Expression Of actA Research Articles

Activin A-Targeted Therapy in Cancer: An Updated Review on Challenges and Opportunities in Clinical Translation.

Activin A (ActA) is a cytokine from the TGF-β superfamily that mediates a vast number of physiological mechanisms, mainly through the SMAD signaling pathway. Growing evidence indicates that ActA overexpression is also correlated with poor prognosis in cancer patients and several tumor characteristics, including cancer proliferation, metastasis, immunosuppression, drug resistance, cachexia, and cancer-associated fibroblast activation. As such, ActA-targeted therapy has been viewed as a potential adjuvant therapy alongside other anti-cancer modalities that may result in more efficient anti-cancer effects, such as stronger immune responses, overcoming drug resistance, reversing cachexia, etc. However, despite its interesting concept, targeting ActA is not without certain challenges and considerations. Indeed, ActA has unexpectedly shown anti-tumor effects in some cases, which might be explained by differences in the expression levels of different ActA receptors on the cell surface, activation of non-SMAD pathways, and imbalance in ActA levels. Besides, many of the current ActA antagonists lack enough specificity and, as a result, bind to non-ActA receptors as well. Furthermore, ubiquitous expression of ActA in the body can cause serious adverse effects following systemic administration. Furthermore, to address these issues, anti-ActA monoclonal antibodies and nanoparticle drug delivery systems have recently been suggested to target ActA with better precision in the affected area. In this review, first, we provide the different implications of ActA in cancer. Then, we discuss the recent insights into targeting ActA signaling as an adjuvant therapy alongside other anti-cancer modalities, as well as the possible challenges and novel opportunities on the path of clinical translation.

Activin A Is a Master Regulator of Phenotypic Switch in Adipose Stromal Cells Initiated by Activated Immune Cell-Secreted Interleukin-1β.

Prolonged tissue ischemia and inflammation lead to organ deterioration and are often accompanied by microvasculature rarefaction, fibrosis, and elevated systemic Activin A (ActA), the level of which frequently correlates with disease severity. Mesenchymal stromal cells are prevalent in the perivascular niche and are likely involved in tissue homeostasis and pathology. This study investigated the effects of inflammatory cells on modulation of phenotype of adipose mesenchymal stromal cells (ASC) and the role of ActA in this process. Peripheral blood mononuclear cells were activated with lipopolysaccharide (activated peripheral blood mononuclear cells [aPBMC]) and presented to ASC. Expression of smooth muscle/myofibroblast markers, ActA, transforming growth factors beta 1-3 (TGFβ1-3), and connective tissue growth factor (CTGF) was assessed in ASC. Silencing approaches were used to dissect the signaling cascade of aPBMC-induced acquisition of myofibroblast phenotype by ASC. ASC cocultured with aPBMC or exposed to the secretome of aPBMC upregulated smooth muscle cell markers alpha smooth muscle actin (αSMA), SM22α, and Calponin I; increased contractility; and initiated expression of ActA. Interleukin (IL)-1β was sufficient to replicate this response, whereas blocking IL-1β eliminated aPBMC effects. ASC-derived ActA stimulated CTGF and αSMA expression in ASC; the latter independent of CTGF. Induction of αSMA in ASC by IL-1β or ActA-enriched media relied on extracellular enzymatic activity. ActA upregulated mRNA levels of several extracellular matrix proteins in ASC, albeit to a lesser degree than TGFβ1, and marginally increased cell contractility. In conclusion, the study suggests that aPBMC induce myofibroblast phenotype with weak fibrotic activity in perivascular progenitors, such as ASC, through the IL-1β-ActA signaling axis, which also promotes CTGF secretion, and these effects require ActA extracellular enzymatic processing.

Gene Expression and Phenotypic Assessment of Egg Quality across Developmental Stages of Atlantic Cod throughout the Spawning Season.

