Roland L. Myers

A novel model for brain iron uptake: introducing the concept of regulation

Neurologic disorders such as Alzheimers, Parkinsons disease, and Restless Legs Syndrome involve a loss of brain iron homeostasis. Moreover, iron deficiency is the most prevalent nutritional concern worldwide with many associated cognitive and neural ramifications. Therefore, understanding the mechanisms by which iron enters the brain and how those processes are regulated addresses significant global health issues. The existing paradigm assumes that the endothelial cells (ECs) forming the blood—brain barrier (BBB) serve as a simple conduit for transport of transferrin-bound iron. This concept is a significant oversimplification, at minimum failing to account for the iron needs of the ECs. Using an in vivo model of brain iron deficiency, the Belgrade rat, we show the distribution of transferrin receptors in brain microvasculature is altered in luminal, intracellular, and abluminal membranes dependent on brain iron status. We used a cell culture model of the BBB to show the presence of factors that influence iron release in non-human primate cerebrospinal fluid and conditioned media from astrocytes; specifically apo-transferrin and hepcidin were found to increase and decrease iron release, respectively. These data have been integrated into an interactive model where BBB ECs are central in the regulation of cerebral iron metabolism.

GLUT-1 GLUCOSE TRANSPORTERS IN THE BLOOD-BRAIN BARRIER: DIFFERENTIAL PHOSPHORYLATION

Glucose is the primary metabolic fuel for the mammalian brain, and a continuous supply is required to maintain normal CNS function. The transport of glucose across the blood–brain barrier (BBB) into the brain is mediated by the facilitative glucose transporter GLUT‐1. Prior studies (Simpson et al. [2001] J Biol Chem 276:12725–12729) had revealed that the conformations of the GLUT‐1 transporter were different in luminal (blood facing) and abluminal (brain facing) membranes of bovine cerebral endothelial cells, based on differential antibody recognition. This study has extended these observations and, by using a combination of 2D‐PAGE/Western blotting and immunogold electron microscopy, determined that these different conformations are exhibited in vivo and arise from differential phosphorylation of GLUT‐1 and not from alternative splicing or altered O‐ or N‐linked glycosylation.

Naltrexone accelerates healing without compromise of adhesion complexes in normal and diabetic corneal epithelium.

Naltrexone (NTX) is an opioid antagonist that accelerates wound healing of corneal epithelium in normal and diabetic animals. Junctional complexes (hemidesmosomes) are important in establishing adhesion of the corneal epithelium to the stroma. This study was designed to examine whether NTX, at a concentration that enhances corneal re-epithelialization, influences the appearance and number of hemidesmosomes in Normal, diabetic (DB) (hyperglycemic), and DB animals receiving insulin (DB-IN) (normoglycemic), and treated topically with NTX (10(-4) M) or sterile vehicle (SV) for 7 days following abrasion. Electron microscopic analysis of the peripheral cornea 2 weeks after removal of the epithelium indicated hemidesmosomes that could be classified into four sectional profiles. No differences were detected in either the structure or the number of junctional complexes in the cornea between Normal, DB, or DB-IN groups receiving vehicle or treated with NTX. Moreover, the fine structure of the basal and suprabasal layers of the corneal epithelium in all groups--including those treated with NTX--were comparable. These results indicate that topical application of NTX accelerates diabetic corneal epithelial healing without causing morphologic abnormalities in the reassembly of adhesion structures. Furthermore, controlled and uncontrolled diabetes for up to 3 months does not affect corneal adhesion complexes when compared to normal corneas. Thus, recurrent erosion following abrasion of the diabetic cornea, with preservation of the basal lamina, cannot be explained by structural abnormalities in the reformation of the epithelial adhesion complex.

Dynamic changes appearing in collagen fibers during intrinsic tendon repair.

Intrinsic healing of severed tendons shows a delay in a gain in breaking strength and the tendon becomes translucent. The cause of tendon translucence was investigated in suture-repaired rat Achilles tendon. The repair site with adjacent translucent tendon were evaluated histologically on day 10 by immunofluorescence and transmission electron microscopy. The healing tendon translucent region by hematoxylin–eosin staining had few inflammatory cells, polarized light birefringence showed thinner collagen fibers, immunofluorescence showed few myofibroblasts, and transmission electron microscopy revealed frayed, irregular thin collagen fibers. During embryogenesis, tendon fibers grow by the addition of discreet collagen fibril segment structures. The speculation is that collagen fibril segment structures are released from collagen fibers within the translucent tendon region for reuse during the regeneration of tendon collagen fibers during intrinsic tendon repair. Healing tendon translucence is related to a decrease in the diameter of collagen fibers by the release of collagen fibril segments within tendon bundles/fascicles.

