Culture Inserts Research Articles

Nicotine directly affects milk production in lactating mammary epithelial cells concurrently with inactivation of STAT5 and glucocorticoid receptor in vitro

Nicotine from tobacco smoke is absorbed into the bloodstream and transferred into breast milk in breastfeeding mothers. Smoking causes a decrease in breast milk volume, adverse changes to the milk composition, and a shortened lactation period. Breast milk is produced by mammary epithelial cells (MECs) in mammary glands during lactation. However, it remains unclear whether nicotine directly affects milk production in lactating MECs. To address this issue, we prepared a culture model with high milk production ability and less-permeable tight junctions (TJs) by seeding mouse MECs on a cell culture insert. Lactating MECs showed expression of α2, α3, β2, and β4 of nicotinic acetylcholine receptors. The high concentration of nicotine at 10–100 μM inhibited β-casein secretion and caused abnormal localization of TJ proteins. We subsequently investigated whether nicotine at a physiological concentration could affect lactating MECs. Nicotine at 1.0 μM directly inhibited α- and β-casein secretion in lactating MECs concurrently with inactivation of STAT5 and glucocorticoid receptor without affecting the TJ barrier. Nicotine treatment also induced MEC apoptosis concurrently with inactivation of Akt. These results support the adverse effects of nicotine on breastfeeding in smoking mothers.

Guided Polarization of iPSC-Derived CD4SP Helper T Cells By CRISPR/Cas9-Based Genome-Editing

Guided Polarization of iPSC-Derived CD4SP Helper T Cells By CRISPR/Cas9-Based Genome-Editing

TMIC-33. INVESTIGATING S1PR3-MEDIATED GLIOBLASTOMA INVASION MECHANISMS USING 3D HYDROGEL - TISSUE CULTURE INSERT MODEL

Abstract Glioblastoma (GBM) is the most common malignant brain tumor and is characterized by its ability to invade into the surrounding microenvironment of the brain. The invasiveness of GBM makes this cancer extremely hard to treat, leading to a median patient survival of less than 16 months. Interactions between the tumor and surrounding tumor microenvironment (TME) play a key role in glioma invasion. Previous data from our xenograft mouse tumor implant model displays increased invasion in regions of fluid flow. Using this model, we identified an upregulation of sphingosine-1-phosphate receptor 3 (S1PR3) in the TME in regions of flow. We used a syngeneic GL261 mouse model and found S1PR3-/- mice display decreased flow-mediated glioma invasion in comparison to wild type mice. To further understand the individual contributions of the S1PR3-presenting cells in the TME, we have examined the role of S1PR3 in our in vitro system. This system is based on patient derived cellular ratios and incorporates collagen-hyaluronan hydrogels placed within 96 well tissue culture insert plates. The tunability of this model allows for interactions between various cell types and the impact of fluid flow on invasion to be examined. To examine the role of S1PR3 on invasion, TY52156 (an S1PR3 inhibitor) was applied to different cellular combinations including: G34 alone (a patient-specific cell line), +astrocytes, +microglia, +TME (astrocytes and microglia). A significant decrease in G34 flow stimulated invasion was observed with TY52156 but only in the presence of the TME or microglia alone. This data suggests that TY52156 thwarts the effects of flow and the microglia contributions to invasion. To further this work we plan to identify and evaluate other S1PR3-expressing cell types from mouse tumor implant samples using immunohistochemistry staining. This information will be used to determine further components that can be examined in our in vitro model.

TMIC-44. IL-1 ANTAGONIST ANAKINRA INHIBITS GLIOBLASTOMA PROLIFERATION BY ANTAGONIZING THE PROINFLAMMATORY MICROENVIRONMENT

