Structured wound angiogenesis instructs mesenchymal barrier compartments in the regenerating nerve.

Organ injury stimulates the formation of new capillaries to restore blood supply raising questions about the potential contribution of neoangiogenic vessel architecture to the healing process. Using single-cell mapping, we resolved the properties of endothelial cells that organize a polarized scaffold at the repair site of lesioned peripheral nerves. Transient reactivation of an embryonic guidance program is required to orient neovessels across the wound. Manipulation of this structured angiogenic response through genetic and pharmacological targeting of Plexin-D1/VEGF pathways within an early window of repair has long-term impact on configuration of the nerve stroma. Neovessels direct nerve-resident mesenchymal cells to mold a provisionary fibrotic scar by assembling an orderly system of stable barrier compartments that channel regenerating nerve fibers and shield them from the persistently leaky vasculature. Thus, guided and balanced repair angiogenesis enables the construction of a "bridge" microenvironment conducive for axon regrowth and homeostasis of the regenerated tissue.

Simple Design for Membrane-Free Microphysiological Systems to Model the Blood-Tissue Barriers.

Microphysiological systems (MPS) incorporate physiologically relevant microanatomy, mechanics, and cells to mimic tissue function. Reproducible and standardized in vitro models of tissue barriers, such as the blood-tissue interface (BTI), are critical for next-generation MPS applications in research and industry. Many models of the BTI are limited by the need for semipermeable membranes, use of homogenous cell populations, or 2D culture. These factors limit the relevant endothelial-epithelial contact and 3D transport, which would best mimic the BTI. Current models are also difficult to assemble, requiring precise alignment and layering of components. The work reported herein details the engineering of a BTI-on-a-chip (BTI Chip) that addresses current disadvantages by demonstrating a single layer, membrane-free design. Laminar flow profiles, photocurable hydrogel scaffolds, and human cell lines were used to construct a BTI Chip that juxtaposes an endothelium in direct contact with a 3D engineered tissue. A biomaterial composite, gelatin methacryloyl and 8-arm polyethylene glycol thiol, was used for in situ fabrication of a tissue structure within a Y-shaped microfluidic device. To produce the BTI, a laminar flow profile was achieved by flowing a photocurable precursor solution alongside phosphate buffered saline. Immediately after stopping flow, the scaffold underwent polymerization through a rapid exposure to UV light (<300 mJ·cm-2). After scaffold formation, blood vessel endothelial cells were introduced and allowed to adhere directly to the 3D tissue scaffold, without barriers or phase guides. Fabrication of the BTI Chip was demonstrated in both an epithelial tissue model and blood-brain barrier (BBB) model. In the epithelial model, scaffolds were seeded with human dermal fibroblasts. For the BBB models, scaffolds were seeded with the immortalized glial cell line, SVGP12. The BTI Chip microanatomy was analyzed post facto by immunohistochemistry, showing the uniform production of a patent endothelium juxtaposed with a 3D engineered tissue. Fluorescent tracer molecules were used to characterize the permeability of the BTI Chip. The BTI Chips were challenged with an efflux pump inhibitor, cyclosporine A, to assess physiological function and endothelial cell activation. Operation of physiologically relevant BTI Chips and a novel means for high-throughput MPS generation was demonstrated, enabling future development for drug candidate screening and fundamental biological investigations.

Passing of Nanocarriers across the Histohematic Barriers: Current Approaches for Tumor Theranostics.

Over the past several decades, nanocarriers have demonstrated diagnostic and therapeutic (i.e., theranostic) potencies in translational oncology, and some agents have been further translated into clinical trials. However, the practical application of nanoparticle-based medicine in living organisms is limited by physiological barriers (blood-tissue barriers), which significantly hampers the transport of nanoparticles from the blood into the tumor tissue. This review focuses on several approaches that facilitate the translocation of nanoparticles across blood-tissue barriers (BTBs) to efficiently accumulate in the tumor. To overcome the challenge of BTBs, several methods have been proposed, including the functionalization of particle surfaces with cell-penetrating peptides (e.g., TAT, SynB1, penetratin, R8, RGD, angiopep-2), which increases the passing of particles across tissue barriers. Another promising strategy could be based either on the application of various chemical agents (e.g., efflux pump inhibitors, disruptors of tight junctions, etc.) or physical methods (e.g., magnetic field, electroporation, photoacoustic cavitation, etc.), which have been shown to further increase the permeability of barriers.

