Response Of Human Cells Research Articles

Abstract 6519: Haploinsufficiency for BRCA2 leads to common fragile site instability in human mammary epithelial cells

Abstract Individuals with germline heterozygous mutations of breast cancer susceptibility genes BRCA1 or BRCA2 have a 50 to 80% lifetime risk of developing breast cancer, yet the mechanisms of these cancers remain largely unknown. There is growing evidence that BRCA1 and BRCA2 heterozygosity confer haploinsufficiency in normal human mammary epithelial cells for their multiple known functions. Haploinsufficiency for BRCA1 leads to defects in differentiation, replication stress response, premature senescence and genomic instability in human mammary epithelial cells. Deficiencies in error-free DNA damage repair has been observed in genetically engineered cells as well as primary human mammary epithelial cells carrying BRCA1 heterozygous mutations. There is relatively less research on understanding the role of haploinsufficiency for BRCA2 in human mammary epithelial cells. It has been reported that haploinsufficiency for BRCA2 leads to genomic instability via unscheduled R-loops. We previously showed that accumulation of sub-chromosomal copy number variations (CNVs) and replication stress-induced DNA damage, together with attenuated checkpoint and apoptotic responses, constitute a haplo-insufficient phenotype in mammary epithelial cells of BRCA2 mutation carrier patients. Common fragile sites (CFS) are found in all individuals and represent a normal component of chromosome structure. Numerous studies have shown that CFSs are prone to deletions and rearrangements in many cancers, a situation referred as CFS instability. Importantly, Fanconi Anemia/BRCA pathway has been previously reported to have a role in regulating CFS instability. In this study, we further explored a link between BRCA2 haploinsufficiency and CFS instability. We induced replication stress with DNA polymerase inhibitor aphidicolin (APH) treatment in control and BRCA2- deficient human mammary epithelial cells. We then performed DAPI banding to map and score the cytogenic lesions in metaphase spreads cells, in the absence or presence of APH treatment. Our results demonstrated that BRCA2- deficient human mammary epithelial cells showed increased frequency of CFS regions such as FRA18B. Correspondingly, BRCA2-deficient cells exhibited a failure to activate CHK1, a central coordinator of the response to replication stress and DNA damage, in response to APH treatment, despite normal levels of total CHK1 protein. ATR/CHK1 pathway is a major regulator of CFS stability. Thus, our results provide support for BRCA2 haploinsufficiency leading to CFS instability in human mammary epithelial cells. These studies will yield unanticipated opportunities for improved risk assessment and prevention strategies in high-risk patients. Citation Format: Mihriban Karaayvaz, Kavya Vipparthi, Antony Caputo. Haploinsufficiency for BRCA2 leads to common fragile site instability in human mammary epithelial cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6519.

Abstract 6133: Growth response and migration of brain cancer cell lines to treatment with agents targeting different members of the HER family, CDKs and other signalling pathways

