Microglial NF-κB Signaling Deficiency Protects Against Metabolic Disruptions Caused by Volatile Organic Compound via Modulating the Hypothalamic Transcriptome.

Prolonged exposure to benzene, a prevalent volatile organic compound (VOC), at concentrations found in smoke, triggers hyperglycemia, and inflammation in mice. Corroborating this with existing epidemiological data, we show a strong correlation between environmental benzene exposure and metabolic impairments in humans. To uncover the underlying mechanisms, we employed a controlled exposure system and continuous glucose monitoring (CGM), revealing rapid blood glucose surges and disturbances in energy homeostasis in mice. These effects were attributed to alterations in the hypothalamic transcriptome, specifically impacting insulin and immune response genes, leading to hypothalamic insulin resistance and neuroinflammation. Moreover, benzene exposure activated microglial transcription characterized by heightened expression of IKKß/NF-κB-related genes. Remarkably, selective removal of IKKß in immune cells or adult microglia in mice alleviated benzene-induced hypothalamic gliosis, and protected against hyperglycemia. In summary, our study uncovers a crucial pathophysiological mechanism, establishing a clear link between airborne toxicant exposure and the onset of metabolic diseases.

Acarbose protects from central and peripheral metabolic imbalance induced by benzene exposure.

Benzene is a well-known human carcinogen that is one of the major components of air pollution. Sources of benzene in ambient air include cigarette smoke, e-cigarettes vaping, and evaporation of benzene containing petrol processes. While the carcinogenic effects of benzene exposure have been well studied, less is known about the metabolic effects of benzene exposure. We show that chronic exposure to benzene at low levels induces a severe metabolic imbalance in a sex-specific manner, and is associated with hypothalamic inflammation and endoplasmic reticulum (ER) stress. Benzene exposure rapidly activates hypothalamic ER stress and neuroinflammatory responses in male mice, while pharmacological inhibition of ER stress response by inhibiting IRE1α-XBP1 pathway significantly alleviates benzene-induced glial inflammatory responses. Additionally, feeding mice with Acarbose, a clinically available anti-diabetes drug, protected against benzene induced central and peripheral metabolic imbalance. Acarbose imitates the slowing of dietary carbohydrate digestion, suggesting that choosing a diet with a low glycemic index might be a potential strategy for reducing the negative metabolic effect of chronic exposure to benzene for smokers or people living/working in urban environments with high concentrations of exposure to automobile exhausts.

Effects of mast cell modulation on early host response to implanted synthetic meshes.

INTRODUCTION: Mast cells (MCs) and their products (e.g., histamine, serotonin, heparin, prostaglandins, cytokines, etc.) play key roles in controlling local inflammation, wound healing, and foreign body reactions in vivo. Investigation of the role of MCs in mediating local tissue responses to synthetic hernia meshes has been very limited to date. We aimed to determine the effects of MCs/MC products in mice undergoing synthetic mesh implantation. MATERIALS AND METHODS: Circular samples (5 mm) of heavyweight microporous polypropylene (Trelex), midweight microporous polypropylene (ProLite), lightweight macroporous polypropylene with poliglecaprone (Ultrapro), and 3-dimensional macroporous polyester (Parietex) meshes were implanted subcutaneously in C57BL/6 J mice with and without cromolyn (MC stabilizer/suppressant) treatment (50 mg/kg, daily IP). Two weeks post-implantation, all meshes were explanted and evaluated histologically using H&E and trichrome stains. RESULTS: Chronic inflammation was focused around individual mesh fibers; inter-fiber inflammation and fibrosis diminished as mesh porosity increased. MC accumulation was seen at the periphery of inflammatory reactions, and in association with mesh-induced fibrosis and neovascularization. Cromolyn treatment resulted in significantly decreased fibrotic responses to all four meshes and reduced inflammation induced by Trelex, ProLite, and Parietex meshes but not Ultrapro. CONCLUSION: We demonstrated that MCs play important roles in mesh-induced host tissue reactions. Blocking MC degranulation decreased early inflammation and fibrosis induced by most synthetic meshes in this study. Further evaluation and understanding of the role of MCs in mesh-induced tissue reactions will provide new therapeutic approaches to enhance the biocompatibility of surgical meshes and ultimately improve clinical outcomes in patients undergoing hernia repair with synthetic biomaterials.