Development of a Novel Tool To Demystify Drug Distribution at Tissue-Blood Barriers.

Tissue endothelial cells express ABC-transporter enzymes that change the concentration of small molecules within different tissue compartments. These "blood-tissue barriers" have been shown to directly affect the efficacy and toxicity of anticancer, antimicrobial, psychiatric, and anti-epileptic drugs. Currently this phenomenon is best studied for the blood-brain barrier, but remains enigmatic for most other tissues. In addition, canonical pharmacokinetic theory specifically assumes an equal concentration of free drug within all tissue compartments. Inspired by Lipinski's "rule of 5," we here clarify current knowledge on drug-tissue distribution by: 1) curating the in-vivo literature on 73 drugs across 23 tissues and 2) developing two graphical web-based applications to visually describe and interpret data. These curated in-vivo dataset and visualization tools enabled us to achieve new insights into the logic of the barrier-tissue organization and showed remarkable correspondence to whole-body imaging of radiolabeled molecules.

Ultrathin and handleable nanofibrous net as a novel biomimetic basement membrane material for endothelial barrier formation.

Many strategies have been adopted to develop porous membranes to reconstitute basement membrane in vitro, which play a key role in the development of in vitro biomimetic models. However, the development of an artificial basement membrane combines cytocompatibility and nano-thickness is still challenging. Herein, a monolayer nanofibrous net patch was fabricated by combining microfabrication and electrospinning as a biomimetic basement membrane material, which was demonstrated for endothelial barrier formation. The nanofibrous net patches with different fiber densities were obtained by controlling electrospinning time. The net was with high porosity and ultrathin thickness approximate to the diameter of nanofibers, which is comparable to that of the native basement membrane. The morphology, proliferation and cell-cell/cell-substrate interactions of endothelial cells on the nanofibrous nets were studied and compared with track-etched polycarbonate membrane and traditional multilayer nanofibers membrane. In addition, the results of TEER measurement and permeability test demonstrated that the endothelial barrier formed on the nanofibrous net patch displayed stronger barrier integrity and function. Therefore, the proposed nanofibrous net patch shows great potential as a novel biomimetic basement membrane, which is promising to be applied for in vitro tissue mimetic applications.

Tissue Barrier-on-Chip: A Technology for Reproducible Practice in Drug Testing.

One key application of organ-on-chip systems is the examination of drug transport and absorption through native cell barriers such the blood-brain barrier. To overcome previous hurdles related to the transferability of existing static cell cultivation protocols and polydimethylsiloxane (PDMS) as the construction material, a chip platform with key innovations for practical use in drug-permeation testing is presented. First, the design allows for the transfer of barrier-forming tissue into the microfluidic system after cells have been seeded on porous polymer or Si3N4 membranes. From this, we can follow highly reproducible models and cultivation protocols established for static drug testing, from coating the membrane to seeding the cells and cell analysis. Second, the perfusion system is a microscopable glass chip with two fluid compartments with transparent embedded electrodes separated by the membrane. The reversible closure in a clamping adapter requires only a very thin PDMS sealing with negligible liquid contact, thereby eliminating well-known disadvantages of PDMS, such as its limited usability in the quantitative measurements of hydrophobic drug molecule concentrations. Equipped with tissue transfer capabilities, perfusion chamber inertness and air bubble trapping, and supplemented with automated fluid control, the presented system is a promising platform for studying established in vitro models of tissue barriers under reproducible microfluidic perfusion conditions.

Engineering Tissue Barrier Models on Hydrogel Microfluidic Platforms.