Abstract INTRODUCTION The recombinant IL-1 receptor antagonist anakinra prevents binding of IL-1 to its receptor and blocks IL-1β-mediated proinflammatory signaling. It is currently used in the treatment of autoinflammatory diseases. As inflammation is a major driver of malignancy, we hypothesized that anakinra – by ameliorating the inflammatory tumor environment – might be capable to mitigate glioblastoma (GBM) progression. METHODS T98G GBM cells were cultivated with PBMC, separated by cell culture inserts with or without anakinra and incubated 48h in 5% O2 mimicking the GBM microenvironment. T-cells were obtained by magnetic bead isolation. mRNA expression levels were measured by quantitative Real-Time-PCR (qRT-PCR). Cytokine production was assessed by ELISA. Cell proliferation was analyzed by flow cytometric quantification of Ki-67 protein expression. Anakinra has been provided by Swedish Orphan Biovitrum AB (Sweden). Results are presented as mean±SEM, p-Values were calculated using Student’s t-test. RESULTS After administration of anakinra, mRNA expression levels of the inflammatory and proangiogenic genes IL-1β, COX-2, CCL2 and IL-8 were reduced in T98G (-64%±19.1%, -56.2%±21.3%, -87.1%±28.8%, -91.7%±27.2%, n=7, p< 0.05). Surprisingly, no changes of these targets were detectable in PBMC. However, in T-cells, anakinra induced mRNA expression of Th2 transcription factor GATA3 and the antiinflammatory cytokines IL-4 and IL-10 (+6,7%±4,6%, +36,1%±22,9%, +69,1%32,8%, n=10, p< 0.05). The expression of the protumorigenic genes IL-22, IL-17 as well as IFNγ was dramatically repressed (-63.2%±21.9%, -88.1%±21.7%, -85.6%±45.2%, n=10, p< 0.05), protein secretion of IL-22 and IFNγ was strongly blocked (-36.3%±9.1%, -13.9%±0.5%, n=6, p< 0.05). Importantly, treatment with anakinra markedly attenuated GBM cell proliferation (-20.1%±5%, n=4, p< 0.05). CONCLUSION Anakinra indeed puts the brake on proinflammatory signaling pathways in GBM cells and simultaneously promotes a shift towards an anti-inflammatory T-cell phenotype. As a result, GBM proliferation is diminished. Hence, administration of anakinra might be an interesting and novel therapeutic approach to reduce Glioblastoma aggressiveness.

Chips-on-a-plate device for monitoring cellular migration in a microchannel-based intestinal follicle-associated epithelium model.

This paper describes a chips-on-a-plate (COP) device for monitoring the migration of Raji cells in the Caco-2/Raji coculture. To generate a model of the human intestinal follicle-associated epithelium (FAE), the coculture method using a conventional Transwell cell culture insert was established. Due to the structural limitations of the Transwell insert, live-cell tracking studies have not been performed previously using the existing FAE model. In this study, we designed a COP device to conduct long-term live-cell tracking of Raji cell migration using a microchannel-based FAE model. The COP device incorporates microfluidic chips integrated on a standard well plate, consistent humidity control to allow live-cell microscopy for 2 days, and microchannels connecting the two cell culture chambers of the COP device, which serve as a monitoring area for cellular migration. Using the COP device, we provide the first analysis of various migratory characteristics of Raji cells, including their chemotactic index in the microchannel-based FAE model. We showed that the migration of Raji cells could be controlled by modulating the geometry of the connecting microchannels. Cellular treatments with cytokines revealed that the cytokines increased the permeability of an FAE model with a detachment of Caco-2 cells. Live-cell monitoring of Raji cells treated with a fluorescent reagent also indicated exocytosis as a key agent of the Caco-2/Raji interaction. The COP device allows live-cell tracking analyses of cocultured cells in the microchannel-based FAE model, providing a promising tool for investigating cellular behavior associated with the recruitment of Raji to Caco-2 cells.

Regulatory T cells promote corneal endothelial cell survival following transplantation via interleukin-10.

The functional competence of corneal endothelial cells (CEnCs) is critical for survival of corneal allografts, but these cells are often targets of the immune response mediated by graft-attacking effector T cells. Although regulatory T cells (Tregs) have been studied for their role in regulating the host's alloimmune response towards the graft, the cytoprotective function of these cells on CEnCs has not been investigated. The aim of this study was to determine whether Tregs suppress effector T cell-mediated and inflammatory cytokine-induced CEnC death, and to elucidate the mechanism by which this cytoprotection occurs. Using 2 well-established models of corneal transplantation (low-risk and high-risk models), we show that Tregs derived from low-risk graft recipients have a superior capacity in protecting CEnCs against effector T cell-mediated and interferon-γ and tumor necrosis factor-α-induced cell death compared to Tregs derived from high-risk hosts. We further demonstrate that the cytoprotective function of Tregs derived from low-risk hosts occurs independently of direct cell-cell contact and is mediated by the immunoregulatory cytokine IL-10. Our study is the first to report that Tregs provide cytoprotection for CEnCs through secretion of IL-10, indicating potentially novel therapeutic targets for enhancing CEnC survival following corneal transplantation.