Abstract Glioblastoma (GB) is one of the most frequent primary brain tumors in adults with an extremely poor prognosis and a very high demand for novel therapies. Poor prognosis has been linked to its complex biology, tumor heterogeneity and an increased activation of signaling pathways through various growth factor receptors signaling pathway. In particular, aberrant expression and activation of epidermal growth factor receptor (EGFR) expression has been found to be common in patients with GBM. However, no EGFR inhibitor has yet been approved for the treatment of patients with brain tumor and there is currently no comprehensive study of all members of the HER family in the progression brain tumors. The aim of this study was to investigate the role of all members of the HER family (EGFR, HER2 HER3 and HER4), other growth factor receptors (e.g., C-MET) and downstream cell signaling pathways in the growth of a panel of human brain cancer cell lines (HBCCLs) and response to therapeutic interventions. Using Sulforhodamine B colorimetric assay, the growth response of four HBCCLs to treatment with 18 agents targeting different members of the HER family and other growth receptors (e.g. C-met/ALK 7, PDGFRs, FGFRs, C-kit), cyclin dependent kinases inhibitors (e.g. CDKs 1/2/5/9, CDK4/6) and downstream signaling molecules (e.g. STAT3, Src, Abl) was examined. We determined the expression level of all members of the HER family in brain cancer cells by flow cytometry and Western blot and the effect of these agents on migration of brain cancer cells were determined using Incucyte. Of the targeted agents examined, the CDK 1/2/5/9 inhibitor dinaciclib was the most potent and inhibited growth for all four HBCCLs with an IC50 below 10nM. Of the HER inhibitors, neratinib and afatinib were more effective than erlotinib and lapatinib and inhibiting the growth of all HBCCLs at IC50 values below 700nM and 1.9 µM, respectively. Treatment with Src/Abl/c-kit inhibitor dasatinib, STAT3 inhibitor static, Abl/PDGFRα/VEGFR2/FGFR1 inhibitor Ponatinib and the TRK/ROS/ALK inhibitor entrecitinb also inhibited the growth of HBCCLs with IC50 value ranging from 0.06 - 2.96 µM, 1 - 3.8µM, 0.19- 0.42 μM and 2.85- 3.47μM, respectively. Interestingly, these agents were more effective in inhibiting growth of HBCCLs when proliferating at a slower rate. In addition to inhibiting the proliferation of HBCCLs, treatment with neratinib, dinaciclib, dasatanib, static and trametinib inhibited the migration of brain tumor cells. Finally, treatment with neratinib in combination with Palbociclib, AZD4547, trametinib and miransertib inhibited the growth of HBCCLs synergistically. Taken together, our results support further investigation on the therapeutic potential of irreversible pan HER in combination with the CDK inhibitor dinaciclib and other targeted agents in brain cancer. Citation Format: Ermira Mulliqi, Said A. Khelwatty, Anna L. Morgan, Keyoumars Ashkan, Helmout Modjtahedi. Growth response and migration of brain cancer cell lines to treatment with agents targeting different members of the HER family, CDKs and other signalling pathways. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6133.

A stress-induced cilium-to-PML-NB route drives senescence initiation

Cellular senescence contributes to tissue homeostasis and age-related pathologies. However, how senescence is initiated in stressed cells remains vague. Here, we discover that exposure to irradiation, oxidative or inflammatory stressors induces transient biogenesis of primary cilia, which are then used by stressed cells to communicate with the promyelocytic leukemia nuclear bodies (PML-NBs) to initiate senescence responses in human cells. Mechanistically, a ciliary ARL13B-ARL3 GTPase cascade negatively regulates the association of transition fiber protein FBF1 and SUMO-conjugating enzyme UBC9. Irreparable stresses downregulate the ciliary ARLs and release UBC9 to SUMOylate FBF1 at the ciliary base. SUMOylated FBF1 then translocates to PML-NBs to promote PML-NB biogenesis and PML-NB-dependent senescence initiation. Remarkably, Fbf1 ablation effectively subdues global senescence burden and prevents associated health decline in irradiation-treated mice. Collectively, our findings assign the primary cilium a key role in senescence induction in mammalian cells and, also, a promising target in future senotherapy strategies.

SARS-CoV-2 Omicron boosting induces de novo B cell response in humans.

The primary two-dose SARS-CoV-2 mRNA vaccine series are strongly immunogenic in humans, but the emergence of highly infectious variants necessitated additional doses and the development of vaccines aimed at the new variants1-4. SARS-CoV-2 booster immunizations in humans primarily recruit pre-existing memory B cells5-9. However, it remains unclear whether the additional doses induce germinal centre reactions whereby re-engaged B cells can further mature, and whether variant-derived vaccines can elicit responses to variant-specific epitopes. Here we show that boosting with an mRNA vaccine against the original monovalent SARS-CoV-2 mRNA vaccine or the bivalent B.1.351 and B.1.617.2 (Beta/Delta) mRNA vaccine induced robust spike-specific germinal centre B cell responses in humans. The germinal centre response persisted for at least eight weeks, leading to significantly more mutated antigen-specific bone marrow plasma cell and memory B cell compartments. Spike-binding monoclonal antibodies derived from memory B cells isolated from individuals boosted with either the original SARS-CoV-2 spike protein, bivalent Beta/Delta vaccine or a monovalent Omicron BA.1-based vaccine predominantly recognized the original SARS-CoV-2 spike protein. Nonetheless, using a more targeted sorting approach, we isolated monoclonal antibodies that recognized the BA.1 spike protein but not the original SARS-CoV-2 spike protein from individuals who received the mRNA-1273.529 booster; these antibodies were less mutated and recognized novel epitopes within the spike protein, suggesting that they originated from naive B cells. Thus, SARS-CoV-2 booster immunizations in humans induce robust germinal centre B cell responses and can generate de novo B cell responses targeting variant-specific epitopes.