Egg quality in fishes is commonly determined by fertilisation success and cleavage patterns as a phenotypic outcome of underlying regulatory mechanisms. Although these phenotypic estimators of egg quality are useful in farming conditions, these "good quality" egg batches do not always translate to good larval growth and survival. The identification of genes involved in embryonic development may help find links between genetic factors of maternal origin and egg quality. Herein, the relative expression of seven stage-specific developmental genes of Atlantic cod was analysed using quantitative PCR to understand the function during embryogenesis and its relationship with egg quality. Genes ccnb2 and pvalb1 showed significant differential expression between developmental stages and significant upregulation from blastula and somite stages, respectively. The comparison of spawning batches showed that the relative gene expression of genes ccnb2, acta, tnnt3 and pvalb1 was significantly higher from the middle of the spawning season where phenotypic quality estimators establish the best egg quality. Moreover, a positive significant correlation was observed between quality estimators based on egg morphology and the genetic expression of genes acta and acta1 during somitogenesis. This study suggests that the combination of quality estimators, genetics and batch timing could help optimise reproductive protocols for commercial stocks of Atlantic cod.

Abstract 3057: Vascular Smooth Muscle Cell Cd47 Contributes To Atherosclerosis Development

Aims: Thrombospondin-1 (TSP1), a secretory protein, has been associated with various cardiovascular diseases. TSP1 mainly functions via its cognate receptors CD47 and CD36. Recently, we observed elevated TSP1 expression in human and murine atherosclerotic arteries. However, the role of vascular smooth muscle cell (VSMC) TSP1-CD47 signaling in regulating VSMC phenotype and atherogenesis is unclear. Methods and Results: Human arterial SMCs were treated with human TSP1 and analyzed for SMC markers and proliferation. The data revealed decreased expression of SMC markers, ACTA, CNN1, and SM22α in TSP1-exposed VSMCs compared with vehicle-treated control cells. Further, TSP1 treatment increased VSMC proliferation in vitro , suggesting TSP1 induces VSMC hyperproliferative phenotype. Additional experiments showed CD47 as the major TSP1’s receptor in VSMCs. To investigate the in vivo role of VSMC Cd47 in atherosclerosis, we generated tamoxifen-inducible SMC-specific Cd47 knockout mice ( Cd47 f/f Myh11 Cre +/– , Cd47 ΔVSMC ) by crossing female Cd47 f/f mice with male Myh11 Cre +/- mice. Male tamoxifen-injected Cd47 ΔVSMC and corn oil-administered Cd47 WT mice were given a single injection of AAV8- hPCSK9 (1х10 11 viral genomes/mouse, IP) and fed a Western diet for 12 weeks. En face oil red O (ORO) staining of whole aortas exhibited reduced atherosclerosis in Cd47 ΔVSMC mice compared with control Cd47 WT mice. Further histochemical staining performed on aortic root sections revealed attenuated neointimal lesion area, lipid accumulation and necrotic area in Cd47 ΔVSMC mice. However, there were no significant differences in weight gain, body composition (fat and lean mass), plasma total cholesterol, and fasting blood glucose between Cd47 ΔVSMC and Cd47 WT mice. Conclusions: Taken together, these findings suggest that TSP1 promotes VSMC phenotype switch and VSMC-specific Cd47 deletion in hypercholesterolemic mice suppresses atherosclerosis progression.

Efficacy of disinfectant and bacteriophage mixture against planktonic and biofilm state of Listeria monocytogenes to control in the food industry

Fresh produce and animal-based products contaminated with Listeria monocytogenes have been the main cause of listeriosis outbreaks for many years. The present investigation explored the potential of combination treatment of disinfectants with a bacteriophage cocktail to control L. monocytogenes contamination in the food industry. A mixture of 1 minimal inhibitory concentration (MIC) of disinfectants (sodium hypochlorite [NaOCl], hydrogen peroxide [H2O2], and lactic acid [LA]) and multiplicity of infection (MOI) 100 of phage cocktail was applied to both planktonic cells in vitro and already-formed biofilm cells on food contact materials (FCMs; polyethylene, polypropylene, and stainless steel) and foods (celery and chicken meat). All the combinations significantly lowered the population, biofilm-forming ability, and the expression of flaA, motB, hlyA, prfA, actA, and sigB genes of L. monocytogenes. Additionally, in the antibiofilm test, approximately 4 log CFU/cm2 was eradicated by 6 h treatment on FCMs, and 3 log CFU/g was eradicated within 3 days on celery. However, <2 log CFU/g was eradicated in chicken meat, and regrowth of L. monocytogenes was observed on foods after 5 days. The biofilm eradication efficacy of the combination treatment was proven through visualization using scanning electron microscopy (SEM) and confocal microscopy. In the SEM images, the unusual behavior of L. monocytogenes invading from the surface to the inside was observed after treating celery with NaOCl+P or H2O2 + P. These results suggested that combination of disinfectants (NaOCl, H2O2, and LA) with Listeria-specific phage cocktail can be employed in the food industry as a novel antimicrobial and antibiofilm approach, and further research of L. monocytogenes behavior after disinfection is needed.