Design, fabrication and testing of an electrical cell stimulation and recording apparatus (ECSARA) for cells in electroculture

A new dual-function electrical cell stimulation and recording apparatus (ECSARA) for simultaneously electrically stimulating cellular behavior within programmed stand-off electric fields (EFs) and monitoring cellular responses via AC electrical impedance spectroscopy (EIS) is reported. ECSARA is designed to have a footprint similar to that of a common 24-well cell culture plate within which each well is electrified via a pair of opposing planar titanium electrodes, within the cover (0.10 cm2) and base (0.50 cm2) of each well. Porous cell culture inserts established a 3-D milieu for bathing cells while keeping them away from unfavorable fields and forces in the vicinity of the electrodes. ECSARA was tested for its temporal stability, well-to-well variability, and responses in different media. EF modeling showed the field strength to be uniform in the subtending plane of the insert and the magnitude to be influenced by the porosity of the insert membrane. HUVECs were exposed to EF (162 mV/mm at 1.2 Hz) and monitored with standard viability Blue assay and EIS with equivalent circuit modeling. During the first 24 h, the viability (population) of EF-stimulated cells was smaller than non-stimulated control (0.8) but after 72 h they outnumbered the control (1.2) indicating that stimulation initially inhibited growth but resulted in eventual adaptive proliferation. EIS monitoring showed an increase in RCell of EF stimulated and control HUVECs after 54 h and 78 h, respectively. This was in accord with viability data that showed faster growth of EF-stimulated HUVECSs. Confluence was confirmed by VE-cadherin staining. The potential to explore the stimulatory influences of electric fields on cellular processes in tissue and regenerative engineering is now easily possible.

Cytotoxicity Evaluation of Three Biocompatible and Bioactive Retrofilling Materials Using Periodontal Ligament Stem Cells: An In-Vitro Study

This study aimed to: evaluate the biocompatibility of Mineral Trioxide Aggregate (MTA) versus Biodentine and Retro MTA extracts at three observation periods (24h, 48h, and 72h) using MTT-Essay.Methods: 72 wells in three culture insert plates were used in this study. MTA, Biodentine and Retro MTA cements were evaluated in a fresh state. Cells at passage four of human periodontal ligament cells were included in this study. After cells preparation and culturing, an extract of the three tested materials was prepared and cultured with the complete media in addition to 1×104 cells were implanted into each well 24 h earlier and incubated in a stem cell incubator. MTT assay was used to evaluate in vitro cytotoxicity, calculated the viable cells by hundred percent using ELISA system and the inverted light microscope used to reassure their confluence. After three different storage times (24h, 48h, and 72h) evaluation of cell viability was done after exposure to the material extract. Results: MTA and Biodentine cement showed no statistically significant differences in all the observation periods. But Retro MTA cement resulted in a statistically significant difference in all the observation periods but none of them below the cut level. All the materials extract resulted in a statistically significant increase in cytotoxicity at 24h and then decrease at 48h and finally a little increase in cytotoxicity at 72h. Conclusions: All the tested materials showed low cytotoxicity and are comparable to the old standard MTA.

Biomimetic In Vitro Model of Cell Infiltration into Skin Scaffolds for Pre-Screening and Testing of Biomaterial-Based Therapies.

Due to great clinical need, research where different biomaterials are tested as 3D scaffolds for skin tissue engineering has increased. In vitro studies use a cell suspension that is simply pipetted onto the material and cultured until the cells migrate and proliferate within the 3D scaffold, which does not mimic the in vivo reality. Our aim was to engineer a novel biomimetic in vitro model that mimics the natural cell infiltration process occurring in wound healing, thus offering a realistic approach when pre-screening and testing new skin substitutes. Our model consists of porous membrane cell culture inserts coated with gelatin and seeded with human dermal fibroblasts, inside which two different commercially available dermal substitutes were placed. Several features relevant to the wound healing process (matrix contraction, cell infiltration and proliferation, integration of the biomaterial with the surrounding tissue, and secretion of exogenous cytokines and growth factors) were evaluated. Our results showed that cells spontaneously infiltrate the materials and that our engineered model is able to induce and detect subtle differences between different biomaterials. The model allows for room for improvements or “adds-on” and miniaturization and can contribute to the development of functional and efficient skin substitutes for burns and chronic wounds.