Biochemical Response of Human Endothelial and Fibroblast Cells to Silver Nanoparticles

Biochemical Response of Human Endothelial and Fibroblast Cells to Silver Nanoparticles

Primary human osteoblast and mesenchymal stem cell responses to apatite/tricalcium phosphate bone cement modified with polyacrylic acid and bioactive glass.

In this work, three different modified cements, control apatite/beta-tricalcium phosphate cement (CPC), polymeric CPC (p-CPC), and bioactive glass added polymeric cement (p-CPC/BG) were evaluated regarding their physical properties and the responses of primary human osteoblast cells (HObs) and mesenchymal stem cells (MSCs). Although polyacrylic acid (PAA) increased compressive strength and Young's modulus of the cement, it could cause poor apatite phase formation, a prolonged setting time, and a lower degradation rate. Consequently, bioactive glass (BG) was added to PAA/cement to improve its physical properties, such as compressive strength, Young's modulus, setting time, and degradation. For in vitro testing, HObs viability was assessed under two culture systems with cement-preconditioned medium (indirect) and with cement (direct). HObs viability was examined in direct contact with cements treated by different prewashing conditions. HObs presented a more well spread morphology on cement soaked in medium overnight, as compared to other cements with no treatment and washing in PBS. In addition, the proliferation, differentiation, and total collagen production of both HObs and MSCs adhered to the cement were detected. Cells showed excellent proliferation on PAA/cement and PAA/BG/cement. Furthermore, the higher released Si ion and lower acidosis of PAA/BG/cement-conditioned medium resulted in an increase in osteogenic differentiation (HObs and MSCs) and enhanced collagen production (HObs in osteogenic medium and MSCs in control medium). Therefore, our findings suggest that BG incorporated PAA/apatite/β-TCP cement could be a promising formula for bone repair applications.

Toxicity of polystyrene nanoplastics to human embryonic kidney cells and human normal liver cells: Effect of particle size and Pb2+ enrichment

Nanoplastics pollution in drinking water has aroused wide concern, but their effects on human health are still poorly understood. Herein we explore the responses of human embryonic kidney 293T cells and human normal liver LO2 cells to polystyrene nanoplastics, mainly focusing on the effects of particle sizes and enrichment of Pb2+. When the exposed particle size is higher than 100 nm, there is no obvious death for these two different cell lines. As the particle size decreases from 100 nm, cell mortality goes up. Although the internalization of polystyrene nanoplastics in LO2 cells is at least 5 times higher than that in 293T cells, the mortality of LO2 cells is lower than that of 293T cells, illustrating that LO2 cells are more resistant to polystyrene nanoplastics than 293T cells. Additionally, the Pb2+ enrichment on polystyrene nanoplastics in water can further enhance their toxicity, which should be taken seriously. The cytotoxicity of polystyrene nanoplastics to cell lines works through a molecular mechanism involving oxidative stress-induced damage of mitochondria and cell membranes, resulting in a decrease in ATP production and an increase in membrane permeability. Referenced to nanoplastics pollution in drinking water, there is no necessary to panic about the adverse effects of plastic itself on human health, but the enrichment of contaminants should get more attention. This work provides a reference for the risk assessment of nanoplastics in drinking water to human health.

Ectopic expression of cGAS in Salmonella typhimurium enhances STING-mediated IFN-β response in human macrophages and dendritic cells

BackgroundInterferon (IFN)-β induction via activation of the stimulator of interferon genes (STING) pathway has shown promising results in tumor models. STING is activated by cyclic dinucleotides such as cyclic GMP–AMP...

Angiotensin II Increases Oxidative Stress and Inflammation in Female, But Not Male, Endothelial Cells.