The apoptotic effects of soybean agglutinin were induced through three different signal pathways by down-regulating cytoskeleton proteins in IPEC-J2 cells

Soybean agglutinin (SBA) is a main anti-nutritional factor in soybean. SBA exhibits its anti-nutritional functions by binding to intestinal epithelial cells. Keratin8 (KRT8), Keratin18 (KRT18) and Actin (ACTA) are the representative SBA-specific binding proteins. Such cytoskeletal proteins act a crucial role in different cell activities. However, limited reports reveal what the signal transduction pathway of apoptosis caused by SBA when binding to KRT8, KRT18 and ACTA. We aimed to evaluate the effects of SBA on cell apoptosis and the expression of the cytoskeletal protein (KRT8, KRT18 and ACTA), reveal the roles of these cytoskeletal proteins or their combinations on SBA-induced cell apoptosis in IPEC-J2 cell line, evaluate the influences of SBA on the mitochondria, endoplasmic reticulum stress and death receptor-mediated apoptosis signal pathway and to show the roles of KRT8, KRT18 and ACTA in different apoptosis signal pathways induced by SBA. The results showed that SBA induced the IPEC-J2 cell apoptosis and decreased the mRNA expression of KRT8, KRT18 and ACTA (p < 0.05). The degree of effect of three cytoskeleton proteins on cell apoptosis was ACTA > KRT8 > KRT18. The roles of these three cytoskeletal proteins on IPEC-J2 apoptotic rates had a certain accumulation effect. SBA up-regulated mitochondrial fission variant protein (FIS1) and fusion protein (Mfn2) promoted CytC and AIF in mitochondria to enter the cytoplasm, activated caspase-9 and caspase-3, damaged or declined mitochondrial function and reduced ATP synthesis (p < 0.05). Also, SBA up-regulated the expression of GRP78, XBP-1, eIF2α, p-eIF2α and CHOP (p < 0.05), down-regulated the expression level of ASK1 protein (p < 0.05). SBA led to the recruitment of FADD to the cytoplasmic membrane and increased the expression of FasL, resulting in caspase-8 processing. SBA up-regulated the expression level of Bax protein and decreased cytosolic Bcl-2 and Bid (p < 0.05). In addition, there was a significant negative correlation between the gene expression of cytoskeleton proteins and apoptosis, as well as the expression of key proteins of apoptosis-related signal transduction pathways. In conclusion, SBA induced the activation of the mitochondria, endoplasmic reticulum stress and the death receptor-mediated apoptosis signal pathway and the crosstalk between them. The effect of SBA on these three pathways was mainly exhibited via down-regulation of the mRNA expression of the three cytoskeletal expressions. This study elucidates the molecular mechanism and signaling pathway of SBA that lead to apoptosis from the perspective of cell biology and molecular biology and provides a new perspective on the toxicity mechanism of other food-derived anti-nutrients, medical gastrointestinal health and related cancer treatment.

Infection behavior of Listeria monocytogenes on iceberg lettuce (Lactuca sativa var. capitata)

Iceberg lettuce among leafy vegetables is susceptible to contamination with foodborne pathogens, posing a risk of food microbial safety. Listeria monocytogenes (L. monocytogenes) is a highly lethal pathogen that can survive and proliferate on leafy vegetables. In this paper, the contamination stage, attachment site, internalization pathway, proliferation process, extracellular substance secretion and virulence factors expression of L. monocytogenes on iceberg lettuce were researched. Results showed that the contamination stage of L. monocytogenes on iceberg lettuce was 0–20 min, the proliferation stage was after 20 min. The attachment tissues were stomata and winkles. The internalization distance of L. monocytogenes in the midrib was farther than that in the leaf blade. They enhanced the movement ability of cells by up-regulating the expression of flaA and motA genes, and enhanced the adhesion ability of cells by up-regulating the expression of actA and inla genes, which was beneficial to the proliferation. During proliferation, cells gradually secreted extracellular substances to promote the biofilm formation on iceberg lettuce. The formation of biofilms experienced: individual bacteria, cell aggregation and biofilm maturation. Biofilms were more likely to form on the leaf blade of iceberg lettuce.