Activation of human mononuclear cells by porcine biologic meshes in vitro.

INTRODUCTION: While porcine-based biologic meshes are increasingly used for hernia repair, little data exist on tissue responses to such products. Host foreign body reaction, local inflammation, and wound healing are principally controlled by monocytes/macrophages (M/MØs). Exaggerated activation of M/MØs may deleteriously influence mesh integration and remodeling. We hypothesized that common porcine meshes induce the differential activation of M/MØs in vitro. MATERIALS AND METHODS: Samples of four acellular porcine-derived meshes, CollaMend (CM; C.R. Bard/Davol), Permacol (PC; TSL/Covidien), Strattice (ST; LifeCell), and Surgisis (SS; Cook Biotech), were exposed to mononuclear cells derived from the peripheral blood of six healthy subjects. Following a 7-day incubation period, supernatants were assayed for interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), and vascular endothelial growth factor (VEGF) using a multiplex bead-based immunoassay system. The four groups were compared using analysis of variance (ANOVA) and Student's t-test. RESULTS: Each mesh type induced differential mononuclear cell activation in vitro. The mean IL-1beta expressions for CM (7,195 pg/ml) and PC (4,215 pg/ml) were significantly higher compared to ST and SS (123 and 998 pg/ml, respectively; P < 0.05). Similar trends were also seen for IL-6 (range 445-70,729 pg/ml), IL-8 (range 11,640-1,045,938 pg/ml), and VEGF (range 686-7,133 pg/ml). CONCLUSION: For the first time, we demonstrated that porcine meshes induce M/MØ activation in vitro. CM and PC (chemically crosslinked dermis) induced significantly higher cytokine expression compared to ST (non-crosslinked dermis) and SS (small intestine submucosa). These differences are likely related to proprietary processing methods and/or the extent of collagen crosslinking. Further understanding of immunologic effects of porcine-derived biologic meshes will not only allow for a comparison between existing products, but it may also lead to mesh modifications and improvement of their clinical performance.

Use of vascular endothelial cell growth factor gene transfer to enhance implantable sensor function in vivo.

In the current study, we developed and validated a simple, rapid and safe in vivo model to test gene transfer and sensor function in vivo. Using the model, we tested the specific hypothesis that in vivo gene transfer of angiogenic factors at sites of biosensor implantation would induce neovascularization surrounding the sensor and thereby enhance biosensor function in vivo. As the in vivo site for testing of our gene transfer cell and biosensor function systems, the developing chorioallantoic membrane (CAM) of the embryo was utilized. Vascular endothelial cell growth factor (VEGF) was used as a prototype for angiogenic factor gene transfer. A helper-independent retroviral vector derived from Rous sarcoma virus (RSV), designated RCAS, was used for gene transfer of the murine VEGF (mVEGF) gene (mVEGF:RCAS) into the DF-1 chicken cell line (designated mVEGF:DF-1). Initially, the ability of VEGF:DF-1 cells to produce VEGF and RCAS viral vectors containing the mVEGF gene (mVEGF:RCAS) was validated in vitro and in vivo, as was the ability of the mVEGF:DF-1 cells to induce neovascularization in the ex ova CAM model. Using the system, we determined the ability of mVEGF:DF-1 cells to enhance acetaminophen sensor function in vivo, by inducing neovascularization at sites of sensor implantation in the ex ova CAM model. For these studies, acetaminophen sensors were placed on 8-day-old ex ova CAMs, followed by addition of media or cells (mVEGF:DF-1 cells or GFP:DF-1 cells) at the sites of biosensor implantation on the CAM. At 4 to 10 days after sensor placement, the biosensor function was determined by measuring sensor response to an intravenous injection of acetaminophen. Sensors implanted on CAMs with buffer or control cells (GFP:DF-1 cells) displayed no induced neovascularization around the sensor and had minimal/baseline sensor responses to intravenous acetaminophen injection (media, 133.33 +/- 27.64 nA; GFP:DF-1, 187.50 +/- 55.43 nA). Alternatively, the sensors implanted with mVEGF:DF-1 cells displayed massive neovascularization and equally massive sensor response to intravenous injection of acetaminophen (VEGF:DF-1, 1387.50 +/- 276.42 nA). These data clearly demonstrate that enhancing vessel density (i.e., neovascularization) around an implanted sensor dramatically enhances sensor function in vivo.