Women are at elevated risk for certain cardiovascular diseases, including pulmonary arterial hypertension, Alzheimer's disease, and vascular complications of diabetes. Angiotensin II (AngII), a circulating stress hormone, is elevated in cardiovascular disease; however, our knowledge of sex differences in the vascular effects of AngII are limited. We therefore analyzed sex differences in human endothelial cell response to AngII treatment. Male and female endothelial cells were treated with AngII for 24h and analyzed by RNA sequencing. We then used endothelial and mesenchymal markers, inflammation assays, and oxidative stress indicators to measure female and male endothelial cell functional changes in response to AngII. Our data show that female and male endothelial cells are transcriptomically distinct. Female endothelial cells treated with AngII had widespread gene expression changes related to inflammatory and oxidative stress pathways, while male endothelial cells had few gene expression changes. While both female and male endothelial cells maintained their endothelial phenotype with AngII treatment, female endothelial cells showed increased release of the inflammatory cytokine interleukin-6 and increased white blood cell adhesion following AngII treatment concurrent with a second inflammatory cytokine. Additionally, female endothelial cells had elevated reactive oxygen species production compared to male endothelial cells after AngII treatment, which may be partially due to nicotinamide adenine dinucleotide phosphate oxidase-2 (NOX2) escape from X-chromosome inactivation. These data suggest that endothelial cells have sexually dimorphic responses to AngII, which could contribute to increased prevalence of some cardiovascular diseases in women. The online version contains supplementary material available at 10.1007/s12195-023-00762-2.

Graphene monolayer as an appropriate substrate for mesenchymal stem cells support in regenerative medicine

Synergistic effect of mesenchymal stem cells and graphene monolayer is an undiscovered topic and crucial in the context of creating a graphene skin dressing. Moreover, capturing early changes in the cytoskeleton may prove to be a valuable indicator of cell condition after direct contact with the biomaterial. In this context, here, we investigated the response of human bone marrow mesenchymal stem cells (hBM-MSCs) to graphene monolayer as a scaffold, based on cytoskeleton architecture. Three main components of the cytoskeleton — microfilaments, intermediate filaments and microtubules were characterized as well as analyzed its entire cell morphology (focal adhesions). Based on microscopic analysis and cell area measurements, it can be stated that graphene did not cause a damage or disturbance of any cytoskeleton members and linker proteins (plectin, vinculin) and gap junction protein (connexin 43). hBM-MSCs growing on the graphene monolayer were more spread than cells growing on the glass but without any other significant difference. This suggested that graphene substrate as a monolayer could be a structural reinforcement for MSCs cultivation without affecting their cytoskeleton, which determines cell integrity, connections and migration, all important cell properties in the process of skin wound healing.

Effects of Exhaustive Exercise on Inflammatory, Apoptotic, and Antioxidative Signaling Pathways in Human Peripheral Blood Mononuclear Cells.

In the present study, we investigated the effects of exhaustive exercise and recovery on inflammatory, pro-apoptotic, and anti-oxidative responses in human peripheral blood mononuclear cells (PBMCs). Sixteen volunteers participated in a guided physical activity program in which they were subjected to progressive exercise on the treadmill until they were exhausted followed by an 1-hour recovery period. Isolated human PBMCs were collected before exercise, immediately after exercise, and after 1-hour recovery. Exhaustive exercise induced expression of heme oxygenase-1 and glutamate cysteine ligase catalytic subunit and activation of NF-κB and NF-E2 related factor 2 (Nrf2). Apoptosis, as measured by activity and cleavage of caspase-3 and its substrate PARP also significantly increased. However, induction of redox signaling and the pro-apoptotic response fully returned to the baseline level during the 1-hour recovery period. On the other hand, COX-2 expression was continuously elevated after exercise cessation throughout the 1-hour recovery period. Taking all these findings into account, we conclude that exhaustive exercise transiently induces Nrf2-mediated antioxidant gene expression and eliminates damaged cells through apoptosis as part of an adaptive cytoprotective response against oxidative and inflammatory stress.