Tissue barriers play a crucial role in human physiology by establishing tissue compartmentalization and regulating organ homeostasis. At the interface between the extracellular matrix (ECM) and flowing fluids, epithelial and endothelial barriers are responsible for solute and gas exchange. In the past decade, microfluidic technologies and organ-on-chip devices became popular as in vitro models able to recapitulate these biological barriers. However, in conventional microfluidic devices, cell barriers are primarily grown on hard polymeric membranes within polydimethylsiloxane (PDMS) channels that do not mimic the cell-ECM interactions nor allow the incorporation of other cellular compartments such as stromal tissue or vascular structures. To develop models that accurately account for the different cellular and acellular compartments of tissue barriers, researchers have integrated hydrogels into microfluidic setups for tissue barrier-on-chips, either as cell substrates inside the chip, or as self-contained devices. These biomaterials provide the soft mechanical properties of tissue barriers and allow the embedding of stromal cells. Combining hydrogels with microfluidics technology provides unique opportunities to better recreate in vitro the tissue barrier models including the cellular components and the functionality of the in vivo tissues. Such platforms have the potential of greatly improving the predictive capacities of the in vitro systems in applications such as drug development, or disease modeling. Nevertheless, their development is not without challenges in their microfabrication. In this review, we will discuss the recent advances driving the fabrication of hydrogel microfluidic platforms and their applications in multiple tissue barrier models.

Drosophila melanogaster as a model for arbovirus infection of adult salivary glands.

Arboviruses are an emerging threat to public health. Arbovirus transmission to vertebrates hinges on dissemination from the arthropod gastrointestinal tract, and ultimately infection of the arthropod salivary glands. Therefore, salivary gland immunity impacts arbovirus transmission; however, these immune responses are poorly understood. Here, we describe the utility of Drosophila melanogaster as a salivary gland infection model. First, we describe the use of a salivary gland-specific driver to launch RNA interference or virus replicon transgenes. Next, we infect flies with an arbovirus panel and find multiple viruses that infect Drosophila salivary glands, albeit inefficiently. We find that this infection is not controlled by antiviral RNA silencing; thus, we silence a panel of immune genes in the salivary glands, but do not observe changes in infection. These data suggest that Drosophila may be used to study salivary gland infection, and that there are likely unexplored pathways controlling infection of this tissue.

Giardia spp. and the Gut Microbiota: Dangerous Liaisons.

Alteration of the intestinal microbiome by enteropathogens is commonly associated with gastrointestinal diseases and disorders and has far-reaching consequences for overall health. Significant advances have been made in understanding the role of microbial dysbiosis during intestinal infections, including infection with the protozoan parasite Giardia duodenalis, one of the most prevalent gut protozoa. Altered species composition and diversity, functional changes in the commensal microbiota, and changes to intestinal bacterial biofilm structure have all been demonstrated during the course of Giardia infection and have been implicated in Giardia pathogenesis. Conversely, the gut microbiota has been found to regulate parasite colonization and establishment and plays a critical role in immune modulation during mono and polymicrobial infections. These disruptions to the commensal microbiome may contribute to a number of acute, chronic, and post-infectious clinical manifestations of giardiasis and may account for variations in disease presentation within and between infected populations. This review discusses recent advances in characterizing Giardia-induced bacterial dysbiosis in the gut and the roles of dysbiosis in Giardia pathogenesis.

Parabronchial remodeling in chicks in response to embryonic hypoxia.

The embryonic development of parabronchi occurs mainly during the second half of incubation in precocious birds, which makes this phase sensitive to possible morphological modifications induced by O2 supply limitation. Thus, we hypothesized that hypoxia during the embryonic phase of parabronchial development induces morphological changes that remain after hatching. To test this hypothesis, chicken embryos were incubated entirely (21 days) under normoxia or partially under hypoxia (15% O2 during days 12 to 18). Lung structures, including air capillaries, blood capillaries, infundibula, atria, parabronchial lumen, bronchi, blood vessels larger than capillaries and interparabronchial tissue, in 1- and 10-day-old chicks were analyzed using light microscopy-assisted stereology. Tissue barrier and surface area of air capillaries were measured using electron microscopy-assisted stereology, allowing for calculation of the anatomical diffusion factor. Hypoxia increased the relative volumes of air and blood capillaries, structures directly involved in gas exchange, but decreased the relative volumes of atria in both groups of chicks, and the parabronchial lumen in older chicks. Accordingly, the surface area of the air capillaries and the anatomical diffusion factor were increased under hypoxic incubation. Treatment did not alter total lung volume, relative volumes of infundibula, bronchi, blood vessels larger than capillaries, interparabronchial tissue or the tissue barrier of any group. We conclude that hypoxia during the embryonic phase of parabronchial development leads to a morphological remodeling, characterized by increased volume density and respiratory surface area of structures involved in gas exchange at the expense of structures responsible for air conduction in chicks up to 10 days old.

