Claudin-4 is an unusual member of the claudin family; in addition to its role in epithelial tight... more Claudin-4 is an unusual member of the claudin family; in addition to its role in epithelial tight junction barrier function, it is a receptor for the Clostridium perfringens enterotoxin. We have also found that claudin-4 is regulated in mucosal epithelium M cells, both in increased expression of the protein and in redistribution into endocytosis vesicles. Our ongoing studies are studying the potential for developing ligands specific to claudin-4 for targeted delivery of cargo such as proteins and poly(DL-lactide-co-glycolide) nanoparticles to mucosal M cells. Methods for the study of claudin-4 movement within epithelial cells, and delivery of nanoparticles through targeted binding of claudin-4 are described.
Polymer-based microparticles are in clinical use mainly for their ability to provide controlled r... more Polymer-based microparticles are in clinical use mainly for their ability to provide controlled release of peptides and compounds, but they are also being explored for their potential to deliver vaccines and drugs as suspensions directly into mucosal sites. It is generally assumed that uptake is mediated by epithelial M cells, but this is often not directly measured. To study the potential for optimizing M cell uptake of polymer microparticles in vivo, we produced sub-micron size PLGA particles incorporating a recombinant protein. This recombinant protein was produced with or without a cterminal peptide previously shown to have high affinity binding to Claudin 4, a protein associated with M cell endocytosis. While the PLGA nanoparticles incorporate the protein throughout the matrix, much of the protein was also displayed on the surface, allowing us to take advantage of the binding activity of the targeting peptide. Accordingly, we found that instillation of these nanoparticles into the nasal passages or stomach of mice was found to significantly enhance their uptake by upper airway and intestinal M cells. Our results suggest that a reasonably simple nanoparticle manufacture method can provide insight into developing an effective needle-free delivery system.
In mucosal tissues, epithelial M cells capture and transport microbes across the barrier to under... more In mucosal tissues, epithelial M cells capture and transport microbes across the barrier to underlying immune cells. Previous studies suggested that high affinity ligands targeting M cells may be used to deliver mucosal vaccines; here, we show that particle composition and dispersion buffer ionic strength can independently influence their uptake in vivo. First, addition of a poloxamer 188 to nanoparticle formulations increased uptake of intranasally administered nanoparticles in vivo, but the effect was dependent on the presence of the M cell-targeting ligand. Second, solvent ionic strength is known to effect electrostatic interactions; accordingly, reduced ionic strength increased the electrostatic potential between the epithelium and the particles. Interestingly, below a critical ionic strength, intranasal particle uptake in vivo significantly was increased even when controlled for osmolarity. Similar results were obtained for uptake of bacterial particles. Surprisingly, at low ionic strength, the specific enhancement effect by the targeting peptide was negligible. Modeling of the electrostatic forces predicted that the enhancing effects of the M cell-targeting ligand only are enabled at high ionic strength, as particle electrostatic forces are reduced through Debye screening. Thus, electrostatic forces can have a dramatic effect on the in vivo M cell particle uptake independent of the action of targeting ligands. Examination of these forces will be helpful to optimizing mucosal vaccine and drug delivery. . 2 The abbreviations used are: M cells, microfold cells; plx, poloxamer 188;
Use of a turkey-breast phantom for developing freehand ultrasound (US)-guided core-needle biopsy ... more Use of a turkey-breast phantom for developing freehand ultrasound (US)-guided core-needle biopsy skills was evaluated. Thirteen diagnostic radiology trainees with varied experience in freehand US-guided breast core-needle biopsy were given instruction and allowed to practice the technique in a turkey-breast phantom. Three attempts were made before and after instruction and practice, and a questionnaire regarding experience, confidence, and anxiety was administered after these attempts. Technique, accuracy, and completion time were evaluated. Confidence related to procedure performance increased (P < .01), but the change in anxiety was not statistically significant. Accuracy improved, with the target being obtained in 87% of passes performed after instruction and practice versus 56% initially. Difficulty with visualizing the core needle sonographically during phantom biopsy decreased from 49% to 5% of attempts. Needle positioning perpendicular to the chest wall was observed initially in 38% of passes but was not observed after instruction and practice. There was no statistically significant change in time to complete biopsy. For teaching US-guided breast core-needle biopsy, use of a turkey-breast phantom helps improve technique, accuracy, and confidence of diagnostic radiology trainees.
