Sirt6 protects retinal ganglion cells and optic nerve from degeneration during aging and glaucoma.

Glaucoma is characterized by the progressive degeneration of retinal ganglion cells (RGCs) and their axons, and its risk increases with aging. Yet comprehensive insights into the complex mechanisms are largely unknown. Here, we found that anti-aging molecule Sirt6 was highly expressed in RGCs. Deleting Sirt6 globally or specifically in RGCs led to progressive RGC loss and optic nerve degeneration during aging, despite normal intraocular pressure (IOP), resembling a phenotype of normal-tension glaucoma. These detrimental effects were potentially mediated by accelerated RGC senescence through Caveolin-1 upregulation and by the induction of mitochondrial dysfunction. In mouse models of high-tension glaucoma, Sirt6 level was decreased after IOP elevation. Genetic overexpression of Sirt6 globally or specifically in RGCs significantly attenuated high tension-induced degeneration of RGCs and their axons, whereas partial or RGC-specific Sirt6 deletion accelerated RGC loss. Importantly, therapeutically targeting Sirt6 with pharmacological activator or AAV2-mediated gene delivery ameliorated high IOP-induced RGC degeneration. Together, our studies reveal a critical role of Sirt6 in preventing RGC and optic nerve degeneration during aging and glaucoma, setting the stage for further exploration of Sirt6 activation as a potential therapy for glaucoma.

Hexavalent sperm-binding IgG antibody released from vaginal film for development of potent on-demand nonhormonal female contraception.

Nonhormonal products for on-demand contraception are a global health technology gap; this unmet need motivated us to pursue the use of sperm-binding monoclonal antibodies to enable effective on-demand contraception. Here, using the cGMP-compliant Nicotiana-expression system, we produced an ultrapotent sperm-binding IgG antibody possessing 6 Fab arms per molecule that bind a well-established contraceptive antigen target, CD52g. We term this hexavalent antibody "Fab-IgG-Fab" (FIF). The Nicotiana-produced FIF had at least 10-fold greater sperm-agglutination potency and kinetics than the parent IgG, while preserving Fc-mediated trapping of individual spermatozoa in mucus. We formulated the Nicotiana-produced FIF into a polyvinyl alcohol-based water-soluble contraceptive film and evaluated its potency in reducing progressively motile sperm in the sheep vagina. Two minutes after vaginal instillation of human semen, no progressively motile sperm were recovered from the vaginas of sheep receiving FIF Film. Our work supports the potential of multivalent contraceptive antibodies to provide safe, effective, on-demand nonhormonal contraception.

Oxidative stress induces Z-DNA-binding protein 1-dependent activation of microglia via mtDNA released from retinal pigment epithelial cells.

Oxidative stress, inflammation, and aberrant activation of microglia in the retina are commonly observed in ocular pathologies. In glaucoma or age-related macular degeneration, the chronic activation of microglia affects retinal ganglion cells and photoreceptors, respectively, contributing to gradual vision loss. However, the molecular mechanisms that cause activation of microglia in the retina are not fully understood. Here we show that exposure of retinal pigment epithelial (RPE) cells to chronic low-level oxidative stress induces mitochondrial DNA (mtDNA)-specific damage, and the subsequent translocation of damaged mtDNA to the cytoplasm results in the binding and activation of intracellular DNA receptor Z-DNA-binding protein 1 (ZBP1). Activation of the mtDNA/ZBP1 pathway triggers the expression of proinflammatory markers in RPE cells. In addition, we show that the enhanced release of extracellular vesicles (EVs) containing fragments of mtDNA derived from the apical site of RPE cells induces a proinflammatory phenotype of microglia via activation of ZBP1 signaling. Collectively, our report establishes oxidatively damaged mtDNA as an important signaling molecule with ZBP1 as its intracellular receptor in the development of an inflammatory response in the retina. We propose that this novel mtDNA-mediated autocrine and paracrine mechanism for triggering and maintaining inflammation in the retina may play an important role in ocular pathologies. Therefore, the molecular mechanisms identified in this report are potentially suitable therapeutic targets to ameliorate development of ocular pathologies.