Caprate Modulates Intestinal Barrier Function in Porcine Peyer's Patch Follicle-Associated Epithelium.

BACKGROUND: Many food components influence intestinal epithelial barrier properties and might therefore also affect susceptibility to the development of food allergies. Such allergies are triggered by increased antibody production initiated in Peyer's patches (PP). Usually, the presentation of antigens in the lumen of the gut to the immune cells of the PP is strongly regulated by the follicle-associated epithelium (FAE) that covers the PP. As the food component caprate has been shown to impede barrier properties in villous epithelium, we hypothesized that caprate also affects the barrier function of the PP FAE, thereby possibly contributing a risk factor for the development of food allergies. METHODS: In this study, we have focused on the effects of caprate on the barrier function of PP, employing in vitro and ex vivo experimental setups to investigate functional and molecular barrier properties. Incubation with caprate induced an increase of transepithelial resistance, and a marked increase of permeability for the paracellular marker fluorescein in porcine PP to 180% of control values. These effects are in accordance with changes in the expression levels of the barrier-forming tight junction proteins tricellulin and claudin-5. CONCLUSIONS: This barrier-affecting mechanism could be involved in the initial steps of a food allergy, since it might trigger unregulated contact of the gut lumen with antigens.

Xenopus oocytes as a heterologous expression system for analysis of tight junction proteins.

Claudins (cldns) represent the largest family of transmembrane tight junction (TJ) proteins, determining organ-specific epithelial barrier properties. Because methods for the analysis of multiple cldn interaction are limited, we have established the heterologous Xenopus laevis oocyte expression system for TJ protein assembly and interaction analysis. Oocytes were injected with cRNA encoding human cldn-1, -2, or -3 or with a combination of these and were incubated in pairs for interaction analysis. Immunoblotting and immunohistochemistry were performed, and membrane contact areas were analyzed morphometrically and by freeze fracture electron microscopy. Cldns were specifically detected in membranes of expressing oocytes, and coincubation of oocytes resulted in adhesive contact areas that increased with incubation time. Adjacent membrane areas revealed specific cldn signals, including "kissing-point"-like structures representing homophilic trans-interactions of cldns. Contact areas of oocytes expressing a combination markedly exceeded those expressing single cldns, indicating effects on adhesion. Ultrastructural analysis revealed a self-assembly of TJ strands and a cldn-specific strand morphology.-Vitzthum, C., Stein, L., Brunner, N., Knittel, R., Fallier-Becker, P., Amasheh, S. Xenopus oocytes as a heterologous expression system for analysis of tight junction proteins.

Chlorophyllin Modulates Gut Microbiota and Inhibits Intestinal Inflammation to Ameliorate Hepatic Fibrosis in Mice.