Mucosal immune surveillance depends on M cells that reside in the epithelium overlying Peyer's pa... more Mucosal immune surveillance depends on M cells that reside in the epithelium overlying Peyer's patch and nasopharyngeal associated lymphoid tissue to transport particles to underlying lymphocytes. M cell development is associated with B lymphocytes in a basolateral pocket, but the interactions between these cells are poorly understood. In a cell culture model of M cell differentiation, we found lymphotoxin/tumor necrosis factor ␣ induction of CD137 (TNFRSF9) protein on intestinal epithelial cell lines, raising the possibility that CD137 on M cells in vivo might interact with CD137L expressed by B cells. Accordingly, while CD137-deficient mice produced UEA-1؉ M cell progenitors in nasopharyngeal associated lymphoid tissue and Peyer's patch epithelium, they showed an abnormal morphology, including the absence of basolateral B cell pockets. More important, CD137-deficient nasopharyngeal associated lymphoid tissue M cells were defective in microparticle transcytosis.
Mild traumatic brain injuries can lead to long-lasting cognitive and motor deficits, increasing t... more Mild traumatic brain injuries can lead to long-lasting cognitive and motor deficits, increasing the risk of future behavioral, neurological, and affective disorders. Our study focused on long-term behavioral deficits after repeated injury in which mice received either a single mild CHI (mCHI), a repeated mild CHI (rmCHI) consisting of one impact to each hemisphere separated by 3 days, or a moderate controlled cortical impact injury (CCI). Shams received only anesthesia. Behavioral tests were administered at 1, 3, 5, 7, and 90 days post-injury (dpi). CCI animals showed significant motor and sensory deficits in the early (1-7 dpi) and long-term (90 dpi) stages of testing. Interestingly, sensory and subtle motor deficits in rmCHI animals were found at 90 dpi. Most importantly, depression-like behaviors and social passiveness were observed in rmCHI animals at 90 dpi. These data suggest that mild concussive injuries lead to motor and sensory deficits and affective disorders that are not observed after moderate TBI.
Claudin-4 is an unusual member of the claudin family; in addition to its role in epithelial tight... more Claudin-4 is an unusual member of the claudin family; in addition to its role in epithelial tight junction barrier function, it is a receptor for the Clostridium perfringens enterotoxin. We have also found that claudin-4 is regulated in mucosal epithelium M cells, both in increased expression of the protein and in redistribution into endocytosis vesicles. Our ongoing studies are studying the potential for developing ligands specific to claudin-4 for targeted delivery of cargo such as proteins and poly(DL-lactide-co-glycolide) nanoparticles to mucosal M cells. Methods for the study of claudin-4 movement within epithelial cells, and delivery of nanoparticles through targeted binding of claudin-4 are described.
Polymer-based microparticles are in clinical use mainly for their ability to provide controlled r... more Polymer-based microparticles are in clinical use mainly for their ability to provide controlled release of peptides and compounds, but they are also being explored for their potential to deliver vaccines and drugs as suspensions directly into mucosal sites. It is generally assumed that uptake is mediated by epithelial M cells, but this is often not directly measured. To study the potential for optimizing M cell uptake of polymer microparticles in vivo, we produced sub-micron size PLGA particles incorporating a recombinant protein. This recombinant protein was produced with or without a cterminal peptide previously shown to have high affinity binding to Claudin 4, a protein associated with M cell endocytosis. While the PLGA nanoparticles incorporate the protein throughout the matrix, much of the protein was also displayed on the surface, allowing us to take advantage of the binding activity of the targeting peptide. Accordingly, we found that instillation of these nanoparticles into the nasal passages or stomach of mice was found to significantly enhance their uptake by upper airway and intestinal M cells. Our results suggest that a reasonably simple nanoparticle manufacture method can provide insight into developing an effective needle-free delivery system.