Tauopathy induces degeneration and impairs regeneration of sensory nerves in the cornea.

The cornea is transparent and innervated by a dense collection of sensory nerves originating from the ocular branch of the trigeminal nerve. This study was designed to comprehensively analyze alterations of corneal sub-basal nerve plexus in a mouse model of tauopathy (P301L transgenic mice) to test the possibility of using corneal nerves as a biomarker for tauopathy. Corneal sensitivity, thickness and epithelial wound healing were measured non-invasively by aeshesiometer, optical coherence tomography and fluorescein staining, respectively. Tau, corneal nerves and immune cells were examined by immunohistochemistry or Western blot. At the early stage of tauopathy, although corneal sensitivity, thickness and nerve fiber density were not greatly altered, corneal nerve abnormalities were observed in the peripheral region of young P301L mice. With aging, the density of abnormal nerves increased, while corneal sensitivity, epithelial thickness, nerve fiber density and length decreased in middle-aged P301L mice compared with WT mice. After corneal epithelial injury in young mice, no difference in reepithelialization was observed between two groups of mice, however, the regeneration of corneal nerves in P301L mice lagged behind WT mice, which was reflected by delayed recovery of corneal sensitivity, decreased corneal nerve density and length and density of CD45+ dendriform cells in P301L mice. In conclusion, our data provide compelling evidence that corneal nerves were changed in a mouse model of tauopathy in an age-dependent manner. Moreover, tau overexpression impairs corneal nerve regeneration. These results suggest that cornea may serve as a promising ocular site for the early diagnosis of tauopathy.

Age dependence of retinal vascular plexus attenuation in the triple transgenic mouse model of Alzheimer's disease.

The influence of Alzheimer's disease (AD) progression and severity on the structural and functional integrity of the cerebral vasculature is well recognized. The retina is an extension of the brain; thus, changes in retinal vascular features may serve as markers of AD cerebrovascular pathologies. However, differentiating normal aging-versus AD-induced retinal vascular changes is unresolved. Therefore, we compared and quantified changes in superficial (SVP), intermediate (IVP), and deep (DVP) retinal vascular plexuses in young, middle-age, and old triple transgenic mouse model of AD (3xT-AD) to the changes that occur in age-matched controls (C57BL/6j). We used immunostaining combined with a novel tissue optical clearing approach along with a computational tool for quantitative analysis of vascular network alterations (vessel length and density) in SVP, IVP, and DVP. All three layers had comparable structural features and densities in young 3xTg-AD and control animals. In controls, IVP and DVP densities decreased with aging (-14% to -32% change from young to old, p < 0.05), while no changes were observed in SVP. In contrast, vascular parameters in the transgenic group decreased in all three layers with aging (-12% to -49% change from young to old, p < 0.05). Furthermore, in the old group, SVP and DVP vascular parameters were lower in the transgenics compared to age-matched controls (p < 0.05). Our analysis demonstrates that normal aging and progression of AD lead to various degrees of vascular alterations in the retina. Specifically, compared to normal aging, changes in vascular features of SVP and DVP regions of the retina are accelerated during AD progression. Considering recent advances in the field of depth-resolved imaging of retinal capillary network and microangiography, noninvasive quantitative monitoring of changes in retinal vascular network parameters of SVP and DVP may serve as markers for diagnosis and staging of Alzheimer's disease and discriminating AD-induced vascular attenuation from age-related vasculopathy.

Longitudinal characterization of retinal vasculature alterations with optical coherence tomography angiography in a mouse model of tauopathy.