Binding and orientation of fibronectin on polystyrene surfaces using immobilized bacterial adhesin-related peptides.

Fibronectin (FN) is known to bind to bacteria via high affinity receptors on bacterial surfaces known as adhesins. The binding of bacteria to FN is thought to have a key role in foreign device associated infections. For example, previous studies have indicated that Staphylococcus aureus adhesins bind to the 29 kDa NH(3) terminus end of FN, and thereby promote bacteria adherence to surfaces. Recently, the peptide sequences within the S. aureus adhesin molecule that are responsible for FN binding have been identified. Based on these observations, we hypothesize that functional FN can be bound and specifically oriented on polystyrene surfaces using bacterial adhesin-related (BRP-A) peptide. We further hypothesize that monoclonal antibodies that react with specific epitopes on the FN can be used to quantify both FN binding and orientation on these surfaces. Based on this hypothesis, we initiated a systematic investigation of the binding and orientation of FN on polystyrene surfaces using BRP-A peptide. To test this hypothesis, the binding and orientation of the FN to immobilized BRP-A was quantified using (125)I-FN, and monoclonal antibodies. (125)I-FN was used to quantitate FN binding to peptide-coated polystyrene surfaces. The orientation of bound FN was demonstrated by the use of monoclonal antibodies, which are reactive with the amine (N) or carboxyl (C) termini of the FN. The results of our studies demonstrated that when the BRP-A peptide was used to bind FN to surfaces that: 1. functional FN was bound to the peptide; 2. anti-C terminus antibodies bound to the peptide FN; and 3. only limited binding of anti-N terminus antibodies to peptide-bound FN occurred. We believe that the data that indicate an enhanced binding of anti-C antibodies reactive to anti-N antibodies are a result of the FN binding in an oriented manner with the N termini of FN bound tightly to the BRP-A on the polystyrene surface.

Ex ova chick chorioallantoic membrane as a novel in vivo model for testing biosensors.

A major problem with implantable sensors is their short in vivo lifetime, due to strong tissue reactions (i.e., inflammation and fibrosis) caused by the implant and the failure of sensor components. The tissue reactions to the sensor, the biocompatibility of components, and the function of the sensor must be evaluated by using in vivo models. Current methods of in vivo biosensor testing are time- and labor- intensive and expensive. In addition, the results often vary on the basis of the surgical skills of the investigator. The in ova chorioallantoic membrane (CAM) of the developing chicken embryo was previously developed in our laboratory as a novel in vivo system to test biomaterials. In this new article, we describe a novel approach for testing biosensors in vivo using the ex ova CAM model as an alternative to the traditional mammalian models. Fertilized chicken eggs were incubated for 3 days in ova and then transferred into a petri dish (ex ova) for further incubation at 37 degrees C and 80% humidity. After 1 week of incubation, acetaminophen biosensors, used as model sensors, were placed on top of the CAM and allowed to incorporate for 1 week. Biosensors were then tested for their sensitivity to acetaminophen. CAM venules were injected with 0.2 mL of a 3.6 mM acetaminophen solution. The current produced by the sensor reflected the change in blood acetaminophen levels. Sensors were also assessed by using gross and histological evaluations. We previously reported on the similarity of the tissue response of the CAM with the mammalian models. The low cost, simplicity, and possibility to continuously visualize the sensor test site through a cell culture dish make this animal model particularly attractive for the rapid in vivo screening of biosensors.

