerance to drought stress than the rglg1rglg2 double mutant. In conclusion, RGLG2 negatively regulates the drought stress response by mediating AtERF53 transcriptional activity in Arabidopsis.

Natural variation and persistent developmental instabilities in geographically diverse accessions of the allopolyploid Arabidopsis suecica

Allopolyploids arise from the hybridization of two species concomitant to genome doubling. While established allopolyploids are common in nature and vigorous in growth, early generation allopolyploids are often less fertile than their progenitors and display frequent phenotypic instabilities. It is commonly assumed that new allopolyploid species must pass through a bottleneck from which only those lines emerge that have reconciled genomic incompatibilities inherited from their progenitors in their combined genome, yet little is known about the processes following allopolyploidization over evolutionary time. To address the question if a single allopolyploidization event leads to a single new homogeneous species or may result in diverse offspring lines, we have investigated 13 natural accessions of Arabidopsis suecica, a relatively recent allopolyploid derived from a single hybridization event. The studied accessions display low genetic diversity between lines, yet show evidence of heritable phenotypic diversity of traits, some of which may be adaptive. Furthermore, our data show that contrary to the notion that unstable phenotypes in neoallopolyploids are eliminated rapidly in the new species, some instabilities are carried along throughout the species' evolution, persisting in the established allopolyploid. In summary, our results suggest that a single allopolyploidization event may lay the foundation for diverse populations of the new allopolyploid species.

A complex multilevel attack on Pseudomonas aeruginosa algT/U expression and AlgT/U activity results in the loss of alginate production

Infection by the opportunistic pathogen Pseudomonas aeruginosa is a leading cause of morbidity and mortality seen in cystic fibrosis (CF) patients. This is mainly due to the genotypic and phenotypic changes of the bacteria that cause conversion from a typical nonmucoid to a mucoid form in the CF lung. Mucoid conversion is indicative of overproduction of a capsule-like polysaccharide called alginate. The alginate-overproducing (Alg+) mucoid phenotype seen in the CF isolates is extremely unstable. Low oxygen tension growth of mucoid variants readily selects for nonmucoid variants. The switching off mechanism has been mapped to the algT/U locus, and the molecular basis for this conversion was partially attributed to mutations in the algT/U gene itself. To further characterize molecular changes resulting in the unstable phenotype, an isogenic PAO1 derivative that is constitutively Alg+ due to the replacement of the mucA with mucA22 (PDO300) was used. The mucA22 allele is common in mucoid CF isolates. Thirty-four spontaneous nonmucoid variants, or sap (suppressor of alginate production) mutants, of PDO300 were isolated under low oxygen tension. About 40% of the sap mutants were rescued by a plasmid carrying algT/U (Group A). The remaining sap mutants were not (Group B). The members of Group B fall into two subsets: one similar to PAO1, and another comparable to PDO300. Sequence analysis of the algT/U and mucA genes in Group A shows that mucA22 is intact, whereas algT/U contains mutations. Genetic complementation and sequencing of one Group B sap mutant, sap22, revealed that the nonmucoid phenotype was due to the presence of a mutation in PA3257. PA3257 encodes a putative periplasmic protease. Mutation of PA3257 resulted in decreased algT/U expression. Thus, inhibition of algT/U is a primary mechanism for alginate synthesis suppression.

IL-2 Controls the Stability of Foxp3 Expression in TGF-β–Induced Foxp3+ T Cells In Vivo