Tauopathies are a family of neurodegenerative diseases which predominately afflict the rapidly growing aging population suffering from various brain disorders including Alzheimer's disease, frontotemporal dementia with parkinsonism-17 and Pick disease. As the only visually accessible region of the central nervous system, in recent years, the retina has attracted extensive attention for its potential as a target for visualizing and quantifying emerging biomarkers of neurodegenerative diseases. Our previous study has found that retinal vascular inflammation and leakage occur at the very early stage of tauopathic mouse model. Here, we aimed to non-invasively visualize age-dependent alterations of retinal vasculature assessing the potential for using changes in retinal vasculature as the biomarker for the early diagnosis of tauopathy. Optical coherence tomography angiography (OCTA), a non-invasive depth-resolved high-resolution imaging technique was used to visualize and quantify tauopathy-induced alterations of retinal vasculature in P301S transgenic mice overexpressing the P301S mutant form of human tau and age-matched wild type littermate mice at 3, 6 and 10 months of age. We observed significant alterations of vascular features in the intermediate capillary plexus (ICP) and deep capillary plexus (DCP) but not in the superficial vascular complex (SVC) of P301S mice at early stages of tauopathy. With aging, alterations of vascular features in P301S mice became more prominent in all three vascular plexuses. Staining of retinal vasculature in flatmounts and trypsin digests of P301S mice at 10 months of age revealed decreased vessel density and increased acellular capillary formation, indicating that vascular degeneration also occurs during tauopathy. Overall, our results demonstrate that the changes in retinal vascular features accelerate during the progression of tauopathy. Vessels in the ICP and DCP may be more susceptible to tauopathy than vessels in the SVC. Since changes in retinal vasculature often precede tau pathology in the brain, non-invasive identification of retinal vascular alterations with OCTA may be a useful biomarker for the early diagnosis of tauopathy and monitoring its progression.

Concentration-associated pathology of alkali burn in a mouse model using anterior segment optical coherence tomography with angiography.

Pathological features of alkali concentration-associated burn were studied using non-invasive anterior segment optical coherence tomography (AS-OCT) and OCT angiography (OCTA). Alkali burn was induced in C57BL/6J mice (n = 20) by placing filter paper soaked in 0.1, 0.25, 0.5, and 1 M NaOH for 30s on the right eye (left eye control). Longitudinal imaging was performed with AS-OCT/OCTA and fluorescein angiography over 14 days, after which eyes were enucleated at 7 and 14 days for histology and immunofluorescence. Concentration-associated corneal swelling was maximal at 0.5M, increasing linearly in a concentration-dependent fashion at 0.1, 0.25, and 0.5 M NaOH, to levels of 50%, 100%, and 175% of control, respectively. At 0.1M, corneal swelling and surface erosions were prominent, while at 0.25M, deep tissue damage, limbal neovascularization, and stromal haze were evident at 7 days. At 0.5M and 1M, severe exacerbation of the corneal swelling, angle closure, Descemet's membrane detachment, hyphema, and profuse central neovascularization were noted as early as day 3, which further progressed to inflammation, fibrosis, and opacity by day 7. We conclude that alkali concentration-dependent burn intensity biomarkers can be assessed by non-invasive AS-OCT/OCTA, distinguishing between mild, moderate, and severe ocular injury, with potential relevance toward clinical utilization in human eyes.

CXCR3 deletion aggravates corneal neovascularization in a corneal alkali-burn model.

Corneal neovascularization can cause devastating consequences including vision impairment and even blindness. Corneal inflammation is a crucial factor for the induction of corneal neovascularization. Current anti-inflammatory approaches are of limited value with poor therapeutic effects. Therefore, there is an urgent need to develop new therapies that specifically modulate inflammatory pathways and inhibit neovascularization in the cornea. The interaction of chemokines and their receptors plays a key role in regulating leukocyte migration during inflammatory response. CXCR3 is essential for mediating the recruitment of activated T cells and microglia/macrophages, but the role of CXCR3 in the initiation and promotion of corneal neovascularization remains unclear. Here, we showed that the expression of CXCL10 and CXCR3 was significantly increased in the cornea after alkali burn. Compared with WT mice, CXCR3-/- mice exhibited significantly increased corneal hemangiogenesis and lymphangiogenesis after alkali burn. In addition, exaggerated leukocyte infiltration and leukostasis, and elevated expression of inflammatory cytokines and angiogenic factor were also found in the corneas of CXCR3-/- mice subjected to alkali burn. With bone marrow (BM) transplantation, we further demonstrated that the deletion of CXCR3 in BM-derived leukocytes plays a key role in the acceleration of alkali burn-induced corneal neovascularization. Taken together, our results suggest that upregulation of CXCR3 does not exhibit its conventional action as a proinflammatory cytokine but instead serves as a self-protective mechanism for the modulation of inflammation and maintenance of corneal avascularity after corneal alkali burn.