Loss of regulation of protein synthesis and turnover underpins an attenuated stress response in senescent human mesenchymal stem cells

Cells respond to stress by synthesizing chaperone proteins that seek to correct protein misfolding and maintain function. However, abrogation of protein homeostasis is a hallmark of aging, leading to loss of function and the formation of proteotoxic aggregates characteristic of pathology. Consequently, discovering the underlying molecular causes of this deterioration in proteostasis is key to designing effective interventions to disease or to maintaining cell health in regenerative medicine strategies. Here, we examined primary human mesenchymal stem cells, cultured to a point of replicative senescence and subjected to heat shock, as an invitro model of the aging stress response. Multi -omics analysis showed how homeostasis components were reduced in senescent cells, caused by dysregulation of a functional network of chaperones, thereby limiting proteostatic competence. Time-resolved analysis of the primary response factors, including those regulating heat shock protein 70 kDa (HSPA1A), revealed that regulatory control is essentially translational. Senescent cells have a reduced capacity for chaperone protein translation and misfolded protein (MFP) turnover, driven by downregulation of ribosomal proteins and loss of the E3 ubiquitin ligase CHIP (C-terminus of HSP70 interacting protein) which marks MFPs for degradation. This limits the cell's stress response and subsequent recovery. A kinetic model recapitulated these reduced capacities and predicted an accumulation of MFP, a hypothesis supported by evidence of systematic changes to the proteomic fold state. These results thus establish a specific loss of regulatory capacity at the protein, rather than transcript, level and uncover underlying systematic links between aging and loss of protein homeostasis.

Perturbation of arachidonic acid and glycerolipid metabolism promoted particulate matter-induced inflammatory responses in human bronchial epithelial cells

Particulate matter (PM) has become the main risk factor for public health, being linked with an increased risk of respiratory diseases. However, the potential mechanisms underlying PM-induced lung injury have not been well elucidated. In this study, we systematically integrated the metabolomics, lipidomics, and transcriptomics data obtained from the human bronchial epithelial cells (HBECs) exposed to PM to reveal metabolic disorders in PM-induced lung injury. We identified 170 differentially expressed metabolites (82 upregulated and 88 downregulated metabolites), 218 differentially expressed lipid metabolites (125 upregulated and 93 downregulated lipid metabolites), and 1417 differentially expressed genes (643 upregulated and 774 downregulated genes). Seven key metabolites (prostaglandin E2, inosinic acid, L-arginine, L-citrulline, L-leucine, adenosine, and adenosine monophosphate), and two main lipid subclasses (triglyceride and phosphatidylcholine) were identified in PM-exposed HBECs. The amino acid metabolism, lipid metabolism, and carbohydrate metabolism were the significantly enriched pathways of identified differentially expressed genes. Then, conjoint analysis of these three omics data and further qRT-PCR validation showed that arachidonic acid metabolism, glycerolipid metabolism, and glutathione metabolism were the key metabolic pathways in PM-exposed HBECs. The knockout of AKR1C3 in arachidonic acid metabolism or GPAT3 in glycerolipid metabolism could significantly inhibit PM-induced inflammatory responses in HBECs. These results revealed the potential metabolic pathways in PM-exposed HBECs and provided a new target to protect from PM-induced airway damage.

Magnitude and kinetics of the human immune cell response associated with severe dengue progression by single-cell proteomics.

Approximately 5 million dengue virus-infected patients progress to a potentially life-threatening severe dengue (SD) infection annually. To identify the immune features and temporal dynamics underlying SD progression, we performed deep immune profiling by mass cytometry of PBMCs collected longitudinally from SD progressors (SDp) and uncomplicated dengue (D) patients. While D is characterized by early activation of innate immune responses, in SDp there is rapid expansion and activation of IgG-secreting plasma cells and memory and regulatory T cells. Concurrently, SDp, particularly children, demonstrate increased proinflammatory NK cells, inadequate expansion of CD16+ monocytes, and high expression of the FcγR CD64 on myeloid cells, yet a signature of diminished antigen presentation. Syndrome-specific determinants include suppressed dendritic cell abundance in shock/hemorrhage versus enriched plasma cell expansion in organ impairment. This study reveals uncoordinated immune responses in SDp and provides insights into SD pathogenesis in humans with potential implications for prediction and treatment.

Comparative analysis of hypoxic response of human microvascular and umbilical vein endothelial cells in 2D and 3D cell culture systems.