A A ATUALIDADE DOS DEZ PRINCÍPIOS DA MAFALDA DE “QUINO” PARA UMA EDUCAÇÃO ÉTICA E DA RAZÃO CRIATIVA

Objetiva-se discutir nesse artigo a atualidade dos Dez princípios da Mafalda, como princípios norteadores de uma ética e uma razão criativa na infância, uma contribuição de “Quino” – Joaquín Salvador Lavado Tejón (1968) para a Educação. Esses Dez princípios são relevantes no aprendizado coletivo das crianças e no processo de sensibilização dos adultos. As críticas ao mundo do adulto são críticas de uma personagem criança, envolvida nas discussões diárias, nos problemas e dilemas dos adultos, causados por eles mesmos. Destaca-se valores como fundamentais nos dez princípios: direito à vida de criança cidadã, respeito ao ser humano e à criança e sua infância, direito ao cuidado, à inserção social, cultural e educacional, proteção, cidadania, direito para expressar criatividade e curiosidade, obter conhecimento de saberes com qualidade para garantir emancipação e autonomia, educar-se sem constrangimentos, discriminação, preconceito de classe, étnico, cultural, aprender a cultivar valores pessoais e coletivos sem imposição e sem dogmatismo. Enfim, Quino parece assinalar para uma educação que preze por uma construção de conhecimentos de modo aberto e que o despertar da curiosidade e da razão criativa sejam valorizados no processo formativo, um processo de mimesis, de troca e de revide entre as crianças e os adultos. A infância em Quino permite discutir sobre comportamentos sociais e educação ética mediante exercícios e posicionamentos críticos e criativos respaldados por uma sensibilidade e uma razão criativa, como elementos formadores e norteadores de uma educação para o pensar e para a valorização da vida.

Novel pendrin inhibitor attenuates airway hyperresponsiveness and mucin expression in experimental murine asthma

Novel pendrin inhibitor attenuates airway hyperresponsiveness and mucin expression in experimental murine asthma

Endocannabinoids and endocannabinoid-like compounds modulate hypoxia-induced permeability in CaCo-2 cells via CB1, TRPV1, and PPARα

Background and purposeWe have previously reported that endocannabinoids modulate permeability in Caco-2 cells under inflammatory conditions and hypothesised in the present study that endocannabinoids could also modulate permeability in ischemia/reperfusion. Experimental approachCaco-2 cells were grown on cell culture inserts to confluence. Trans-epithelial electrical resistance (TEER) was used to measure permeability. To generate hypoxia (0% O2), a GasPak™ EZ anaerobe pouch system was used. Endocannabinoids were applied to the apical or basolateral membrane in the presence or absence of receptor antagonists. Key resultsComplete hypoxia decreased TEER (increased permeability) by ~35% after 4 h (recoverable) and ~50% after 6 h (non-recoverable). When applied either pre- or post-hypoxia, apical application of N-arachidonoyl-dopamine (NADA, via TRPV1), oleamide (OA, via TRPV1) and oleoylethanolamine (OEA, via TRPV1) inhibited the increase in permeability. Apical administration of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) worsened the permeability effect of hypoxia (both via CB1). Basolateral application of NADA (via TRPV1), OA (via CB1 and TRPV1), noladin ether (NE, via PPARα), and palmitoylethanolamine (PEA, via PPARα) restored permeability after 4 h hypoxia, whereas OEA increased permeability (via PPARα). After 6 h hypoxia, where permeability does not recover, only basolateral application PEA sustainably decreased permeability, and NE decreased permeability. Conclusions and implicationsA variety of endocannabinoids and endocannabinoid-like compounds modulate Caco-2 permeability in hypoxia/reoxygenation, which involves multiple targets, depending on whether the compounds are applied to the basolateral or apical membrane. CB1 antagonism and TRPV1 or PPARα agonism may represent novel therapeutic targets against several intestinal disorders associated with increased permeability.