Fibroblasts Mediate Ectopic Bone Formation of Calcium Phosphate Ceramics.

Heterogeneity of fibroblasts directly affects the outcome of tissue regeneration; however, whether bioactive ceramics regulate bone regeneration through fibroblasts is unclear. Ectopic bone formation model with biphasic calcium phosphate (BCP) implantation was used to investigate the temporal and spatial distribution of fibroblasts around ceramics. The effect of BCP on L929 fibroblasts was evaluated by EdU assay, transwell assay, and qRT-PCR. Further, the effect of its conditioned medium on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) was confirmed by ALP staining. SEM and XRD results showed that BCP contained abundant micro- and macro-pores and consisted of hydrogen-apatite (HA) and β-tricalcium phosphate (β-TCP) phases. Subsequently, BCP implanted into mice muscle successfully induced osteoblasts and bone formation. Fibroblasts labelled by vimentin gathered around BCP at 7 days and peaked at 14 days post implantation. In vitro, BCP inhibited proliferation of L929 fibroblast but promoted its migration. Moreover, expression of Col1a1, Bmp2, and Igf1 in L929 treated by BCP increased significantly while expression of Tgfb1 and Acta did not change. ALP staining further showed conditioned media from L929 fibroblasts treated by BCP could enhance osteogenic differentiation of BMSCs. In conclusion, fibroblasts mediate ectopic bone formation of calcium phosphate ceramics.

Differentiation of adipose‐derived stem cells into urothelial and smooth muscle cell lines within the structure of collagen/hyaluronan scaffold

Many urethral pathologies require surgical interventions. Although the commonly used surgical techniques are still being developed, complications such as the urethral strictures often occur and repeated surgery is needed. Tissue engineering of the urethral tissue, using autologous stem cells and appropriate scaffolds, represents promising technique to overcome these complications. In our experiment, we focused on the differentiation of adipose tissue‐derived stem cells seeded on the collagen/hyaluronan scaffolds into urothelial and smooth muscle cells. Differentiation culture media containing keratinocyte serum‐free medium with 2 % fetal bovine serum (FBS) and 30 ng/mL EGF, and D‐MEM with 10 % FBS, 2.5 ng/mL TGF‐β and 5 ng/mL PDGF were used to induce urothelial and smooth muscle differentiation, respectively. After two weeks of cultivation, cells were analyzed by scanning electron microscope (JEOL 7500F) for morphological features. Expression of the uroplakin 2 (UPK2) gene, keratin 2 gene (KRT2), ACTA 2 gene, and calponin 1 gene (CNN1) was tested in order to determine successful differentiation by quantitative Real‐Time PCR. Obtained results showed attachment of the cells on scaffold surface. The increased levels of UPK2 and KRT2 confirmed urothelial differentiation and expression of ACTA 2 and CNN1 confirmed smooth muscle differentiation. On the basis of obtained results, it can be emphasized that collagen/ hyaluronan scaffolds colonized by adipose tissue‐derived stem cells after performing further tests dealing with biosafety may have promising potential for tissue engineering of the urethra.Support or Funding InformationSupported by grant APVV no. 15‐0111.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

P1.07-026 Activin A is Associated with Poor Prognosis and Promotes Metastatic Growth in Small Cell Lung Cancer