Binding and orientation of fibronectin to silanated glass surfaces using immobilized bacterial adhesin-related peptides.

Previously, we have demonstrated the suitability of bacterial adhesin-related peptides, directly immobilized on polystyrene surfaces, to bind and orient fibronectin (FN). For these studies a method to bind the large protein FN in a desired orientation on a solid substratum was developed which utilizes a bacterial adhesin-related peptide (designated BRP-A), which is known to bind specifically to the NH3-terminus end of FN. Glass substrata was first coated with an amine-terminated silane, followed by streptavidin (SA), which was used as an intermediate tether to bind the biotinylated bacterial adhesin-related peptide. The BRP-A peptide, used for these studies was synthesized with a terminal biotin to assure irreversible coupling of the BRP-A to the streptavidin. The biotinylated BRP-A was next immobilized on the SA-silanated glass surfaces. 125I-FN was used to quantify the amount of FN binding to the (BRP-A):SA-silanated glass surface. Monoclonal antibodies, which react with specific epitopes at either the NH3- or -COOH-termini of FN, were used to quantify the binding and orientation of FN. The results of these studies indicated: (1) FN bound to the BRP-A:SA-silanated glass surface; and (2) the bound FN was oriented such that NH2-terminal region of FN was bound towards the glass surface and the COOH-terminus was oriented away from the glass surface. These studies demonstrate that small peptides can be used to specifically bind and orient large proteins such as FN on the surfaces.

Binding and orientation of fibronectin on surfaces with collagen-related peptides. Student Research Award in the Masters Science Degree Candidate category, 27th annual meeting of the Society for Biomaterials, St. Paul, MN, April 24-29, 2001.

Although fibronectin (FN) has been used in a variety of in vitro studies to enhance cell and bacteria adhesion, relatively little is known about the molecular interactions of FN with surfaces, particularly the interactions that can control the binding, conformation, and functionality of FN on these surfaces. Even less is known about approaches needed to control binding, orientation, and functionality of FN bound on surfaces. To begin to fill this gap in our knowledge, we hypothesized that functional FN can be bound and specifically oriented on polystyrene surfaces with FN-specific collagen-related peptides (CRPs). We further hypothesized that monoclonal antibodies that react with specific epitopes on FN can be used to quantify both FN binding and orientation on these surfaces. On the basis of these hypotheses, we initiated a systematic investigation of the binding and orientation of FN on polystyrene surfaces with CRPs. To bind FN to surfaces, we used two different CRPs: CRP-I (TLQPVYEYMVGV) and CRP-II (TGLPVGVGYVVTVLT). The binding and orientation of the FN molecule to these immobilized CRPs was quantified with (125)I-FN and monoclonal antibodies. Monoclonal antibodies used for this study were reactive with specific regions of the FN molecule, that is, the amino (N) terminus (anti-N antibodies) and carboxyl (C) terminus (anti-C antibodies). The results of our studies demonstrated that although CRP-I and CRP-II could be bound directly to polystyrene, these directly immobilized CRPs failed to bind (125)I-FN . Thus, to facilitate FN binding to the CRPs, we used bovine serum albumin (BSA) as a spacer to physically elevate the CRPs away from the polystyrene surface. Thus, CRP-I and CRP-II were covalently linked to BSA via the N and C termini of each CRP (CRP-I-BSA and CRP-II-BSA). (125)I-CRP-BSAs were all found to bind to equivalent levels on polystyrene (1.60-2.60 microg/cm2). When CRP-BSAs were immobilized on polystyrene, they all successfully bound (125)I-FN in a range of 34-72 ng/cm2 (mean). Using monoclonal antibodies to FN to characterize the orientation of FN bound to the various CRP-BSAs, we demonstrated that (1) FN consistently bound to either CRP-I-BSA or CRP-II-BSA; (2) bound FN reacted significantly more with anti-C antibodies than with anti-N antibodies; and (3) the increased reactivity of bound FN to anti-C antibodies was consistent, whether FN was bound by CRP-I or CRP-II or the CRPs were bound to BSA by the C or N termini. These data demonstrated an enhanced binding of anti-C antibodies to immobilized CRP-BSA relative to anti-N antibodies. We interpreted the data to be the result of FN binding in an oriented fashion with N termini of FN bound tightly to the BSA-polystyrene surface. In this position, the C termini of FN are exposed and available for binding by the anti-C antibodies. Alternatively, in this orientation the N termini of the FN would not be available to bind the anti-N antibodies, thereby explaining the decreased reactivity of the CRP immobilized FN to the anti-N antibodies. These studies not only demonstrate the utility of peptides in binding and orienting large molecular weight proteins such as FN on surfaces but underscore the need for well-characterized reagents (e.g., monomeric/functional FN and antibodies) to specifically bind, orient, and characterize large molecular weight proteins immobilized on various surfaces.