Optical biosensing of markers of mucosal inflammation.

We report the design and adaptation of iron/iron oxide nanoparticle-based optical nanobiosensors for enzymes or cytokine/chemokines that are established biomarkers of lung diseases. These biomarkers comprise ADAM33, granzyme B, MMP-8, neutrophil elastase, arginase, chemokine (C-C motif) ligand 20 and interleukin-6. The synthesis of nanobiosensors for these seven biomarkers, their calibration with commercially available enzymes and cytokines/chemokines, as well as their validation using bronchoalveolar lavage (BAL) obtained from a mouse model of TLR3-mediated inflammation are discussed here. Exhaled Breath Condensate (EBC) is a minimally invasive approach for sampling airway fluid in the diagnosis and management of various lung diseases in humans (e.g., asthma, COPD and viral infections). We report the proof-of-concept of using human EBC in conjunction with nanobiosensors for diagnosis/monitoring airway inflammation. These findings suggest that, with nanosensor technology, human EBC can be utilized as a liquid biopsy to monitor inflammation/remodeling in lung disease.

Spatial-Temporal Speckle Variance in the En-Face View as a Contrast for Optical Coherence Tomography Angiography (OCTA).

Optical Coherence Tomography (OCT) is an adaptable depth-resolved imaging modality capable of creating a non-invasive 'digital biopsy' of the eye. One of the latest advances in OCT is optical coherence tomography angiography (OCTA), which uses the speckle variance or phase change in the signal to differentiate static tissue from blood flow. Unlike fluorescein angiography (FA), OCTA is contrast free and depth resolved. By combining high-density scan patterns and image processing algorithms, both morphometric and functional data can be extracted into a depth-resolved vascular map of the retina. The algorithm that we explored takes advantage of the temporal-spatial relationship of the speckle variance to improve the contrast of the vessels in the en-face OCT with a single frame. It also does not require the computationally inefficient decorrelation of multiple A-scans to detect vasculature, as used in conventional OCTA analysis. Furthermore, the spatial temporal OCTA (ST-OCTA) methodology tested offers the potential for post hoc analysis to improve the depth-resolved contrast of specific ocular structures, such as blood vessels, with the capability of using only a single frame for efficient screening of large sample volumes, and additional enhancement by processing with choice of frame averaging methods. Applications of this method in pre-clinical studies suggest that the OCTA algorithm and spatial temporal methodology reported here can be employed to investigate microvascularization and blood flow in the retina, and possibly other compartments of the eye.

Simvastatin-loaded liposome nanoparticles treatment for uterine leiomyoma in a patient-derived xenograft mouse model: a pilot study.

Uterine leiomyomas are complex tumours with limited medical treatment options. Simvastatin is used to treat hypercholesterolaemia and has shown promising effects as a treatment option for leiomyomas. Previously, our group demonstrated a promising effect of simvastatin treatment in a patient-derived xenograft mouse model. Here, we tested the efficacy of simvastatin liposomal nanoparticles (NPs). After bilateral leiomyoma xenograft implantation, mice (N = 12) were divided into three treatment arms: control, simvastatin and simvastatin-loaded liposome NPs (simvastatin-NPs). Treatment with simvastatin significantly reduced tumour volume and inhibited the Ki67 expression when compared to the control group. There was a trend of reduced tumour volume and Ki67 expression after treatment with simvastatin-NP; however, the results were not significant. Due to low bioavailability and short half-life of simvastatin, liposomal NPs have the potential to enhance drug delivery, however, in this study NP did not provide improvement over simvastatin, but did demonstrate their potential for the delivery of simvastatin.Impact statementWhat is already known on this subject? Simvastatin treatment in a patient-derived xenograft mouse model reduced tumour growth and decreased proliferation.What do the results of this study add? Treatment with simvastatin significantly reduced tumour volume and inhibited the Ki67 expression when compared to the control group. There was a trend of reduced tumour volume and Ki67 expression after treatment with simvastatin-NP, however, it did not improve the efficacy of simvastatin at reducing tumour growth and proliferation.What are the implications of these findings for clinical practice and/or further research? More studies are needed to optimise the formulation of NPs to further enhance the sustainable delivery of simvastatin.