Small cell lung cancer (SCLC) is a devastating malignancy characterized by resistance to therapy and poor clinical outcome. Therefore, identification of novel therapeutic strategies and non-invasive biomarkers that facilitate early detection and predict prognosis is urgently needed. Expression of the growth factor activin A (ActA), a member of the TGF beta superfamily, is deregulated in a number of malignancies. However, to date there is no data on the role of ActA in SCLC. In a cohort of SCLC patients (n=79) and in sex- and age-matched controls (n=66), plasma levels of ActA were measured by ELISA. The diagnostic value of plasma ActA was evaluated by ROC curve analysis. The mRNA and protein expression levels of ActA were analyzed in SCLC cell lines by qRT-PCR and by ELISA, respectively, and one of the cell lines with low baseline ActA expression was transfected with ActA and a control vector. The effect of ActA overexpression on the in vivo growth of SCLC cells was investigated in an orthotopic xenograft model. Increased plasma ActA levels were found in patients with SCLC (vs. controls) and ActA levels were elevated in a TNM stage-dependent manner. Moreover, high ActA levels were associated with significantly shorter overall survival and multivariate analysis revealed that plasma ActA concentration is an independent negative prognostic factor in this patient cohort. With an area under the curve of 0.81 (95% CI: 0.74-0-0.88), circulating ActA was identified as a useful biomarker for the diagnosis of SCLC. Expression of ActA in SCLC cell lines was detected in vitro. Furthermore, ActA overexpression increased the metastatic capacity of SCLC cells in our xenograft model. Our findings suggest that the measurement of circulating ActA can support the diagnosis and staging of SCLC and, moreover, that it can help to predict the clinical outcome. We also conclude that ActA has a role in the aggressive behavior of this tumor type and that its potential therapeutic relevance needs to be further investigated.

Strain-Specific Interactions of Listeria monocytogenes with the Autophagy System in Host Cells

Listeria monocytogenes is an intracellular bacterial pathogen that can replicate in the cytosol of host cells. These bacteria undergo actin-based motility in the cytosol via expression of ActA, which recruits host actin-regulatory proteins to the bacterial surface. L. monocytogenes is thought to evade killing by autophagy using ActA-dependent mechanisms. ActA-independent mechanisms of autophagy evasion have also been proposed, but remain poorly understood. Here we examined autophagy of non-motile (ΔactA) mutants of L. monocytogenes strains 10403S and EGD-e, two commonly studied strains of this pathogen. The ΔactA mutants displayed accumulation of ubiquitinated proteins and p62/SQSTM1 on their surface. However, only strain EGD-e ΔactA displayed colocalization with the autophagy marker LC3 at 8 hours post infection. A bacteriostatic agent (chloramphenicol) was required for LC3 recruitment to 10403S ΔactA, suggesting that these bacteria produce a factor for autophagy evasion. Internalin K was proposed to block autophagy of L. monocytogenes in the cytosol of host cells. However, deletion of inlK in either the wild-type or ΔactA background of strain 10403S had no impact on autophagy evasion by bacteria, indicating it does not play an essential role in evading autophagy. Replication of ΔactA mutants of strain EGD-e and 10403S was comparable to their parent wild-type strain in macrophages. Thus, ΔactA mutants of L. monocytogenes can block killing by autophagy at a step downstream of protein ubiquitination and, in the case of strain EGD-e, downstream of LC3 recruitment to bacteria. Our findings highlight the strain-specific differences in the mechanisms that L. monocytogenes uses to evade killing by autophagy in host cells.

Effects of tripterygium wilfordii polyglucoside on expression of Activin A and influence of transdifferentiation in renal tubulointerstitium of rats with diabetic nephropathy

