Epigenetic alterations differ in phenotypically distinct human neuroblastoma cell lines

<p>Abstract</p> <p>Background</p> <p>Epigenetic aberrations and a CpG island methylator phenotype have been shown to be associated with poor outcomes in children with neuroblastoma (NB). Seven cancer related genes (<it>THBS-1, CASP8, HIN-1, TIG-1, BLU, SPARC</it>, and <it>HIC-1</it>) that have been shown to have epigenetic changes in adult cancers and play important roles in the regulation of angiogenesis, tumor growth, and apoptosis were analyzed to investigate the role epigenetic alterations play in determining NB phenotype.</p> <p>Methods</p> <p>Two NB cell lines (tumorigenic LA1-55n and non-tumorigenic LA1-5s) that differ in their ability to form colonies in soft agar and tumors in nude mice were used. Quantitative RNA expression analyses were performed on seven genes in LA1-5s, LA1-55n and 5-Aza-dC treated LA1-55n NB cell lines. The methylation status around <it>THBS-1, HIN-1, TIG-1 </it>and <it>CASP8 </it>promoters was examined using methylation specific PCR. Chromatin immunoprecipitation assay was used to examine histone modifications along the <it>THBS-1 </it>promoter. Luciferase assay was used to determine <it>THBS-1 </it>promoter activity. Cell proliferation assay was used to examine the effect of 5-Aza-dC on NB cell growth. The soft agar assay was used to determine the tumorigenicity.</p> <p>Results</p> <p>Promoter methylation values for <it>THBS-1</it>, <it>HIN-1</it>, <it>TIG-1</it>, and <it>CASP8 </it>were higher in LA1-55n cells compared to LA1-5s cells. Consistent with the promoter methylation status, lower levels of gene expression were detected in the LA1-55n cells. Histone marks associated with repressive chromatin states (H3K9Me3, H3K27Me3, and H3K4Me3) were identified in the <it>THBS-1 </it>promoter region in the LA1-55n cells, but not the LA1-5s cells. In contrast, the three histone codes associated with an active chromatin state (acetyl H3, acetyl H4, and H3K4Me3) were present in the <it>THBS-1 </it>promoter region in LA1-5s cells, but not the LA1-55n cells, suggesting that an accessible chromatin structure is important for <it>THBS-1 </it>expression. We also show that 5-Aza-dC treatment of LA1-55n cells alters the DNA methylation status and the histone code in the <it>THBS-1 </it>promoter modifies cell morphology, and inhibits their ability to form colonies in soft agar.</p> <p>Conclusion</p> <p>Our results suggest that epigenetic aberrations contribute to NB phenotype, and that tumorigenic properties can be inhibited by reversing the epigenetic changes with 5-Aza-dC.</p

The Origin and Contribution of Cancer-Associated Fibroblasts in Colorectal Carcinogenesis

Background & Aims: Cancer-associated fibroblasts (CAFs) play an important role in colorectal cancer (CRC) progression and predict poor prognosis in CRC patients. However, the cellular origins of CAFs remain unknown, making it challenging to therapeutically target these cells. Here, we aimed to identify the origins and contribution of colorectal CAFs associated with poor prognosis. Methods: To elucidate CAF origins, we used a colitis-associated CRC mouse model in 5 different fate-mapping mouse lines with 5-bromodeoxyuridine dosing. RNA sequencing of fluorescence-activated cell sorting–purified CRC CAFs was performed to identify a potential therapeutic target in CAFs. To examine the prognostic significance of the stromal target, CRC patient RNA sequencing data and tissue microarray were used. CRC organoids were injected into the colons of knockout mice to assess the mechanism by which the stromal gene contributes to colorectal tumorigenesis. Results: Our lineage-tracing studies revealed that in CRC, many ACTA2+ CAFs emerge through proliferation from intestinal pericryptal leptin receptor (Lepr)+ cells. These Lepr-lineage CAFs, in turn, express melanoma cell adhesion molecule (MCAM), a CRC stroma-specific marker that we identified with the use of RNA sequencing. High MCAM expression induced by transforming growth factor β was inversely associated with patient survival in human CRC. In mice, stromal Mcam knockout attenuated orthotopically injected colorectal tumoroid growth and improved survival through decreased tumor-associated macrophage recruitment. Mechanistically, fibroblast MCAM interacted with interleukin-1 receptor 1 to augment nuclear factor κB–IL34/CCL8 signaling that promotes macrophage chemotaxis. Conclusions: In colorectal carcinogenesis, pericryptal Lepr-lineage cells proliferate to generate MCAM+ CAFs that shape the tumor-promoting immune microenvironment. Preventing the expansion/differentiation of Lepr-lineage CAFs or inhibiting MCAM activity could be effective therapeutic approaches for CRC