In vitro cultivation conditions play a crucial role in cell physiology and the cellular response to external stimuli. Oxygen concentrations represent an essential microenvironmental factor influencing cell physiology and behaviour both in vivo and in vitro. Therefore, new approaches are urgently needed to monitor and control oxygen concentrations in 2D and 3D cultures, as well as cell reactions to these concentrations. In this work, we modified two types of human endothelial cells-human microvascular (huMECs) and umbilical vein endothelial cells (huVECs) with genetically encoded hypoxia biosensors and monitored cell reactions in 2D to different oxygen concentrations. Moreover, we fabricated 3D cell spheroids of different cell numbers and sizes to reveal the onset of hypoxia in huVECs and huMECs. We could demonstrate a quantitative sensor response of two cell types to reduced oxygen supply in 2D and reveal different thresholds for hypoxic response. In 3D cell spheroids we could estimate critical construct sizes for the appearance of a hypoxic core. This work for the first time directly demonstrates different hypoxic signatures for huVECs and huMECs in 2D and 3D cell culture systems.

Radiotherapy exposure directly damages the uterus and causes pregnancy loss.

Female cancer survivors are significantly more likely to experience infertility than the general population. It is well established that chemotherapy and radiotherapy can damage the ovary and compromise fertility, yet the ability of cancer treatments to induce uterine damage, and the underlying mechanisms, have been understudied. Here, we show that in mice total-body γ-irradiation (TBI) induced extensive DNA damage and apoptosis in uterine cells. We then transferred healthy donor embryos into ovariectomized adolescent female mice that were previously exposed to TBI to study the impacts of radiotherapy on the uterus independent from effects to ovarian endocrine function. Following TBI, embryo attachment and implantation were unaffected, but fetal resorption was evident at midgestation in 100% of dams, suggesting failed placental development. Consistent with this hypothesis, TBI impaired the decidual response in mice and primary human endometrial stromal cells. TBI also caused uterine artery endothelial dysfunction, likely preventing adequate blood vessel remodeling in early pregnancy. Notably, when pro-apoptotic protein Puma-deficient (Puma-/-) mice were exposed to TBI, apoptosis within the uterus was prevented, and decidualization, vascular function, and pregnancy were restored, identifying PUMA-mediated apoptosis as a key mechanism. Collectively, these data show that TBI damages the uterus and compromises pregnancy success, suggesting that optimal fertility preservation during radiotherapy may require protection of both the ovaries and uterus. In this regard, inhibition of PUMA may represent a potential fertility preservation strategy.

Newly detected, virulent Toxoplasma gondii COUG strain causing fatal steatitis and toxoplasmosis in southern sea otters (Enhydra lutris nereis)

From February 2020 to March 2022, four southern sea otters (Enhydra lutris nereis) stranded in California with severe protozoal steatitis and systemic toxoplasmosis. Three of the infected otters stranded within 26 km of each other, and all four animals died during periods of increased rainfall-driven surface water runoff. High parasite burdens were observed in all tissues except the central nervous system, and toxoplasmosis with severe protozoal steatitis was the primary cause of death for all cases. This lesion pattern differs substantially from all prior reports of toxoplasmosis in sea otters. All cases were T. gondii-positive via serology, immunohistochemistry, and PCR. Multilocus sequence typing at 13 loci revealed that all were infected with the same strain of T. gondii, previously characterized as an atypical and rare genotype in North America (TgCgCa1, or COUG). The COUG genotype was first isolated from mountain lions in British Columbia, Canada during investigation of a waterborne outbreak of toxoplasmosis in humans. This genotype has not been previously reported from sea otters, nor any aquatic species. All prior T. gondii strains obtained from >140 southern sea otters represent Type II or Type X strains, or variants of these genotypes. Archival necropsy data (>1,000 animals over 24 years) were negative for prior cases of severe T. gondii-associated steatitis prior to the cases described herein, and no sublethal COUG T. gondii infections have been previously indentified in sea otters. According to prior studies, the T. gondii COUG genotype is highly virulent in mice and is unusual among T. gondii isolates in eliciting a Type I interferon response in murine and human cells in vitro; this unusual immunomodulatory response could explain the apparent high virulence of this atypical T. gondii strain. Our findings reveal a novel and concerning lesion pattern for sea otters with toxoplasmosis. Due to high zoonotic potential and the risk of infection via shared marine food resources, these findings may also indicate potential health threats for other animals and humans.