To explore the effects of tripterygium wilfordii polyglucoside (TWP) on expression of Activin A (Act-A) and influence of transdifferentiation in renal tubulointerstitium of rats with diabetes nephropathy (DN). A total of 40 male Wistar rats were randomly divided into normal control, DN model, irbesartan treatment and TWP treatment groups (n = 10 each). The animal model of DN was established by an injection of streptozotocin. After modeling, irbesartan treatment and TWP treatment groups received an intragastric injection of corresponding drugs. The body weights were recorded and the 24 h Pro quantitative levels detected at Weeks 4, 8, 12 and 16 respectively. After sacrificing at Week 16, serum creatinine (Scr) was detected. The renal tissues were excised and examined after HE and PAS staining. The expressions of Act-A, alpha-smooth muscle actin (α-SMA), collagen type IV (Co-IV) and E-cad in renal tubulointerstitium were detected by immunohistochemistry. And the expressions of Act-A, α-SMA, Co-IV and mRNA of E-cad in renal tissue were detected by real-time quantitative fluorescent polymerase chain reaction (PCR). Compared with DN model group, 24 hPro ((67.1 ± 9.6), (61.8 ± 8.0) vs (158.3 ± 13.3) mg), quantitative Scr ((31.7 ± 4.1), (32.6 ± 6.2) vs (55.3 ± 8.9) µmol/L) and renal hypertrophic index ((9.14 ± 0.65), (7.50 ± 0.34) vs (11.28 ± 0.70) mg/g) of irbesartan treatment and TWP treatment groups decreased obviously at Week 16 (all P < 0.05). However, no statistical significance existed between irbesartan treatment and TWP treatment groups. The body weights of irbesartan treatment and TWP treatment groups increased versus DN model group. Pathological sections showed that lesion degree of renal tubulointerstitium became alleviated in each group. Yet no statistical significance existed between irbesartan treatment and TWP treatment groups. Both immunohistochemistry and real-time quantitative fluorescent PCR showed that the expressions of Act-A, α-SMA and Co-IV significantly decreased in renal tubulointerstitium (P < 0.01) while the expression E-cad significantly increased (P < 0.01) in inirbesartan treatment and TWP treatment groups versus DN model group. And no statistical significance existed between irbesartan treatment and TWP treatment groups. TWP may down-regulate the expression of Act-A and inhibit the transdifferentiation of renal tubulointerstitial so as to delay renal fibrosis.

The effects of Smad6 via small RNA pathways on the ischemic injury in PC12 cells.

Though we have reported the neuroprotective effect of exogenous ActA on oxygen-glucose deprivation (OGD) injury, the endogenous role of Smad6 remains not well understood. Smad6 is an important regulator of the ActA/smads signaling via a negative feedback circuit. In this study, nerve growth factor (NGF) and OGD were used to stimulate (rat adrenal pheochromocytoma) PC12 cells converting them into neurons to establish an ischemia in vitro model. Combined with the small interfering technology of Smad6 and FCM, Hoechst and Western blot were used to identify the apoptosis rate. The effect of silencing of Smad6 with siRNA was observed. These results showed that the apoptosis rate was 21.54% by 16-h OGD. For the combined Smad6-siRNA, the apoptosis rate was 10.55%. The expression of procaspase-3 protein was increased by Smad6-siRNA.The expression of ActA and p300 was also increased. The apoptosis rate was decreased in the ischemic injury with Smad6-siRNA. At the same time, it provided a reference to study the mechanism of Smad6 and its signaling in response to the acute ischemic damage.

Abstract 1811: Cancer/testis antigens: Comprehensive expression analysis and correlation with survival in glioblastoma.

Abstract Glioblastoma (GBM) is a highly invasive and aggressive tumor which confers a dismal prognosis despite advances of current therapy. Cancer vaccines have been utilized as treatment of other tumors and might be an option for brain tumors. Cancer/testis (CTA) genes represent promising targets for immunotherapeutic approaches. They are expressed in tumors and expression in normal tissues is exclusively or preferentially restricted to testis. The expression of 31 CTA genes was investigated in 48 glioblastomas and 5 normal brain samples using RT-PCR. A tissue microarray was made and expression of a CTA was detected by immunohistochemical staining. Among the genes with no expression in normal brain CTA1 (57%), CTA2 (54%), CTA3 (44%) and CTA4 (15%) were the most expressed in glioblastomas. At least one of these four selected CTA genes was found expressed in 86% of the GBM cases. Coexpression of two, three and four CTA genes occurred in 25%, 17% and 5% of the examined GBM cases, respectively. CTA4 protein expression was identified in 13% (12/92) of GBM and was negative in all anaplastic astrocytomas (0/15), diffuse astrocytomas (0/48), pilocytic astrocytomas (0/38) and normal brain samples (0/40) evaluated. We found that CTA2-positive GBMs and tumors expressing 3-4 CTA genes presented a significantly better outcome (P = 0.032; hazards ratio = 0.53; 95% CI; 0.28 to 1.0 and P = 0.017, hazards ratio = 0.36; 95% CI, 0.17 to 0.74; respectively). Furthermore, multivariate survival analysis revealed that coexpression of 3-4 CTA genes (P = 0.044, hazards ratio = 0.3, 95%CI = 0.093-0.963), radiotherapy (P = 0.010, hazards ratio = 0.16, 95%CI = 0.04-0.65) and chemotherapy (P = 0.001, hazards ratio = 0.10, 95% CI = 0.03-0.41) remained as independent predictors of overall survival. In conclusion, CTAs are potential targets for immunotherapy in patients with GBM, 86% of the cases examined expressed at least one of four selected CTA genes. The presence of 3-4 CTA genes was associated with better survival, suggesting that these genes might elicit an immune response against GBM. Supported by FAPESP(Grant 2010/20218-2 and 2011/15118-1). Citation Format: Marcelo Freitas, Thaís P. Biassi, Suzana M.F Malheiros, João N. Stávale, Fernando T. Zamunér, Maria D.F.S Begnami, Fernando A. Soares, André L. Vettore. Cancer/testis antigens: Comprehensive expression analysis and correlation with survival in glioblastoma. [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 1811. doi:10.1158/1538-7445.AM2013-1811