Induction of plasminogen activator inhibitor type-1 (PAI-1) by hypoxia and irradiation in human head and neck carcinoma cell lines

Contains fulltext : 53187.pdf ( ) (Open Access)BACKGROUND: Squamous cell carcinoma of the head and neck (SCCHN) often contain highly radioresistant hypoxic regions, nonetheless, radiotherapy is a common treatment modality for these tumours. Reoxygenation during fractionated radiotherapy is desired to render these hypoxic tumour regions more radiosensitive. Hypoxia additionally leads to up-regulation of PAI-1, a protein involved in tumour progression and an established prognostic marker for poor outcome. However, the impact of reoxygenation and radiation on PAI-1 levels is not yet clear. Therefore, we investigated the kinetics of PAI-1 expression and secretion after hypoxia and reoxygenation, and determined the influence of ionizing radiation on PAI-1 levels in the two human SCCHN cell lines, BHY and FaDu. METHODS: HIF-1alpha immunoblot was used to visualize the degree of hypoxia in the two cell lines. Cellular PAI-1 expression was investigated by immunofluorescence microscopy. ELISA was used to quantify relative changes in PAI-1 expression (cell lysates) and secretion (cell culture supernatants) in response to various lengths (2-4 h) of hypoxic exposure (< 0.66% O2), reoxygenation (24 h, 20% O2), and radiation (0, 2, 5 and 10 Gy). RESULTS: HIF-1alpha expression was induced between 2 and 24 h of hypoxic exposure. Intracellular PAI-1 expression was significantly increased in BHY and FaDu cells as early as 4 h after hypoxic exposure. A significant induction in secreted PAI-1 was seen after 12 to 24 h (BHY) and 8 to 24 h (FaDu) hypoxia, as compared to the normoxic control. A 24 h reoxygenation period caused significantly less PAI-1 secretion than a 24 h hypoxia period in FaDu cells. Irradiation led to an up-regulation of PAI-1 expression and secretion in both, BHY and FaDu cells. CONCLUSION: Our data suggest that both, short-term (approximately 4-8 h) and long-term (approximately 20-24 h) hypoxic exposure could increase PAI-1 levels in SCCHN in vivo. Importantly, radiation itself could lead to PAI-1 up-regulation in head and neck tumours, whereas reoxygenation of hypoxic tumour cells during fractionated radiotherapy could counteract the increased PAI-1 levels

Persistent symptoms and decreased health-related quality of life after symptomatic pediatric COVID-19: A prospective study in a Latin American tertiary hospital