Stimulation of naive mouse CD4(+)Foxp3(-) T cells in the presence of TGF-β results in the induction of Foxp3 expression and T suppressor function. However, Foxp3 expression in these induced regulatory T cells (iTreg) is unstable, raising the possibility that iTreg would not be useful for treatment of autoimmune diseases. To analyze the factors that control the stability of Foxp3 expression in iTreg, we generated OVA-specific iTreg from OT-II Foxp3-GFP knockin mice. Following transfer to normal C57BL/6 mice, OT-II GFP(+) cells maintained high levels of Foxp3 expression for 8 d. However, they rapidly lost Foxp3 expression upon stimulation with OVA in IFA in vivo. This unstable phenotype was associated with a strong methylation of the Treg-specific demethylated region within the Foxp3 locus. Administration of IL-2/anti-IL-2 complexes expanded the numbers of transferred Foxp3(+) iTreg in the absence of Ag challenge. Notably, when the iTreg were stimulated with Ag, treatment with IL-2/anti-IL-2 complexes stabilized Foxp3 expression and resulted in enhanced demethylation of the Treg-specific demethylated region. Conversely, neutralization of IL-2 or disruption of its signaling by deletion of Stat5 diminished the level of Foxp3 expression resulting in decreased suppressor function of the iTreg in vivo. Our data suggest that stimulation with TGF-β in vitro is not sufficient for imprinting T cells with stable expression of Foxp3. Administration of IL-2 in vivo results in stabilization of Foxp3 expression and may prove to be a valuable adjunct for the use of iTreg for the treatment of autoimmune diseases.

Effects of emotional and physiological stress on plaque instability in apolipoprotein E knockout mice

In the present study, we sought to investigate the effects of emotional and physiological stress on plaque instability in atherosclerosis. We used different stress-treated apolipoprotein E (ApoE)-deficient mice, which have been shown to spontaneously develop atherosclerosis with features similar to those seen in humans, as an animal model. Morphology study showed that emotional stress (ES) obviously promoted the development of atherosclerotic plaques and plaque instability evidenced by significantly increasing plaque size, plaque-to-surface ratio and plaque calcification, and enhancing the frequency of large necrotic core and medial erosion compared with control ApoE(-/-) mice (P<0.01). Physiological stress (PS) treatment alone did not affect the plaque stability compared with control ApoE(-/-) mice (P>0.05). However, the combination of ES and PS treatment (CS) initiated much stronger plaque instability compared with ES treatment alone (P<0.01), increased the frequency of thin fibrous caps, and even triggered plaque rupture and buried fibrous cap. Immunohistochemical analysis indicated that both ES and CS treatment led to an increase in the accumulation of macrophages and T cells and a decrease of smooth muscle cells, reflecting an unstable atherosclerotic plaque phenotype, in the atherosclerotic lesions in ApoE(-/-) mice. PS alone did not affect plaque cellular components. Similarly, CS-mediated changes in atherosclerotic plaque composition were stronger than that caused by ES alone (P<0.01). Taken together, ES treatment alone is sufficient to promote plaque instability. PS alone does not affect atherosclerotic plaque development, but can potentiate ES-mediated plaque destabilization.

IL-2 controls the stability of Foxp3 expression in TGF-β-induced Foxp3+ T cells in vivo (115.11)

Abstract Stimulation of naïve mouse CD4+Foxp3- T cells in the presence of TGF-β results in the induction of Foxp3 expression and T suppressor function. However, Foxp3 expression in these induced Treg (iTreg) is unstable raising the possibility that iTreg would not be useful for treatment of autoimmune diseases. To analyze the factors that control the stability of Foxp3 expression in iTreg, we generated OVA-specific iTreg from OT-II Foxp3-GFP knock in mice. Following transfer to normal C57BL/6 mice, OT-II GFP+ cells maintained high levels of Foxp3 for 8 days. However, they rapidly lost Foxp3 expression upon stimulation with OVA in IFA in vivo. This unstable phenotype was associated with a strong methylation of the Treg-specific demethylated region (TSDR) within the Foxp3 locus. Administration of IL-2/anti-IL-2 complexes expanded the numbers of transferred Foxp3+ iTreg in the absence of antigen challenge. Notably, when the iTreg were stimulated with antigen, treatment with IL-2/anti-IL-2 complexes stabilized Foxp3 expression and resulted in enhanced demethylation of the TSDR. Conversely, neutralization of IL-2 diminished Foxp3 expression resulting in decreased suppressor function of the iTreg in vivo. Our data suggest that stimulation with TGF-β in vitro is not sufficient for imprinting T cells with stable Foxp3 expression. Administration of IL-2 in vivo stabilizes Foxp3 expression and may prove to be a valuable adjunct for the use of iTreg for the treatment of autoimmune diseases.