Liver fibrosis is an abnormal wound healing response and a common consequence of chronic liver diseases from infection or alcohol/xenobiotic exposure. At the cellular level, liver fibrosis is mediated by trans-differentiation of hepatic stellate cells (HSCs), which is driven by persistent hepatic and systemic inflammation. However, impaired enterohepatic circulation and gut dysbiosis may indirectly contribute to the liver fibrogenesis. The composition of the gut microbiota depends on diet composition and host factors. In this study, we examined chlorophyllin, derived from green pigment chlorophyll, on gut microbiota, the intestinal mucosal barrier, and liver fibrosis. BALB/c mice received carbon tetrachloride through intraperitoneal injection to induce liver fibrosis and chlorophyllin was administrated in drinking water. The effects of chlorophyllin on liver fibrosis were evaluated for (1) survival rate, (2) hepatic morphologic analysis, (3) inflammatory factors in both the small intestine and liver, and (4) gut microbiota. Our results indicate that oral administration of chlorophyllin could attenuate intestinal and hepatic inflammation and ameliorate liver fibrosis. Importantly, oral administration of chlorophyllin promptly rebalanced the gut microbiota, exhibiting down-regulation of the phylum Firmicutes and up-regulation of the phylum Bacteroidetes. In vitro experiments on intestinal epithelial cells showed that chlorophyllin exposure could inhibit NF-κB pathway via IKK-phosphorylation suppression. In conclusion, this study demonstrates potential application of chlorophyllin to regulate the intestinal microbiota and ameliorate hepatic fibrosis.

Micro-/nano-topography of selective laser melting titanium enhances adhesion and proliferation and regulates adhesion-related gene expressions of human gingival fibroblasts and human gingival epithelial cells.

BACKGROUND: Selective laser melting (SLM) titanium is an ideal option to manufacture customized implants with suitable surface modification to improve its bioactivity. The peri-implant soft tissues form a protective tissue barrier for the underlying osseointegration. Therefore, original microrough SLM surfaces should be treated for favorable attachment of surrounding soft tissues. MATERIAL AND METHODS: In this study, anodic oxidation (AO) was applied on the microrough SLM titanium substrate to form TiO2 nanotube arrays. After that, calcium phosphate (CaP) nanoparticles were embedded into the nanotubes or the interval of nanotubes by electrochemical deposition (AOC). These two samples were compared to untreated (SLM) samples and accepted mechanically polished (MP) SLM titanium samples. Scanning electron microscopy, energy dispersive spectrometry, X-ray diffraction, surface roughness, and water contact angle measurements were used for surface characterization. The primary human gingival epithelial cells (HGECs) and human gingival fibroblasts (HGFs) were cultured for cell assays to determine adhesion, proliferation, and adhesion-related gene expressions. RESULTS: For HGECs, AOC samples showed significantly higher adhesion, proliferation, and adhesion-related gene expressions than AO and SLM samples (P<0.05) and similar exceptional ability in above aspects to MP samples. At the same time, AOC samples showed the highest adhesion, proliferation, and adhesion-related gene expressions for HGFs (P<0.05). CONCLUSION: By comparison between each sample, we could confirm that both anodic oxidation and CaP nanoparticles had improved bioactivity, and their combined utilization may likely be superior to mechanical polishing, which is most commonly used and widely accepted. Our results indicated that creating appropriate micro-/nano-topographies can be an effective method to affect cell behavior and increase the stability of the peri-implant mucosal barrier on SLM titanium surfaces, which contributes to its application in dental and other biomedical implants.

Copper-modified Ti6Al4V alloy fabricated by selective laser melting with pro-angiogenic and anti-inflammatory properties for potential guided bone regeneration applications.

Custom-made biocompatible titanium alloy mesh can be designed to facilitate the regeneration of alveolar bone defects by supporting a protected space and inhibiting bacterial infections. Copper ions are often incorporated into titanium alloy due to their high bioactivity and outstanding antibacterial properties. However, the impacts of copper-bearing alloys on peri-implanted cell behaviors have rarely been systematically explored. In the present study, a copper-bearing alloy (Ti6Al4V-6Cu) was fabricated by selective laser melting (SLM) technology. The characterization of Ti6Al4V-6Cu alloy and its effects on the behaviors of gingival fibroblasts (HGFs), human umbilical vein endothelial cells (HUVECs), osteoblasts and macrophages were evaluated and compared with Ti6Al4V. The diffraction peaks of the Ti2Cu intermetallic phase were observed in the Ti6Al4V-6Cu alloy. Adding Cu enhanced the release of Ti and Al ions. The chemical state of Cu in the Ti6Al4V-6Cu alloy may exist predominantly in Cu2O or TiCuOx. Ti6Al4V-6Cu did not affect the attachment of HGFs or the osteogenic activity of osteoblasts. Furthermore, it inhibited the activation, proliferation, and pro-inflammatory cytokine secretion of macrophages and upregulated angiogenesis-related gene expression and VEGF-A secretion of HUVECs. These results demonstrate that a Ti6Al4V-6Cu alloy was successfully fabricated that did not negatively impact the cell viability of gingival fibroblasts and osteoblasts, inhibited the inflammatory response of macrophages, and increased the angiogenesis of HUVECs. Thus, Ti6Al4V-6Cu has potential applications for the fabrication of titanium alloy mesh to promote alveolar bone regeneration.