OBJECTIVES: To prospectively evaluate demographic, anthropometric and health-related quality of life (HRQoL) in pediatric patients with laboratory-confirmed coronavirus disease 2019 (COVID-19) METHODS: This was a longitudinal observational study of surviving pediatric post-COVID-19 patients (n=53) and pediatric subjects without laboratory-confirmed COVID-19 included as controls (n=52) was performed. RESULTS: The median duration between COVID-19 diagnosis (n=53) and follow-up was 4.4 months (0.8-10.7). Twenty-three of 53 (43%) patients reported at least one persistent symptom at the longitudinal follow-up visit and 12/53 (23%) had long COVID-19, with at least one symptom lasting for &gt;12 weeks. The most frequently reported symptoms at the longitudinal follow-up visit were headache (19%), severe recurrent headache (9%), tiredness (9%), dyspnea (8%), and concentration difficulty (4%). At the longitudinal follow-up visit, the frequencies of anemia (11%&nbsp;versus&nbsp;0%,&nbsp;p=0.030), lymphopenia (42%&nbsp;versus&nbsp;18%,&nbsp;p=0.020), C-reactive protein level of &gt;30 mg/L (35%&nbsp;versus&nbsp;0%,&nbsp;p=0.0001), and D-dimer level of &gt;1000 ng/mL (43%&nbsp;versus&nbsp;6%,&nbsp;p=0.0004) significantly reduced compared with baseline values. Chest X-ray abnormalities (11%&nbsp;versus&nbsp;2%,&nbsp;p=0.178) and cardiac alterations on echocardiogram (33%&nbsp;versus&nbsp;22%,&nbsp;p=0.462) were similar at both visits. Comparison of characteristic data between patients with COVID-19 at the longitudinal follow-up visit and controls showed similar age (p=0.962), proportion of male sex (p=0.907), ethnicity (p=0.566), family minimum monthly wage (p=0.664), body mass index (p=0.601), and pediatric pre-existing chronic conditions (p=1.000). The Pediatric Quality of Live Inventory 4.0 scores, median physical score (69 [0-100]&nbsp;versus&nbsp;81 [34-100],&nbsp;p=0.012), and school score (60 [15-100]&nbsp;versus&nbsp;70 [15-95],&nbsp;p=0.028) were significantly lower in pediatric patients with COVID-19 at the longitudinal follow-up visit than in controls. CONCLUSIONS: Pediatric patients with COVID-19 showed a longitudinal impact on HRQoL parameters, particularly in physical/school domains, reinforcing the need for a prospective multidisciplinary approach for these patients. These data highlight the importance of closer monitoring of children and adolescents by the clinical team after COVID-19

Evaluation of the Physicochemical, Structural, Thermal, and Behavioral Properties of the Energy of Consciousness Healing Treated Zinc Chloride

Zinc chloride is a source of zinc used in various pharmaceutical/nutraceutical formulations. The objective of the current study was to investigate the impact of The Trivedi Effect® - Energy of Consciousness Healing Treatment (Biofield Energy Treatment) on physical, structural, thermal, and behavioral properties of zinc chloride using PXRD, PSD, FT-IR, UV-vis, and DSC analysis. Zinc chloride was divided into two parts – one part was control, while another part was treated with The Trivedi Effect® remotely by twenty renowned Biofield Energy Healers and defined as The Trivedi Effect® Treated sample. A significant alteration of the crystallite size and relative intensities of the PXRD peaks was observed in The Trivedi Effect® treated sample compared with the control sample. The average crystallite size of the treated sample was significantly increased by 23.18% compared with the control sample. The particle size values at d10, d50, and d90 values were significantly decreased by 3.70%, 4.13%, and 6.13%, respectively in the treated sample compared with the control sample. Therefore, the surface area of the treated sample was increased by 4.21% compared with the control sample. The FT-IR spectroscopic analysis revealed that Zn-Cl stretching in the control sample was found at 512 cm-1, whereas it was significantly shifted upward to 520 cm-1 in the treated sample. The UV-vis analysis exhibited that wavelength of the maximum absorbance (λmax) of the control and treated samples were at 197.6 nm and 197.1 nm, respectively. The DSC analysis exhibited that the melting temperature was decreased by 0.22%, while decomposition temperature was increased by 2.56% in the treated sample compared to the control sample. The latent heat of fusion of the treated sample (320.44 J/g) was significantly decreased by 16.70% compared with the control sample (284.67 J/g). Similarly, the enthalpy of decomposition of the treated sample (952.53 J/g) was significantly increased by 122.61% compared with the control sample (427.90 J/g). Thus, the results indicated that the thermal stability of the treated zinc chloride was improved compared with the control sample. The current study anticipated that The Trivedi Effect® - Energy of Consciousness Healing Treatment might lead to produce a thermally stable new polymorphic form of zinc chloride, which would be more soluble and bioavailable compared with the untreated compound. Hence, the treated zinc chloride would be very useful to design better nutraceutical/pharmaceutical formulations that might offer better therapeutic response against inflammatory diseases, immunological disorders, aging, stress, cancer, etc. https://www.trivedieffect.com/science/evaluation-of-the-physicochemical-structural-thermal-and-behavioral-properties-of-the-energy-of-consciousness-healing-treated-zinc-chloride http://www.sciencepublishinggroup.com/journal/paperinfo?journalid=217&doi=10.11648/j.bio.20170502.1