Towards the molecular characterization of the stable producer phenotype of recombinant antibody-producing NS0 myeloma cells

The loss of heterologous protein expression is one of the major problems faced by industrial cell line developers and has been reported by several authors. Therefore, the understanding of the mechanisms involved in the generation of stable and high producer cell lines is a critical issue, especially for those processes based on long term continuous cultures. We characterized two recombinant NS0 myeloma cell lines expressing Nimotuzumab, a humanized anti-human epidermal growth factor receptor (EGFR) antibody. The hR3/H7 clone is a stable producer obtained from the unstable hR3/t16 clone. The unstable clone was characterized by a bimodal distribution of intracellular immunoglobulin staining using flow cytometry. Loss of antibody production was due to the emergence of a non-producer cell subpopulation that increased with cell generation number. Immunoglobulin heavy chain (HC) and light chain (LC) ratio (HC/LC) was lower for the unstable phenotype. Proteomic maps using two dimensional gel electrophoresis (2DE) were obtained for both clones, at initial cell culture time and after 40 generations. Fifteen proteins potentially associated with the phenomenon of production stability were identified. The hR3/H7 stable clone showed an up-regulated expression pattern for most of these proteins. The regulation of recombinant antibody production by the host NS0 myeloma cell line most likely involves simultaneously cellular processes such as DNA transcription, mRNA processing, protein synthesis and folding, vesicular transport, glycolysis and energy production, according to the proteins identified in the present proteomic study.

RADIATION-INDUCED GENOMIC INSTABILITY

Radiation induced genomic instability can be observed in the progeny of irradiated cells multiple generations after irradiation of parental cells. The phenotype is well established both in vivo (Morgan 2003) and in vitro (Morgan 2003), and may be critical in radiation carcinogenesis (Little 2000, Huang et al. 2003). Instability can be induced by both the deposition of energy in irradiated cells as well as by signals transmitted by irradiated (targeted) cells to non-irradiated (non-targeted) cells (Kadhim et al. 1992, Lorimore et al. 1998). Thus both targeted and non-targeted cells can pass on the legacy of radiation to their progeny. However the radiation induced events and cellular processes that respond to both targeted and non-targeted radiation effects that lead to the unstable phenotype remain elusive.

Collagenase matrix metalloproteinase-8 expressed in atherosclerotic carotid plaques is associated with systemic cardiovascular outcome

Atherosclerotic plaque rupture and subsequent thrombus formation are the major cause of acute cardiovascular events. Local plaque markers may facilitate detection of the vulnerable plaque and help identify the patient at risk for cardiovascular events. Matrix metalloproteinases (MMPs) are prevalent in the arterial wall throughout the arterial system and are associated with local plaque destabilization. We hypothesized that local MMP plaque levels are predictive for atherosclerotic cardiovascular events in other vascular territories. Atherosclerotic plaques were obtained from 543 patients undergoing carotid endarterectomy (CEA). Plaques were analysed for the presence of macrophages, lipid-core, smooth muscle cells, collagen, calcification, and presence of plaque haemorrhage. MMP-2, MMP-8, and MMP-9 levels were assessed within the plaque. Following CEA, all patients underwent follow-up during 3 years. The primary outcome was defined as the composite of vascular death, non-fatal vascular event, and surgical or percutaneous vascular intervention. In contrast with MMP-2 plaque levels, MMP-8 and MMP-9 levels in the plaque were associated with an unstable carotid plaque composition and clinical presentation at baseline. Increased plaque MMP-8 level (>4.58) was associated with an increased risk for the occurrence of secondary manifestations of atherosclerotic disease during follow-up [hazard ratio = 1.76, 95% CI (1.25-2.48)] (P= 0.001), whereas plaque MMP-2 and MMP-9 levels were not predictive for systemic cardiovascular events. In contrast with MMP-2, increased carotid MMP-8 and MMP-9 plaque levels are associated with an unstable plaque phenotype. High collagenase MMP-8 levels in the carotid plaque are associated with the occurrence of systemic cardiovascular outcome during follow-up.