Liver-Specific Nonviral Gene Delivery of Fibroblast Growth Factor 21 Protein Expression in Mice Regulates Body Mass and White/Brown Fat Respiration.

Viral-mediated in vivo gene delivery methods currently dominate among therapeutic strategies within the clinical and experimental settings, albeit with well documented limitations arising from immunologic constraints. In this study, we demonstrate the utility of nonviral hepatotropic in vivo gene delivery of unpackaged expression constructs, including one encoding fibroblast growth factor 21 (FGF21). FGF21 is an important hepatokine whose expression positively correlates with therapeutic outcomes across various animal models of obesity. Our data demonstrate that FGF21 expression can be restored into the livers of immunocompetent FGF21 knockout mice for at least 2 weeks after a single injection with an FGF21 expression plasmid. In wild-type C57BL6/J mice, in vivo transfection with an FGF21-expressing plasmid induced weight loss, decreased adiposity, and activated thermogenesis in white fat within 2 weeks. Furthermore, in vivo FGF21 gene delivery protected C57BL6/J mice against diet-induced obesity by decreasing adiposity and increasing uncoupling protein 1-dependent thermogenesis in brown fat and by boosting respiratory capacity in subcutaneous and perigonadal white fat. Together, the data illustrate a facile and effective methodology for delivering prolonged protein expression specifically to the liver. We contend that this method will find utility in basic science research as a practical means to enhance in vivo studies characterizing liver protein function. We further believe our data provide a rationale for further exploring the potential clinical utility of nonviral gene therapy in mouse models of disease. SIGNIFICANCE STATEMENT: This study presents a valuable method for nonviral gene delivery in mice that improves upon existing techniques. The data provide a rationale for further exploring the potential clinical utility of nonviral gene therapy in mouse models of disease and will likely enhance in vivo studies characterizing liver protein function.

Surrogate post-coital testing for contraceptive efficacy against human sperm activity in the ovine vaginal model†.

High unintended pregnancy rates are partially due to lack of effective nonhormonal contraceptives; development of safe, effective topical vaginal methods will address this need. Preclinical product safety and efficacy assessment requires in vivo testing in appropriate models. The sheep is a good model for the evaluation of vaginally delivered products due to its close similarities to humans. The study objective was to develop an ovine model for efficacy testing of female nonhormonal contraceptives that target human sperm. Fresh human semen was pooled from male volunteers. Nonpregnant female Merino sheep were treated with control or vaginal contraceptive product (IgG antibody with action against sperm or nonoxynol-9 [N9]). Pooled semen was added to the sheep vagina and mixed with product and vaginal secretions. Microscopic assessment of samples was performed immediately and progressive motility (PM) of sperm was compared between treatments. Cytokines CXCL8 and IL1B were assessed in vaginal fluid after instillation of human semen. No adverse reactions or elevations in proinflammatory cytokines occurred in response to human semen. N9 produced signs of acute cellular toxicity while there were no cellular changes after IgG treatment. N9 and IgG had dose-related effects with the highest dose achieving complete sperm immobilization (no sperm with PM). Surrogate post-coital testing of vaginally administered contraceptives that target human semen was developed in an ovine model established for vaginal product preclinical testing. This expanded model can aid the development of much needed nonhormonal topical vaginal contraceptives, providing opportunities for rapid iterative drug development prior to costly, time-intensive human testing.

Efficacy of Novel Highly Specific Bromodomain-Containing Protein 4 Inhibitors in Innate Inflammation-Driven Airway Remodeling.