Biological response of an in vitro human 3D lung cell model exposed to brake wear debris varies based on brake pad formulation.

Wear particles from automotive friction brake pads of various sizes, morphology, and chemical composition are significant contributors towards particulate matter. Knowledge concerning the potential adverse effects following inhalation exposure to brake wear debris is limited. Our aim was, therefore, to generate brake wear particles released from commercial low-metallic and non-asbestos organic automotive brake pads used in mid-size passenger cars by a full-scale brake dynamometer with an environmental chamber simulating urban driving and to deduce their potential hazard in vitro. The collected fractions were analysed using scanning electron microscopy via energy-dispersive X-ray spectroscopy (SEM-EDS) and Raman microspectroscopy. The biological impact of the samples was investigated using a human 3D multicellular model consisting of human epithelial cells (A549) and human primary immune cells (macrophages and dendritic cells) mimicking the human epithelial tissue barrier. The viability, morphology, oxidative stress, and (pro-)inflammatory response of the cells were assessed following 24 h exposure to ~ 12, ~ 24, and ~ 48 µg/cm2 of non-airborne samples and to ~ 3.7 µg/cm2 of different brake wear size fractions (2-4, 1-2, and 0.25-1 µm) applying a pseudo-air-liquid interface approach. Brake wear debris with low-metallic formula does not induce any adverse biological effects to the in vitro lung multicellular model. Brake wear particles from non-asbestos organic formulated pads, however, induced increased (pro-)inflammatory mediator release from the same in vitro system. The latter finding can be attributed to the different particle compositions, specifically the presence of anatase.

Expression and significance of interleukin-1β and claudin-5 in lupus mice renal tissue / 中华风湿病学杂志

Objective To study the expression difference and the meaning of interleukin (IL)-1β and Claudin-5 in the kidney and kidney's membrane between lupus mice and the control mice. Methods Gene expression difference of IL-1βand claudin-5 between lupus mice and control mice in their kidney and kidney's membrane was detected with quantitative polymerase chain reaction (Q-PCR). The location of the expression was identified by immunohistochemistry. One-way analysis of variance (ANOVA) was used to compare the means of each group, pair-wise comparison was used to compare the difference between multiple sample means. LSD method was used when the variance was equal, and Tamhane's T2 method was used when the variance was different. Results Q-PCR test results showed that IL-1β expression in lupus mice's kidney membrane (0.0095±0.0052) was statistically higher than lupus mice's kidney parenchyma (0.0057±0.0013) (t=2.137, P=0.0458) and control mice's kidney membrane (0.0045±0.0033) (t=2.709, P=0.0131), however, there's no statistical significant difference between control mice's kidney membrane and parenchyma (0.0065± 0.0011) (P>0.05), and there's no statistical difference between control and lupus mice's kidney parenchyma (P>0.05). Claudin-5 expression was statistically higher in control mice kidney membrane (0.0192 ±0.0048) than its kidney parenchyma (0.01156 ±0.002190) (t=4.009, P=0.0015) but statistically lower in lupus mice kidney membrane (0.0069±0.0004) than its kidney parenchyma (0.0098±0.0027) (t=2.727, P=0.0173);there's no statistical significant difference between control mice's kidney parenchyma and lupus mice's kidney parenchyma (P>0.05), and lupus mice's kidney membrane expression was statistically lower than control mice's kidney membrane (t=6.018, P=0.0001). Immun-ohistochemistry showed that IL-1β expression was mainly around glomerulus and membrane, but not renal tu-bule. Claudin-5 expression was mainly around glomerulus and membrane. Conclusion Immune inflammation induced by IL-1β has mainly shown in blood vessels, while claudin-5 has protective effect on lupus immune inflammation.