Atherosclerotic Plaque Stability Is Affected by the Chemokine CXCL10 in Both Mice and Humans

Background. The chemokine CXCL10 is specifically upregulated during experimental development of plaque with an unstable phenotype. In this study we evaluated the functional consequences of these findings in mice and humans. Methods and Results. In ApoE−/− mice, we induced unstable plaque with using a flow-altering device around the carotid artery. From week 1 to 4, mice were injected with a neutralizing CXCL10 antibody. After 9 weeks, CXCL10 inhibition resulted in a more stable plaque phenotype: collagen increased by 58% (P = 0.002), smooth muscle cell content increased 2-fold (P = 0.03), while macrophage MHC class II expression decreased by 50% (P = 0.005). Also, the size of necrotic cores decreased by 41% (P = 0.01). In 106 human carotid endarterectomy specimens we found that increasing concentrations of CXCL10 strongly associate with an increase in atheromatous plaque phenotype (ANOVA, P = 0.003), with high macrophage, low smooth muscle cell, and low collagen content. Conclusions. In the present study we showed that CXCL10 is associated with the development of vulnerable plaque in human and mice. We conclude that CXCL10 might provide a new lead towards plaque-stabilizing therapy.

Unstable Side Population Phenotype of Mouse Spermatogonial Stem Cells In Vitro

Stem cells of the side population (SP) phenotype are found in many self-renewing tissues and can be identified by their unique ability to effectively exclude the dye Hoechst 33342. We previously established a method for expanding spermatogonial stem cells (SSCs) in vitro, but the frequency of SSCs is only about 1 to 2%, limiting detailed SSC analyses. In this study, we sought to isolate SSCs from in vitro cultures by exploiting their ability to exclude Hoechst 33342. In contrast to the findings of previous in vivo studies, we found that SP cells developed in a stochastic manner in vitro. Moreover, SP cells in culture were not enriched in SSCs, but they were interconvertible with non-SP cells. Although SP cells were consistently found in testes after transplantation of cultured cells, they were not enriched in SSCs. These results show that SSCs have an unstable SP phenotype and provide evidence that SSCs change their phenotype characteristics in response to their microenvironment.

Abstract A32: Identification and characterization of colon cancer-associated antigens

Abstract Colon cancer (CRC) is an immunogenic tumor and adaptive immunity may play a role in inhibiting disease relapse. A vaccine to boost cellular immunity in CRC patients could prevent disease recurrence, but there have been few defined immunogenic proteins identified as vaccine candidates. TVG has shown that overexpressed tumor associated proteins, abundant in the cancer state but expressed at basal levels in normal cells, can be immunogenic. We hypothesized that ideal candidate antigens would be ones already validated as prognostic markers in multivariate analysis. We performed a directed literature review using search words e.g. colon cancer, prognosis, biomarker, multivariate analysis. We selected a panel of eight proteins based on (1) incidence of expression, (2) independent predictor of poor prognosis, (3) independent predictor of early disease recurrence, and (4) known biologic function in CRC pathogenesis. Using a web based algorithm program developed by TVG, we identified peptides that were predicted to be high affinity binders across multiple HLA DR alleles; characteristics we have shown to be associated with native epitopes of self tumor antigens. We selected and synthesized 19 peptides predicted to be the highest binders. We evaluated the potential immunogenicity of peptides with 1. IFN-g ELISPOT assays using PBMC from CRC patients and normal controls and 2. specific IgG antibody response with ELISA using sera from CRC patients and normal controls. We quantitated antigen gene expression using qPCR in cell lines representing microsatellite unstable (MSI), chromosome unstable (CIN) and CpG island methylator phenotype (CIMP) colorectal cancer. The eight proteins are: CDC25B, COX-2, EBAG9, EGFR, Fascin-1, IGF-1R, PRL-3 and VCP. Two of the eight proteins had already been shown to be human tumor antigens. For all candidate antigens there was a greater IFN-g response in CRC patients than in controls; for three proteins, this difference reached statistical significance. Six proteins were positive by ELISA. Seven antigen genes were overexpressed relative to normal colon. We conclude that: 1. biologically relevant CRC associated proteins can be identified that are associated with prognosis, 2. epitopes predicted to bind multiple DR alleles, derived from candidate antigens, elicit T cell responses greater in CRC patients than in normal controls, 3. there was an overexpression of candidate genes across three different CRC phenotypes, and 4. these antigens may represent novel immunologic targets for CRC. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A32.