In vitro Cell Migration, Invasion, and Adhesion Assays: From Cell Imaging to Data Analysis.

Cell migration is a key procedure involved in many biological processes including embryological development, tissue formation, immune defense or inflammation, and cancer progression. How physical, chemical, and molecular aspects can affect cell motility is a challenge to understand migratory cells behavior. In vitro assays are excellent approaches to extrapolate to in vivo situations and study live cells behavior. Here we present four in vitro protocols that describe step-by-step cell migration, invasion and adhesion strategies and their corresponding image data quantification. These current protocols are based on two-dimensional wound healing assays (comparing traditional pipette tip-scratch assay vs. culture insert assay), 2D individual cell-tracking experiments by live cell imaging and three-dimensional spreading and transwell assays. All together, they cover different phenotypes and hallmarks of cell motility and adhesion, providing orthogonal information that can be used either individually or collectively in many different experimental setups. These optimized protocols will facilitate physiological and cellular characterization of these processes, which may be used for fast screening of specific therapeutic cancer drugs for migratory function, novel strategies in cancer diagnosis, and for assaying new molecules involved in adhesion and invasion metastatic properties of cancer cells.

A CELL-CULTURING SYSTEM FOR THE STUDY OF INTERACTION BETWEEN MACROPHAGES INFECTED WITH Leishmania amazonensis

The cell culture insert system is a culturing system for the study of contact-independent cellular communication. Leishmaniasis is a neglect tropical disease with no vaccines and the availabledrugs present toxic side effects. Studies on Leishmania interaction with host macrophages aim to develop strategies for parasite control and drug development. The purpose of this study was to evaluate the effects of interaction between non-infected and L. amazonensis-infected human macrophages, by using the cell culture system. The results showed that the infection index was reduced by 56.2% as compared to controls only when infected macrophages were inserted on both sides of the Transwell membranes. An improvement in macrophage viability was also observed in this cell culture. The levels of interleukin-1β, an inflammatory cytokine, and nitric oxide, a microbicidal molecule, did not increase in L. amazonensis-infected macrophagecultures in the Transwell system; thus other soluble factors were responsible for parasite control.

A novel wound dressing material with prolonged action

The development and creation of non-toxic dressing materials exhibiting pronounced antimicrobial effect and accelerating wound-healing process without increasing pathogenic microorganisms drug resistance is an actual task of modern medicine. Aim. Evaluation of toxicological characteristics of a dressing material containing 6 % and 9 % active chlorine in a single application on the skin as well as the study of dressing material effect on the epithelization rate of the uninfected wound surface and determination of its antimicrobial and wound healing activity in vivo on a model of S. aureus ATCC 6538-infected wound. Materials and methods . An experimental study of the dressings with immobilized polymeric N-chlorosulfonamide sodium and N,N-dichlorosulfonamide was performed to evaluate the safety in use and pharmacological activity in vivo . Evaluation of the dressing material toxicological characteristics in a single application on the skin was carried out on laboratory rats and rabbits according to the generally accepted method, the wound healing effect was studied using the test by L. N. Popova and antimicrobial activity in vivo - on a model of wound infected by the insertion of a 24-hour culture of S. aureus ATCC 6538 contaminated silk thread through the skin. Results . Acute toxicity pharmacological studies of material test-samples containing 6 % and 9 % active chlorine in a single application on the skin of rats and rabbits found that these applications did not lead to deaths, as well as changes in their behavioral pattern, consumption of feed and water. The epithelization rate of uninfected wound surface in application of dressings containing both sodium N-chlorosulfonamide and N, N-dichlorosulfonamide was significantly, on average 1.5 times, faster at the end of the observational period (day 14) in comparison with the control group animals. According to the data obtained, the application of dressings containing sodium N-chlorosulfonamide on the 24-hour culture of S. aureus ATCC 6538 infected wound significantly reduced the intensity of the inflammatory process local manifestations and 4–10 times decreased the number of pathogenic staphylococci under bandages on day 8 of observation. Conclusions . Thus, a single application of native materials on the animal skin does not cause a lethal outcome. Skin application of polymeric materials on uninfected wounds stimulates the processes of natural reparation, and decreases the number of pathogenic microorganisms in wound fluid as well as shortens the healing time in staphylococcus-infected wounds.