Building barriers.

Formation of tissue barriers starts in early development where it is critical for normal cell fate selection, differentiation and organogenesis. Barrier maintenance is critical to the ongoing function of organs during adulthood and aging. Dysfunctional tissue barrier formation and function at any stage of the organismal life cycle underlies many disease states.

Molecular Characterization of Barrier Properties in Follicle-Associated Epithelium of Porcine Peyer's Patches Reveals Major Sealing Function of Claudin-4.

The pig represents a preferred model for the analysis of intestinal immunology. However, the barrier of the follicle-associated epithelium (FAE) covering porcine Peyer's patches (PP) has not yet been characterized in detail. This study aimed to perform this characterization in order to pave the way toward an understanding of the functional contribution of epithelial barrier properties in gut immunology. Porcine tissue specimens were taken from the distal small intestine in order to obtain electrophysiological data of PP FAE and neighboring villous epithelium (VE), employing the Ussing chamber technique. Transepithelial resistance (TER) and paracellular fluorescein flux were measured, and tissues were morphometrically compared. In selfsame tissues, expression and localization of major tight junction (TJ) proteins (claudin-1, -2, -3, -4, -5, and -8) were analyzed. PP FAE specimens showed a higher TER and a lower apparent permeability for sodium fluorescein than VE. Immunoblotting revealed an expression of all claudins within both epithelia, with markedly stronger expression of the sealing TJ protein claudin-4 in PP FAE compared with the neighboring VE. Immunohistochemistry confirmed the expression and localization of all claudins in both PP FAE and VE, with stronger claudin-4 abundance in PP FAE. The results are in accordance with the physiological function of the FAE, which strongly regulates and limits antigen uptake determining a mandatory transcellular route for antigen presentation, highlighting the importance of this structure for the first steps of the intestinal immune response. Thus, this study provides detailed insights into the specific barrier properties of the porcine FAE covering intestinal PP, at the interface of intestinal immunology and barriology.

Biodegradable brain-penetrating DNA nanocomplexes and their use to treat malignant brain tumors.

The discovery of powerful genetic targets has spurred clinical development of gene therapy approaches to treat patients with malignant brain tumors. However, lack of success in the clinic has been attributed to the inability of conventional gene vectors to achieve gene transfer throughout highly disseminated primary brain tumors. Here, we demonstrate ex vivo that small nanocomplexes composed of DNA condensed by a blend of biodegradable polymer, poly(ß-amino ester) (PBAE), with PBAE conjugated with 5kDa polyethylene glycol (PEG) molecules (PBAE-PEG) rapidly penetrate healthy brain parenchyma and orthotopic brain tumor tissues in rats. Rapid diffusion of these DNA-loaded nanocomplexes observed in fresh tissues ex vivo demonstrated that they avoided adhesive trapping in the brain owing to their dense PEG coating, which was critical to achieving widespread transgene expression throughout orthotopic rat brain tumors in vivo following administration by convection enhanced delivery. Transgene expression with the PBAE/PBAE-PEG blended nanocomplexes (DNA-loaded brain-penetrating nanocomplexes, or DNA-BPN) was uniform throughout the tumor core compared to nanocomplexes composed of DNA with PBAE only (DNA-loaded conventional nanocomplexes, or DNA-CN), and transgene expression reached beyond the tumor edge, where infiltrative cancer cells are found, only for the DNA-BPN formulation. Finally, DNA-BPN loaded with anti-cancer plasmid DNA provided significantly enhanced survival compared to the same plasmid DNA loaded in DNA-CN in two aggressive orthotopic brain tumor models in rats. These findings underscore the importance of achieving widespread delivery of therapeutic nucleic acids within brain tumors and provide a promising new delivery platform for localized gene therapy in the brain.