Adipocyte fatty acid binding protein in atherosclerotic plaques is associated with local vulnerability and is predictive for the occurrence of adverse cardiovascular events

There is an increasing need for translational studies identifying molecular targets contributing to atherosclerotic plaque destabilization. Local molecular plaque markers that are related to plaque vulnerability may hold predictive value to identify patients who are at increased risk to suffer from cardiovascular events. Animal studies revealed that adipocyte fatty acid binding protein (FABP4) is associated with the progression of atherosclerosis; however, FABP4 expression studies in human atherosclerotic plaques are lacking. We investigated FABP4 expression in carotid atherosclerotic lesions in relation to plaque composition and future cardiovascular events. Atherosclerotic plaques were obtained from 561 patients undergoing carotid endarterectomy (CEA). Plaques were analysed for the presence of macrophages, lipid core, smooth-muscle cells, collagen, calcification, and intraplaque haemorrhage. Patients were followed for 3 years after CEA. The primary outcome was defined as the composite of vascular death, vascular event, and surgical or percutaneous vascular intervention. Fatty acid binding protein levels correlated with unstable plaque characteristics and symptomatic lesions. Patients with increased FABP4 plaque levels showed a two-fold increased risk [HR = 1.99, 95% confidence interval (95% CI) (1.30-3.04)] (P = 0.005) to reach the primary outcome during follow-up. Increased FABP4 levels related to primary outcome, independent from general cardiovascular risk factors [HR = 1.33, 95% CI (1.08-1.65)] (P = 0.008). FABP4 levels in atherosclerotic lesions are associated with an unstable plaque phenotype and an increased risk for cardiovascular events during follow-up. Besides risk stratification for adverse future cardiovascular events, the outcome of the present study supports the relevance of exploring FABP4 antagonists as a potential pharmaceutical intervention to treat atherosclerotic disease progression.

Chronic unpredictable stress accelerates atherosclerosis through promoting inflammation in apolipoprotein E knockout mice

Introduction Chronic unpredictable stress (CUS) has been suggested to accelerate atherosclerosis. However, the underlying mechanism of this adverse effect is not fully understood. Since chronic stress can promote or even initiate inflammation response, which is thought to be a major contributor to atherogenesis, we postulated that stress-induced inflammatory response might be one important reason for CUS-promoted atherosclerotic disease. Materials and methods We used the CUS treated apolipoprotein E (ApoE)-deficient mice, which have been shown to spontaneously develop atherosclerosis with features similar to those seen in humans, as an animal model. Haematoxylin and eosin staining and immunohistostaining were used to analyze the plaque formation and composition. Results Histological analysis clearly demonstrated that CUS treatment promoted the development of atherosclerotic lesions, such as triggering plaque rupture, increasing plaque size and plaque-to-surface ratio, and also led to profound changes in plaque composition, as evidenced by increased macrophage and T cell infiltration and decreased smooth muscle cell mass, all reflecting an unstable plaque phenotype. Moreover, adhesion molecular vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), acute phase reactant C-reactive protein (CRP), and proinflammatory cytokine interleukin-6 (IL-6) were significantly enhanced in CUS treated ApoE -/- mice compared with untreated control animals ( P < 0.01). Conclusion The involvement of CUS in the pathogenesis of atherosclerosis is at least partially attributable to its acceleration of inflammation.

Abstract 4774: Identification and characterization of colon cancer associated antigens which would be key therapeutic targets in the prevention of disease relapse or progression