NF-κB/RelA triggers innate inflammation by binding to bromodomain-containing protein 4 (BRD4), an atypical histone acetyltransferase (HAT). Although RelA·BRD4 HAT mediates acute neutrophilic inflammation, its role in chronic and functional airway remodeling is not known. We observed that BRD4 is required for Toll-like receptor 3 (TLR3)-mediated mesenchymal transition, a cell-state change that is characteristic of remodeling. We therefore tested two novel highly selective BRD4 inhibitors, ZL0420 and ZL0454, for their effects on chronic airway remodeling produced by repetitive TLR3 agonist challenges, and compared their efficacy with that of two nonselective bromodomain and extraterminal (BET) protein inhibitors, JQ1 and RVX208. We observed that ZL0420 and ZL0454 more potently reduced polyinosinic:polycytidylic acid-induced weight loss and fibrosis as assessed by microcomputed tomography and second harmonic generation microscopy. These measures correlated with the collagen deposition observed in histopathology. Importantly, the ZL inhibitors were more effective than the nonselective BET inhibitors at equivalent doses. The ZL inhibitors had significant effects on lung physiology, reversing TLR3-associated airway hyperresponsiveness and increasing lung compliance in vivo. At the molecular level, ZL inhibitors reduced elaboration of the transforming growth factor-ß-induced growth program, thereby preventing mucosal mesenchymal transition and disrupting BRD4 HAT activity and complex formation with RelA. We also observed that ZL0454 treatment blocked polyinosinic:polycytidylic acid-associated expansion of the α-SMA1+/COL1A+ myofibroblast population and prevented myofibroblast transition in a coculture system. We conclude that 1) BRD4 is a central effector of the mesenchymal transition that results in paracrine activation of myofibroblasts, mechanistically linking innate inflammation to airway hyperresponsiveness and fibrosis, and 2) highly selective BRD4 inhibitors may be effective in reversing the effects of repetitive airway viral infections on innate inflammation-mediated remodeling.

Long-Term Effect of Lactation on Maternal Cardiovascular Function and Adiposity in a Murine Model.

OBJECTIVE: Epidemiological studies suggest that lactation is associated with long-term maternal health benefits. To avoid confounders in human studies, we used a previously characterized murine model to investigate the long-term effect of lactation on both cardiovascular function and adiposity. STUDY DESIGN: After the delivery of the pups, CD-1 female mice were randomly divided into two groups: lactated and nonlactated (NL). Before pregnancy and at 9 months postdelivery, blood pressure was measured using a tail cuff, visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) were assessed by computed tomography (CT), echocardiography was performed using microultrasound, and cholesterol panels and fasting blood glucose were measured. The data were analyzed using Student's t-test (significance at p < 0.05). RESULTS: There were no differences in baseline parameters between the two groups. At 9 months postdelivery, the NL group weighed significantly more (p = 0.03) and demonstrated a significantly lower cardiac output (p = 0.05) and ejection fraction (p = 0.03). The mice in the NL group also had higher VAT (p < 0.01) and SAT percentiles (p = 0.03). Fasting glucose (p = 0.01) and low-density lipoprotein (p = 0.01) were significantly higher in the NL group at 9 months. CONCLUSION: Our results show the benefit of lactation is not just limited to the immediate postpartum period but it also extends into midlife in a murine model.

Critical Role of the CXCL10/C-X-C Chemokine Receptor 3 Axis in Promoting Leukocyte Recruitment and Neuronal Injury during Traumatic Optic Neuropathy Induced by Optic Nerve Crush.

Traumatic optic neuropathy (TON) is an acute injury of the optic nerve secondary to trauma. Loss of retinal ganglion cells (RGCs) is a key pathological process in TON, yet mechanisms responsible for RGC death remain unclear. In a mouse model of TON, real-time noninvasive imaging revealed a dramatic increase in leukocyte rolling and adhesion in veins near the optic nerve (ON) head at 9 hours after ON injury. Although RGC dysfunction and loss were not detected at 24 hours after injury, massive leukocyte infiltration was observed in the superficial retina. These cells were identified as T cells, microglia/monocytes, and neutrophils but not B cells. CXCL10 is a chemokine that recruits leukocytes after binding to its receptor C-X-C chemokine receptor (CXCR) 3. The levels of CXCL10 and CXCR3 were markedly elevated in TON, and up-regulation of CXCL10 was mediated by STAT1/3. Deleting CXCR3 in leukocytes significantly reduced leukocyte recruitment, and prevented RGC death at 7 days after ON injury. Treatment with CXCR3 antagonist attenuated TON-induced RGC dysfunction and cell loss. In vitro co-culture of primary RGCs with leukocytes resulted in increased RGC apoptosis, which was exaggerated in the presence of CXCL10. These results indicate that leukocyte recruitment in retinal vessels near the ON head is an early event in TON and the CXCL10/CXCR3 axis has a critical role in recruiting leukocytes and inducing RGC death.