Adaptation of Listeria monocytogenes in a simulated cheese medium: effects on virulence using the Galleria mellonella infection model

The aim of this study was to evaluate the effect of the acid and salt adaptation in a cheese-based medium on the virulence potential of Listeria monocytogenes strains isolated from cheese and dairy processing environment using the Galleria mellonella model. Four L. monocytogenes strains were exposed to a cheese-based medium in conditions of induction of an acid tolerance response and osmotolerance response (pH 5·5 and 3·5% w/v NaCl) and injected in G. mellonella insects. The survival of insects and the L. monocytogenes growth kinetics in insects were evaluated. The gene expression of hly, actA and inlA genes was determined by real-time PCR. The adapted cells of two dairy strains showed reduced insect mortality (P < 0·05) in comparison with nonadapted cells. Listeria monocytogenes Scott A was the least virulent, whereas the cheese isolate C882 caused the highest insect mortality, and no differences (P > 0·05) was found between adapted and nonadapted cells. The gene expression results evidenced an overexpression of virulence genes in cheese-based medium, but not in simulated insect-induced conditions. Our results suggest that adaptation to low pH and salt in a cheese-based medium can affect the virulence of L. monocytogenes, but this effect is strain dependent. In this study, the impact of adaptation to low pH and salt in a cheese-based medium on L. monocytogenes virulence was tested using the Wax Moth G. mellonella model. This model allowed the differentiation of the virulence potential between the L. monocytogenes strains. The effect of adaptation on virulence is strain dependent. The G. mellonella model revealed to be a prompt method to test food-related factors on L. monocytogenes virulence.

RsbV of Listeria monocytogenes contributes to regulation of environmental stress and virulence

SigmaB factor is an important regulatory factor for stress response in Gram-positive bacteria such as Listeria monocytogenes (L. monocytogenes), Staphylococcus aureus and Bacillus subtilis. However, the activity of SigmaB factor is regulated by RsbV factor. Currently, the functional studies of RsbV factor are mostly focused on non-pathogenic B. subtilis, but the roles of RsbV factor in pathogenic L. monocytogenes during the regulation of environmental stress and virulence are still unclear. In the study, a ∆RsbV mutant of L. monocytogenes was constructed to explore the regulatory role of RsbV in environmental stress and virulence. The environmental stress experiments indicated that the growth and survival capability of ∆RsbV mutant obviously decreased in stress of low temperature, osmotic pressure, alcohol and acid, compared with EGD strain. The macrophage infection experiment indicated that ∆RsbV mutant had weaker survival capability than EGD strain, and the expression of PrfA, actA, PlcA and LLO was down-regulated in infected cells. Animal inoculation experiments indicated that RsbV deletion significantly reduced the pathogenicity of L. monocytogenes. Our data demonstrate that, in addition to regulating tolerance under environmental stress conditions, RsbV also contributes to regulation of L. monocytogenes virulence.

Listeria monocytogenes: No Spreading without NO

In infectious disease immunology, inducible nitric oxide (NO) synthase-derived NO is believed to function primarily as an antimicrobial and immunoregulatory molecule. In this issue of Immunity, Cole etal. (2012) show that NO helps the intracellular bacteria Listeria monocytogenes to spread.