In mucosal tissues, epithelial M cells capture and transport microbes across the barrier to under... more In mucosal tissues, epithelial M cells capture and transport microbes across the barrier to underlying immune cells. Previous studies suggested that high affinity ligands targeting M cells may be used to deliver mucosal vaccines; here, we show that particle composition and dispersion buffer ionic strength can independently influence their uptake in vivo. First, addition of a poloxamer 188 to nanoparticle formulations increased uptake of intranasally administered nanoparticles in vivo, but the effect was dependent on the presence of the M cell-targeting ligand. Second, solvent ionic strength is known to effect electrostatic interactions; accordingly, reduced ionic strength increased the electrostatic potential between the epithelium and the particles. Interestingly, below a critical ionic strength, intranasal particle uptake in vivo significantly was increased even when controlled for osmolarity. Similar results were obtained for uptake of bacterial particles. Surprisingly, at low ionic strength, the specific enhancement effect by the targeting peptide was negligible. Modeling of the electrostatic forces predicted that the enhancing effects of the M cell-targeting ligand only are enabled at high ionic strength, as particle electrostatic forces are reduced through Debye screening. Thus, electrostatic forces can have a dramatic effect on the in vivo M cell particle uptake independent of the action of targeting ligands. Examination of these forces will be helpful to optimizing mucosal vaccine and drug delivery. . 2 The abbreviations used are: M cells, microfold cells; plx, poloxamer 188;
Use of a turkey-breast phantom for developing freehand ultrasound (US)-guided core-needle biopsy ... more Use of a turkey-breast phantom for developing freehand ultrasound (US)-guided core-needle biopsy skills was evaluated. Thirteen diagnostic radiology trainees with varied experience in freehand US-guided breast core-needle biopsy were given instruction and allowed to practice the technique in a turkey-breast phantom. Three attempts were made before and after instruction and practice, and a questionnaire regarding experience, confidence, and anxiety was administered after these attempts. Technique, accuracy, and completion time were evaluated. Confidence related to procedure performance increased (P < .01), but the change in anxiety was not statistically significant. Accuracy improved, with the target being obtained in 87% of passes performed after instruction and practice versus 56% initially. Difficulty with visualizing the core needle sonographically during phantom biopsy decreased from 49% to 5% of attempts. Needle positioning perpendicular to the chest wall was observed initially in 38% of passes but was not observed after instruction and practice. There was no statistically significant change in time to complete biopsy. For teaching US-guided breast core-needle biopsy, use of a turkey-breast phantom helps improve technique, accuracy, and confidence of diagnostic radiology trainees.
Mucosal immune surveillance depends on M cells that reside in the epithelium overlying Peyer's pa... more Mucosal immune surveillance depends on M cells that reside in the epithelium overlying Peyer's patch and nasopharyngeal associated lymphoid tissue to transport particles to underlying lymphocytes. M cell development is associated with B lymphocytes in a basolateral pocket, but the interactions between these cells are poorly understood. In a cell culture model of M cell differentiation, we found lymphotoxin/tumor necrosis factor ␣ induction of CD137 (TNFRSF9) protein on intestinal epithelial cell lines, raising the possibility that CD137 on M cells in vivo might interact with CD137L expressed by B cells. Accordingly, while CD137-deficient mice produced UEA-1؉ M cell progenitors in nasopharyngeal associated lymphoid tissue and Peyer's patch epithelium, they showed an abnormal morphology, including the absence of basolateral B cell pockets. More important, CD137-deficient nasopharyngeal associated lymphoid tissue M cells were defective in microparticle transcytosis.
Mild traumatic brain injuries can lead to long-lasting cognitive and motor deficits, increasing t... more Mild traumatic brain injuries can lead to long-lasting cognitive and motor deficits, increasing the risk of future behavioral, neurological, and affective disorders. Our study focused on long-term behavioral deficits after repeated injury in which mice received either a single mild CHI (mCHI), a repeated mild CHI (rmCHI) consisting of one impact to each hemisphere separated by 3 days, or a moderate controlled cortical impact injury (CCI). Shams received only anesthesia. Behavioral tests were administered at 1, 3, 5, 7, and 90 days post-injury (dpi). CCI animals showed significant motor and sensory deficits in the early (1-7 dpi) and long-term (90 dpi) stages of testing. Interestingly, sensory and subtle motor deficits in rmCHI animals were found at 90 dpi. Most importantly, depression-like behaviors and social passiveness were observed in rmCHI animals at 90 dpi. These data suggest that mild concussive injuries lead to motor and sensory deficits and affective disorders that are not observed after moderate TBI.
Uploads
Papers by Mary Hamer