NF-κB Mediates Mesenchymal Transition, Remodeling, and Pulmonary Fibrosis in Response to Chronic Inflammation by Viral RNA Patterns.

Airway remodeling is resultant of a complex multicellular response associated with a progressive decline of pulmonary function in patients with chronic airway disease. Here, repeated infections with respiratory viruses are linked with airway remodeling through largely unknown mechanisms. Although acute activation of the Toll-like receptor (TLR) 3 pathway by extracellular polyinosinic:polycytidylic acid (poly[I:C]) induces innate signaling through the NF-κB transcription factor in normal human small airway epithelial cells, prolonged (repetitive or tonic) poly(I:C) stimulation produces chronic stress fiber formation, mesenchymal transition, and activation of a fibrotic program. Chronic poly(I:C) stimulation enhanced the expression of core mesenchymal regulators Snail family zinc finger 1, zinc finger E-box binding homeobox, mesenchymal intermediate filaments (vimentin), and extracellular matrix proteins (fibronectin-1), and collagen 1A. This mesenchymal transition was prevented by silencing expression of NF-κB/RelA or administration of a small-molecule inhibitor of the IκB kinase, BMS345541. Acute poly(I:C) exposure in vivo induced profound neutrophilic airway inflammation. When administered repetitively, poly(I:C) resulted in enhanced fibrosis observed by lung micro-computed tomography, second harmonic generation microscopy of optically cleared lung tissue, and by immunohistochemistry. Epithelial flattening, expansion of the epithelial mesenchymal trophic unit, and enhanced Snail family zinc finger 1 and fibronectin 1 expression in airway epithelium were also observed. Repetitive poly(I:C)-induced airway remodeling, fibrosis, and epithelial-mesenchymal transition was inhibited by BMS345541 administration. Based on this novel model of viral inflammation-induced remodeling, we conclude that NF-κB is a major controller of epithelial-mesenchymal transition and pulmonary fibrosis, a finding that has potentially important relevance to airway remodeling produced by repetitive viral infections.

In Vivo Rectal Mucosal Barrier Function Imaging in a Large-Animal Model by Using Confocal Endomicroscopy: Implications for Injury Assessment and Use in HIV Prevention Studies.

Injury occurring on the surface of the rectal mucosal lining that causes defects in barrier function may result in increased risk for transmission of infection by HIV and other pathogens. Such injury could occur from microbicidal or other topical agents, mechanical trauma during consensual or nonconsensual intercourse, or inflammatory conditions. Tools for evaluation of rectal mucosal barrier function for assessing the mucosa under these conditions are lacking, particularly those that can provide in vivo structural and functional barrier integrity assessment and are adaptable to longitudinal imaging. We investigated confocal endomicroscopy (CE) as a means for in vivo imaging of the rectal epithelial barrier in the ovine model following spatially confined injury to the surface at a controlled site using a topical application of the microbicide test agent benzalkonium chloride. Topical and intravenous (i.v.) fluorescent probes were used with CE to provide subcellular resolution imaging of the mucosal surface and assessment of barrier function loss. A 3-point CE grading system based on cellular structure integrity and leakage of dye through the mucosa showed significant differences in score between untreated (1.19 ± 0.53) and treated (2.55 ± 0.75) tissue (P < 0.0001). Histological grading confirmed findings of barrier compromise. The results indicate that CE is an effective means for detecting epithelial injury and barrier loss following localized trauma in a large-animal model. CE is promising for real-time rectal mucosal evaluation after injury or trauma or topical application of emerging biomedical prevention strategies designed to combat HIV.