Effects of HEMA on Nrf2-related gene expression using a newly developed 3D co-culture model of the oral mucosa

Objective2-Hydroxyethyl methacrylate (HEMA) is a component of many resin-modified materials and elutes from dental restorations into the oral cavity. Objective of our investigation was to determine the impact of HEMA on oral keratinocytes (OKF6/TERT2) and gingival fibroblasts (HGFs) in a newly established 3D co-culture model (3D-CCM) and to analyze the permeability of OKF6/TERT2 cells for HEMA. MethodsWell-characterized 3D-CCMs, consisting of confluent OKF6/TERT2 cells on cell culture inserts above HGF-containing collagen gels, were treated supra-epithelial with HEMA. Mass spectrometry was used to measure the supra- and sub-epithelial distribution of HEMA after 24 h. The impact of HEMA on nuclear factor erythroid 2-related factor 2 (Nrf2) target genes was measured by qRT-PCR and western blot analysis. ResultsMass spectrometry showed that HEMA was evenly distributed above and below the keratinocyte layer after 24 h. Analyzed target genes of Nrf2 were induced in both cell types on the mRNA-level but less pronounced in HGFs. On the protein-level, both cell types showed similar effects: At 5 mM HEMA, heme oxygenase-1 was induced 5.1-fold in OKF6/TERT2 cells and 4.1-fold in HGFs. NAD(P)H quinone dehydrogenase-1 was approximately induced 1.85-fold in both cell types. SignificanceOur 3D-CCM is suitable to analyze the biocompatibility of dental materials due to an improved simulation of the oral mucosa compared to monolayer cultures. Our results indicate that HEMA is able to penetrate a dense layer of keratinocytes and to activate the cellular oxidative defense response. This may be due to the activation of the Nrf2-pathway in both cell types.

Organotypic Culture of Neonatal Murine Inner Ear Explants.

The inner ear is a complex organ containing highly specialised cell types and structures that are critical for sensing sound and movement. In vivo, the inner ear is difficult to study due to the osseous nature of the otic capsule and its encapsulation within an intricate bony labyrinth. As such, mammalian inner ear explants are an invaluable tool for the study and manipulation of the complex intercellular connections, structures, and cell types within this specialised organ. The greatest strength of this technique is that the complete organ of Corti, or peripheral vestibular organs including hair cells, supporting cells and accompanying neurons, is maintained in its in situ form. The greatest weakness of in vitro hair cell preparations is the short time frame in which the explanted tissue remains viable. Yet, cochlear explants have proven to be an excellent experimental model for understanding the fundamental aspects of auditory biology, substantiated by their use for over 40 years. In this protocol, we present a modernised inner ear explant technique that employs organotypic cell culture inserts and serum free media. This approach decreases the likelihood of explant damage by eliminating the need for adhesive substances. Serum free media also restricts excessive cellular outgrowth and inter-experimental variability, both of which are side effects of exogenous serum addition to cell cultures. The protocol described can be applied to culture both cochlear and vestibular explants from various mammals. Example outcomes are demonstrated by immunohistochemistry, hair cell quantification, and electrophysiological recordings to validate the versatility and viability of the protocol.

A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain.

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the barrier and the brain parenchyma is still an unmet need. To fill this gap, we designed a 2D in cellulo model, the BBB-Minibrain, by combining a polyester porous membrane culture insert human BBB model with a Minibrain formed by a tri-culture of human brain cells (neurons, astrocytes and microglial cells). The BBB-Minibrain allowed us to test the transport of a neuroprotective drug candidate (e.g., Neurovita), through the BBB, to determine the specific targeting of this molecule to neurons and to show that the neuroprotective property of the drug was preserved after the drug had crossed the BBB. We have also demonstrated that BBB-Minibrain constitutes an interesting model to detect the passage of virus particles across the endothelial cells barrier and to monitor the infection of the Minibrain by neuroinvasive virus particles. The BBB-Minibrain is a reliable system, easy to handle for researcher trained in cell culture technology and predictive of the brain cells phenotypes after treatment or insult. The interest of such in cellulo testing would be twofold: introducing derisking steps early in the drug development on the one hand and reducing the use of animal testing on the other hand.