The Effect of Cholesterol in MCF7 Human Breast Cancer Cells

In the last decade, cholesterol level has been implicated in several types of cancer, including breast cancer. In the current study, we aimed to investigate the condition of lipid depletion, hypocholesterolemia or hypercholesterolemia reproduced in vitro to analyze the response of different human breast cancer cells. Thus, MCF7 as the luminal A model, MB453 as the HER2 model and MB231 as the triple-negative model were used. No effect on cell growth and viability was detected in MB453 and MB231 cells. In MCF7 cells, hypocholesterolemia (1) reduced cell growth, and Ki67 expression; (2) increased ER/PgR expression; (3) stimulated the 3-Hydroxy-3-Methylglutaryl-CoA reductase and neutral sphingomyelinase and; (4) stimulated the expression of CDKN1A gene coding cyclin-dependent kinase inhibitor 1A protein, GADD45A coding growth arrest and DNA-damage-inducible alpha protein and, PTEN gene coding phosphatase and tensin homolog. All these effects were exacerbated by the lipid-depleted condition and reversed by the hypercholesterolemic condition. The relationship between cholesterol level and sphingomyelin metabolism was demonstrated. In summary, our data suggest that cholesterol levels should be controlled in luminal A breast cancer.

Disruption of endosomal trafficking with EGA alters TLR9 cytokine response in human plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) exhibit bifurcated cytokine responses to TLR9 agonists, an IRF7-mediated type 1 IFN response or a pro-inflammatory cytokine response via the activation of NF-κB. This bifurcated response has been hypothesized to result from either distinct signaling endosomes or endo-lysosomal trafficking delay of TLR9 agonists allowing for autocrine signaling to affect outcomes. Utilizing the late endosome trafficking inhibitor, EGA, we assessed the bifurcated cytokine responses of pDCs to TLR9 stimulation. EGA treatment of pDCs diminished both IFNα and pro-inflammatory cytokine expression induced by CpG DNAs (D- and K-type), CpG-DNAs complexed with DOTAP, and genomic DNAs complexed with LL37. Mechanistically, EGA suppressed phosphorylation of IKKα/β, STAT1, Akt, and p38, and decreased colocalization of CpG oligodeoxynucleotides with LAMP+ endo-lysosomes. EGA also diminished type 1 IFN expression by pDCs from systemic lupus erythematosus patients. Therefore, our findings help understand mechanisms for the bifurcated cytokine responses by pDCs and support future examination of the potential benefit of EGA in treating type 1 IFN-associated inflammatory diseases in the future.

Biological Response Evaluation of Human Fetal Osteoblast Cells and Bacterial Cells on Fractal Silver Dendrites for Bone Tissue Engineering.

Prosthetic joint replacement is the most widely used surgical approach to repair large bone defects, although it is often associated with prosthetic joint infection (PJI), caused by biofilm formation. To solve the PJI problem, various approaches have been proposed, including the coating of implantable devices with nanomaterials that exhibit antibacterial activity. Among these, silver nanoparticles (AgNPs) are the most used for biomedical applications, even though their use has been limited by their cytotoxicity. Therefore, several studies have been performed to evaluate the most appropriate AgNPs concentration, size, and shape to avoid cytotoxic effects. Great attention has been focused on Ag nanodendrites, due to their interesting chemical, optical, and biological properties. In this study, we evaluated the biological response of human fetal osteoblastic cells (hFOB) and P. aeruginosa and S. aureus bacteria on fractal silver dendrite substrates produced by silicon-based technology (Si_Ag). In vitro results indicated that hFOB cells cultured for 72 h on the Si_Ag surface display a good cytocompatibility. Investigations using both Gram-positive (S. aureus) and Gram-negative (P. aeruginosa) bacterial strains incubated on Si_Ag for 24 h show a significant decrease in pathogen viability, more evident for P. aeruginosa than for S. aureus. These findings taken together suggest that fractal silver dendrite could represent an eligible nanomaterial for the coating of implantable medical devices.