High-fructose diet in pregnancy leads to fetal programming of hypertension, insulin resistance, and obesity in adult offspring.

BACKGROUND: Consumption of fructose-rich diets in the United States is on the rise and thought to be associated with obesity and cardiometabolic diseases. OBJECTIVE: We sought to determine the effects of antenatal exposure to high-fructose diet on offspring's development of metabolic syndrome-like phenotype and other cardiovascular disease risk factors later in life. STUDY DESIGN: Pregnant C57BL/6J dams were randomly allocated to fructose solution (10% wt/vol, n = 10) or water (n = 10) as the only drinking fluid from day 1 of pregnancy until delivery. After weaning, pups were started on regular chow, and evaluated at 1 year of life. We measured percent visceral adipose tissue and liver fat infiltrates using computed tomography, and blood pressure using CODA nonivasive monitor. Intraperitoneal glucose tolerance testing with corresponding insulin concentrations were obtained. Serum concentrations of glucose, insulin, triglycerides, total cholesterol, leptin, and adiponectin were measured in duplicate using standardized assays. Fasting homeostatic model assessment was also calculated to assess insulin resistance. P values <.05 were considered statistically significant. RESULTS: Maternal weight, pup number, and average weight at birth were similar between the 2 groups. Male and female fructose group offspring had higher peak glucose and area under the intraperitoneal glucose tolerance testing curve compared with control, and higher mean arterial pressure compared to control. Female fructose group offspring were heavier and had higher percent visceral adipose tissue, liver fat infiltrates, homeostatic model assessment of insulin resistance scores, insulin area under the intraperitoneal glucose tolerance testing curve, and serum concentrations of leptin, and lower concentrations of adiponectin compared to female control offspring. No significant differences in these parameters were noted in male offspring. Serum concentrations of triglycerides or total cholesterol were not different between the 2 groups for either gender. CONCLUSION: Maternal intake of high fructose leads to fetal programming of adult obesity, hypertension, and metabolic dysfunction, all risk factors for cardiovascular disease. This fetal programming is more pronounced in female offspring. Limiting intake of high fructose-enriched diets in pregnancy may have significant impact on long-term health.

Topical injury evaluation of the murine colorectal mucosa using confocal endomicrosopy: a valuable method for assessing mucosal injuries associated with risk of pathogen transmission.

BACKGROUND: Concern regarding the effect of epithelial damage to the colorectal surface and possible impact on sexually transmitted infection transmission prompts the need for methods to evaluate the mucosal microscopic surface in preclinical studies examining such injury. This includes determining the effect of topical HIV prevention products on mucosal barrier integrity. In vivo imaging with high-resolution endomicroscopy could reveal defects in the mucosal barrier resulting from injury/surface trauma. METHODS: Confocal endomicroscopy was investigated to assess the ability to image surface injury resulting from topical application of a chemical used in lubricants and microbial products. Mice treated with a 50 µL rectal dose of 0.2% benzalkonium chloride solution, 1% benzalkonium chloride or phosphate-buffered saline control for 20 min were imaged in vivo using confocal endomicroscopy for assessment of epithelial disruption. Following imaging, mice were sacrificed and rectal tissue evaluated by histology. Confocal images were graded based on degree of disruption to crypt and epithelial microstructure. Histology was graded based on percent of epithelial disruption observed in stained sections. Confocal image features were confirmed by high-resolution two-photon microscopy. RESULTS: Based on quantitative grading of in vivo confocal endomicroscopy images, disruption at the microscopic scale was observed following treatment with benzalkonium chloride, with increased injury occurring with higher dose. Epithelial disruption at the lumen surface, evident between crypts and alteration in crypt structure on the luminal side were observed in confocal endomicroscopy and confirmed by histology. CONCLUSIONS: High-resolution imaging by confocal endomicroscopy can be used as a noninvasive tool for rapid visual assessment of rectal epithelial integrity following surface injury, potentially providing valuable indication of epithelial injury or trauma.