5-Aminolevulinic Acid Suppresses Prostaglandin E2 Production by Murine Macrophages and Enhances Macrophage Cytotoxicity Against Glioma

5-Aminolevulinic acid (5-ALA) induces the accumulation of a large amount of protoporphyrin IX (PpIX) in tumors, which has been used in the treatment of several cancers. 5-ALA is commonly used for fluorescence-guided tumor resection in clinical neurosurgery and for photodynamic therapy based on the generation of cytotoxic oxygen. The purpose of this study was to identify the mechanisms of 5-ALA-induced immune response in macrophages in malignant glioma. Intracellular levels of 5-ALA-induced PpIX in C3H/HeN murine peritoneal macrophages were measured by the median fluorescence intensity using flow cytometry and confocal laser scanning microscopy. Macrophages were cultured invitro with or without 0.5 mM 5-ALA, 0.1 μg/mL lipopolysaccharide, and 20% glioma-conditioned medium. Levels of immunosuppressive prostaglandin E2 (PGE2), interleukin-10, and transforming growth factor β were measured using enzyme immunoassay in the culture supernatant. In addition, macrophages and RSV-M mouse glioma cells were co-cultured invitro with cell culture inserts with or without 5-ALA (0.1 and 0.5 mM) and lipopolysaccharide (0.1 μg/mL). We found that 5-ALA-induced PpIX accumulated in macrophages and significantly suppressed PGE2 production and expression of both cyclooxygenase-2 and microsomal prostaglandin E synthase-1. 5-ALA treatment also suppressed PGE2 production by glioma-conditioned medium. 5-ALA suppressed RSV-M glioma cell proliferation in a concentration-dependent manner. These results indicate that 5-ALA suppressed PGE2 production by macrophages via the downregulation of cyclooxygenase-2 and microsomal prostaglandin E synthase-1 expression levels. This is a novel mechanism to induce effective immune response against glioma in macrophages.

Surfactant Protein D Reverses the Gene Signature of Transepithelial HIV-1 Passage and Restricts the Viral Transfer Across the Vaginal Barrier.

Effective prophylactic strategy against the current epidemic of sexually transmitted HIV-1 infection requires understanding of the innate gatekeeping mechanisms at the genital mucosa. Surfactant protein D (SP-D), a member of the collectin family of proteins naturally present in the vaginal tract, is a potential HIV-1 entry inhibitor at the cellular level. Human EpiVaginal tissues compartmentalized in culture inserts were apically exposed to HIV-1 and/or a recombinant fragment of human SP-D (rfhSP-D) and viral passage was assessed in the basal chamber containing mononuclear leukocytes. To map the gene signature facilitating or resisting the transepithelial viral transfer, microarray analysis of the HIV-1 challenged EpiVaginal tissues was performed in the absence or presence of rfhSP-D. Mucosal biocompatibility of rfhSP-D was assessed ex vivo and in the standard rabbit vaginal irritation model. The passage of virus through the EpiVaginal tissues toward the underlying target cells was associated with a global epithelial gene signature including differential regulation of genes primarily involved in inflammation, tight junctions and cytoskeletal framework. RfhSP-D significantly inhibited HIV-1 transfer across the vaginal tissues and was associated with a significant reversal of virus induced epithelial gene signature. Pro-inflammatory NF-κB and mTOR transcripts were significantly downregulated, while expression of the tight junctions and cytoskeletal genes was upheld. In the absence of virus, rfhSP-D directly interacted with the EpiVaginal tissues and upregulated expression of genes related to structural stability of the cell and epithelial integrity. There was no increment in the viral acquisition by the PBMCs present in basal chambers wherein, the EpiVaginal tissues in apical chambers were treated with rfhSP-D. The effective concentrations of rfhSP-D had no effect on lactobacilli, epithelial barrier integrity and were safe on repeated applications onto the rabbit vaginal mucosa. This pre-clinical safety data, coupled with its efficacy of restricting viral passage via reversal of virus-induced gene expression of the vaginal barrier, make a strong argument for clinical trials of rfhSP-D as a topical anti-HIV microbicide.