HLA Class I Restriction as a Possible Driving Force for Chikungunya Evolution

After two decades of quiescence, epidemic resurgence of Chikungunya fever (CHIKF) was reported in Africa, several islands in the Indian Ocean, South-East Asia and the Pacific causing unprecedented morbidity with some cases of fatality. Early phylogenetic analyses based on partial sequences of Chikungunya virus (CHIKV) have led to speculation that the virus behind recent epidemics may result in greater pathogenicity. To understand the reasons for these new epidemics, we first performed extensive analyses of existing CHIKV sequences from its introduction in 1952 to 2009. Our results revealed the existence of a continuous genotypic lineage, suggesting selective pressure is active in CHIKV evolution. We further showed that CHIKV is undergoing mild positive selection, and that site-specific mutations may be driven by cell-mediated immune pressure, with occasional changes that resulted in the loss of human leukocyte antigen (HLA) class I-restricting elements. These findings provide a basis to understand Chikungunya virus evolution and reveal the power of post-genomic analyses to understand CHIKV and other viral epidemiology. Such an approach is useful for studying the impact of host immunity on pathogen evolution, and may help identify appropriate antigens suitable for subunit vaccine formulations

New Model of Macrophage Acquisition of the Lymphatic Endothelial Phenotype

Macrophage-derived lymphatic endothelial cell progenitors (M-LECPs) contribute to new lymphatic vessel formation, but the mechanisms regulating their differentiation, recruitment, and function are poorly understood. Detailed characterization of M-LECPs is limited by low frequency in vivo and lack of model systems allowing in-depth molecular analyses in vitro. Our goal was to establish a cell culture model to characterize inflammation-induced macrophage-to-LECP differentiation under controlled conditions.Time-course analysis of diaphragms from lipopolysaccharide (LPS)-treated mice revealed rapid mobilization of bone marrow-derived and peritoneal macrophages to the proximity of lymphatic vessels followed by widespread (∼50%) incorporation of M-LECPs into the inflamed lymphatic vasculature. A differentiation shift toward the lymphatic phenotype was found in three LPS-induced subsets of activated macrophages that were positive for VEGFR-3 and many other lymphatic-specific markers. VEGFR-3 was strongly elevated in the early stage of macrophage transition to LECPs but undetectable in M-LECPs prior to vascular integration. Similar transient pattern of VEGFR-3 expression was found in RAW264.7 macrophages activated by LPS in vitro. Activated RAW264.7 cells co-expressed VEGF-C that induced an autocrine signaling loop as indicated by VEGFR-3 phosphorylation inhibited by a soluble receptor. LPS-activated RAW264.7 macrophages also showed a 68% overlap with endogenous CD11b(+)/VEGFR-3(+) LECPs in the expression of lymphatic-specific genes. Moreover, when injected into LPS- but not saline-treated mice, GFP-tagged RAW264.7 cells massively infiltrated the inflamed diaphragm followed by integration into 18% of lymphatic vessels.We present a new model for macrophage-LECP differentiation based on LPS activation of cultured RAW264.7 cells. This system designated here as the "RAW model" mimics fundamental features of endogenous M-LECPs. Unlike native LECPs, this model is unrestricted by cell numbers, heterogeneity of population, and ability to change genetic composition for experimental purposes. As such, this model can provide a valuable tool for understanding the LECP and lymphatic biology