Abstract Introduction: Colon cancer (CRC) is an immunogenic tumor and adaptive immunity may play a role in inhibiting disease relapse. A vaccine which could boost cellular immunity in CRC patients would prevent disease recurrence. However, there have been few defined immunogenic proteins, implicated in driving the malignant phenotype, that have been identified as vaccine candidates. Our group has shown that overexpressed tumor associated proteins, abundant in the cancer state but expressed at basal levels in normal cells, can be immunogenic. We hypothesized that ideal candidate antigens would be ones already validated as prognostic markers in multivariate analysis. Methods: We performed a directed literature review using search words e.g. colon cancer, prognosis, biomarker to identify such proteins in CRC. We selected a panel of eight proteins based on (1) incidence of expression, (2) independent predictor of poor prognosis and (3) early disease recurrence, and (4) known biologic function in CRC pathogenesis. Using our algorithm program we identified peptides predicted to be high affinity binders across multiple HLA DR alleles; characteristics shown to be associated with native epitopes of self tumor antigens. We synthesized the 19 peptides that were predicted to be the highest binders. We evaluated the potential immunogencity of the peptides with 1. IFN-g ELISPOT assays using PBMC's from CRC patients and controls and 2. specific IgG antibody response with ELISA using serum from CRC patients and controls. We quantitated antigen gene expression using qPCR in cell lines representing microsatellite unstable (MSI), chromosome unstable (CIN) CpG island methylator phenotype (CIMP) CRC. Summary: The eight proteins are: CDC25B, COX-2, EBAG9, EGFR, Fascin-1, IGF-1R, PRL-3 and VCP. For all candidate antigens there was a greater IFN-g response in CRC patients than in controls; for three proteins, this difference reached statistical significance (CDC25B p=.013, COX-2 p=.013, PRL-3 p=.018). For ELISA positive response, the cut-off point was set at values of the mean of control serum plus two standard deviations. All six proteins were positive. All seven antigen genes were overexpressed relative to normal colon. Conclusions: 1. biologically relevant CRC associated proteins can be identified that are associated with prognosis and potentially expressed in the majority of colorectal cancers, 2. epitopes predicted to bind multiple DR alleles, derived from candidate antigens, elicit T cell responses that are greater in CRC patients than in normal controls, 3. there was an overexpression of candidate genes across three different CRC phenotypes, and 4. these antigens may represent novel immunologic targets for CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4774.

Darapladib and atherosclerosis

This review will focus specifically on lipoprotein-associated phospholipase A 2 (Lp-PLA 2) and its inhibitor darapladib. Lp-PLA 2 is bound predominantly to apolipoprotein B (apoB)-containing lipoproteins and highly expressed in the necrotic core of atherosclerotic lesions. It rapidly degrades oxidatively modified phospholipids in modified LDL, for example, leading to formation of proinflammatory and cytotoxic products (i.e., lysophosphatidylcholine and oxidized nonesterified fatty acids). In a study conducted in diabetic and hypercholesterolemic swines, selective Lp-PLA 2 inhibitors (i.e., darapladib) reduced the development of advanced coronary atherosclerosis. Specifically, darapladib treatment considerably decreased the area of plaques and the necrotic cores, and reduced medial destruction, resulting in fewer lesions with an unstable phenotype. In the IBIS 2 study conducted in humans, darapladib significantly halted the increase of the necrotic core volume (-0.5 ± 13.9 mm 3; P = 0.71) that was observed in the cohort receiving placebo (4.5 ± 17.9 mm 3; P = 0.009), resulting in a significant treatment difference of -5.2 mm3 (P = 0.012). In conclusion, despite standard-of-care treatment, patients with coronary artery disease continue to have recurrent cardiovascular events. PLA 2 inhibition may represent a new approach for the treatment of atherosclerosis if the benefit of this intervention is confirmed by the results of ongoing event-driven outcome trials. Copyright

Opposing Role of Condensin and Radiation-sensitive Gene RAD52 in Ribosomal DNA Stability Regulation

Blocking target of rapamycin signaling by starvation or rapamycin inhibits ribosomal DNA (rDNA) transcription and causes condensin-mediated rDNA condensation and nucleolar contraction. In the absence of condensin, however, repression of rDNA transcription leads to rDNA instability and elevated level of extrachromosomal rDNA circles and nucleolar fragmentation. Here, we show that mutations in the Rad52 homologous recombination machinery block rDNA instability. Rad52 is normally excluded from the nucleolus. In the absence of condensin, however, repression of rDNA transcription results in Rad52 localization to the nucleolus, association with rDNA and subsequent formation of extrachromosomal rDNA circles, and reduced cell survival. In contrast, deletion of RAD52 restores cell viability under the same conditions. These results reveal an opposing role of condensin and Rad52 in the control of rDNA stability under nutrient starvation conditions.