Differential expression of InlB and ActA in Listeria monocytogenes in selective and nonselective enrichment broths

To investigate the effect of selective and nonselective media on the expression of ActA and InlB proteins in Listeria monocytogenes. Polyclonal antibodies to InlB and ActA were used in western blotting to determine the effect of selective (BLEB, UVM, and FB) or nonselective (BHI and LB) enrichment broths or hotdog exudates. Of the 13 L. monocytogenes serotypes tested, 11 and 12 serotypes showed a strong InlB expression in brain heart infusion (BHI) and Luria-Bertani (LB), respectively, while only seven and one serotypes showed a strong ActA expression in these two respective broths, and others showed a weaker or no expression. On the contrary, in selective broths, expression of InlB was either very weak or undetectable. However, ActA expression was stronger in 12 serotypes when grown in buffered Listeria enrichment broth (BLEB), 11 in University of Vermont medium (UVM), and 10 in Fraser broth (FB). When tested in hotdog exudates, InlB and ActA were detected in serotypes grown at 37 degrees C but not at 4 degrees C. Transmission electron microscopy, enzyme-linked immunosorbent assay, and mRNA analysis further supported these observations. Overall, selective enrichment broths promote ActA while nonselective broths promote InlB expression. As commonly recommended enrichment broths show differential InlB and ActA expression, proper media must be selected to avoid false results during antibody-based detection of L. monocytogenes.

Transforming growth factor-β1 and activin A generate antiproliferative signaling in thyroid cancer cells

Transforming growth factor-beta 1 (TGF-beta1) and activin A (ActA) induce similar intracellular signaling mediated by the mothers against decapentaplegic homolog (SMAD) proteins. TGF-beta1 is a potent antimitogenic factor for thyroid follicular cells, while the role of ActA is not clear. In our study, the proliferation of TPC-1, the papillary thyroid carcinoma cell line, was reduced by both recombinant ActA and TGF-beta1. Due to the concomitant expression of TGF-beta1 and ActA in thyroid tumors, we investigated the effects of either TGF-beta1 or ActA gene silencing by RNA interference in TPC-1 cells in order to distinguish the specific participation of each in proliferation and intracellular signaling. An increased proliferation and reduced SMAD2, SMAD3, and SMAD4 mRNA expression were observed in both TGF-beta1 and ActA knockdown cells. Recombinant TGF-beta1 and ActA increased the expression of inhibitory SMAD7, whereas they reduced c-MYC. Accordingly, we detected a reduction in SMAD7 expression in knockdown cells while, unexpectedly, c-MYC was reduced. Our data indicate that both TGF-beta1 and ActA generate SMADs signaling with each regulating the expression of their target genes, SMAD7 and c-MYC. Furthermore, TGF-beta1 and ActA have an antiproliferative effect on thyroid papillary carcinoma cell, exerting an important role in the control of thyroid tumorigenesis.

Evidence implicating the 5' untranslated region of Listeria monocytogenes actA in the regulation of bacterial actin-based motility.

The ActA protein of Listeria monocytogenes is a major virulence factor, essential for the recruitment and polymerization of host actin filaments that lead to intracellular motility and cell-to-cell spread of bacteria within the infected host. The expression of actA is tightly regulated and is strongly induced only when L. monocytogenes is within the host cytosol. Intracellular induction of actA expression is mediated through a single promoter element that directs the expression of a messenger RNA with a long (150 bp) 5' untranslated region (UTR). Deletion of the actA+3 to +130 upstream region was found to result in bacterial mutants that were no longer capable of intracellular actin recruitment or cell-to-cell spread, thus indicating that this region is important for actA expression. L. monocytogenes strains that contained smaller deletions (21-23 bp) within the actA upstream region demonstrated a range of actA expression levels that coincided with the amount of bacterial cell-to-cell spread observed within infected monolayers. A correlation appeared to exist between levels of actA expression and the ability of L. monocytogenes to transition from uniform actin accumulation surrounding individual bacteria (actin clouds) to directional assembly and the formation of actin tails. Bacterial mutants containing deletions that most significantly altered the predicted secondary structure of the actA mRNA 5' UTR had the largest reductions in actA expression. These results suggest that the actA 5' UTR is required for maximal ActA synthesis and that a threshold level of ActA synthesis must be achieved to promote the transition from bacteria-associated actin clouds to directional actin assembly and movement.