Efficacy of silver-coated fabric to prevent bacterial colonization and subsequent device-based biofilm formation.

Efficacy of silver-coated poly(ethylene terephthalate) to prevent bacterial attachment and subsequent infection was quantified in vitro, in both batch- and flowing-fluid experiments. Kinetic analysis of batch suspended cell cultures of Staphylococcus epidermidis (SE), at various growth-limiting nutrient concentrations, in the absence of any fabric, indicated a maximum culture growth rate constant micro(max) = 0.78 +/- 0.02 h(-1). Batch experiments for Control fabric samples indicated that SE cultures exhibited about the same suspended cell growth rate (0.72 +/- 0.02 h(-1)) as observed in batch suspended cultures without fabric. Suspended SE cultures in the presence of silver-coated fabric grew at a considerably lower rate, 0.15 +/- 0.01 h(-1), indicating the inhibitory effect of Ag(+2) ion released from the fabric. Growth rates of suspended SE cultures were 5-6 times higher in the fluid phase in contact with the Control fabric compared to cultures exposed to silver-coated fabric. Maximum suspended cell concentrations attained at time = 24 h were 1-2 orders of magnitude higher for Control fabrics vs. silver-coated fabric. In all batch colonization experiments, both live and dead SE bacterial cells accumulate on the surfaces of both silver-coated and Control fabrics. Adherent viable SE cells accumulated to 1-2 orders of magnitude more ( approximately 5 x 10(+8) cells/cm(2)) on Control fabric than SE cells on the silver-coated fabric ( approximately 1.1 x 10(+6) cells/cm(2)), respectively. Between 70-95% SE cells on the Control fabric were viable, while on the silver-coated fabric samples, at 24 h, viable cells were less than 10% of the adherent community (i.e., greater than 90% nonviable cells). In flow cell colonization experiments, SE cells accumulated on Control fabric to a maximum adherent cell concentration of 6 x 10(+7) - 8 x 10(+7) cells/cm(2) by 24 h with the proportion of viable cells remaining relatively constant at 76% throughout an experiment. Both noninvasive microscopic enumeration and destructive assays gave the same results for adherent cell numbers. Using silver-coated fabric, total cells numbers (live + dead) reached a level of approximately 1.1 x 10(+7) - 3.0 x 10(+7) cells/cm(2) after about 6 h and remained constant. However, while the proportion of viable cells initially on the surface was 63-75%, this fraction dropped continuously during each experiment to less than 6% viable cells at 24 h. Regardless of the criteria, the number of viable or nonviable cells attached to silver-coated fabric were significantly lower than on Control fabric.