Vanadate ingestion increases the gain in wound breaking strength and leads to better organized collagen fibers in rats during healing.

Repair of incision wounds closed by suturing is evaluated by the progressive gain in wound breaking strength. Previously the closure of open wounds in rats ingesting vanadate, an inhibitor of tyrosine phosphate phosphatases, was shown to occur with deposition of more uniformly organized collagen fiber bundles. The hypothesis of this study was that deposition of more uniformly organized collagen fibers would enhance the gain in wound breaking strength of incisional wounds. Six adult rats received vanadate-supplemented saline drinking water for 1 week before placement of two 6-cm, parallel, suture-closed wounds on their backs. Six control rats received identical wounds and were given saline drinking water. The drinking water regimen was continued for 1 week after wounding, and then wound strength was tested with a tensiometer and tissue samples were obtained for histologic evaluation. Wound breaking strength doubled in vanadate-treated rats compared with controls. Bright-field and polarized light microscopy showed that the connective tissue matrix of granulation tissue from control rats was oriented perpendicular to the surface of the skin. In contrast, the connective tissue matrix of granulation tissue from vanadate-treated rats was oriented parallel to the skin surface. The gap in granulation tissue between the edges of the wounds in the vanadate-treated rats was greater than that in controls. Electron microscopy showed that wounds in the vanadate-treated contained uniform collagen fibers that were 20 percent greater in diameter and more evenly spaced than they were in controls. It is proposed that these changes in the organization of collagen fibers within incisional wounds were responsible for the increased wound breaking strength observed in rats ingesting vanadate.

Topical vanadate optimizes collagen organization within granulation tissue

Systemic ingestion of vanadate, a nonspecific inhibitor of tyrosine phosphatases, doubles wound breaking strength, enhances the packing of collagen fibers, and prevents the appearance of myofibroblasts in granulation tissue. Will the local application of vanadate mimic the systemic effects? Pairs of polyvinyl alcohol sponges, each with a central reservoir and attached injection port, were subcutaneously implanted in rats. Daily, one implant received 0.2 ml of saline and the other received 0.2 ml of 0.03 mM vanadate in saline. On day 7, harvested sponges had equivalent wet weights. The vanadate‐treated sponges had fibroblasts separated by connective tissue, with a more intense birefringence of the collagen fibers. Transmission electron microscopy showed collagen more uniformly packed in the vanadate treated sponges where collagen fibers were equally spaced and had equal diameters. By immunohistology, myofibroblasts, defined by the expression of α‐smooth muscle actin within stress fibers, were absent in vanadate‐treated granulation tissue. The expression of α‐smooth muscle actin was restricted to smooth muscle cells of blood vessels. Controls had densely packed α‐smooth muscle actin staining myofibroblasts, weak birefringence, and randomly spaced collagen fibers with irregular diameters. We conclude that the local application of vanadate prevents the appearance of myofibroblasts and optimizes the organization of collagen fibers in developing granulation tissue. (WOUND REP REG 2003;11:204–212)

Mouse papillomavirus infections spread to cutaneous sites with progression to malignancy

We report secondary cutaneous infections in the mouse papillomavirus (MmuPV1)/mouse model. Our previous study demonstrated that cutaneous MmuPV1 infection could spread to mucosal sites. Recently, we observed that mucosal infections could also spread to various cutaneous sites including the back, tail, muzzle and mammary tissues. The secondary site lesions were positive for viral DNA, viral capsid protein and viral particles as determined by in situ hybridization, immunohistochemistry and transmission electron microscopy analyses, respectively. We also demonstrated differential viral production and tumour growth at different secondarily infected skin sites. For example, fewer viral particles were detected in the least susceptible back tissues when compared with those in the infected muzzle and tail, although similar amounts of viral DNA were detected. Follow-up studies demonstrated that significantly lower amounts of viral DNA were packaged in the back lesions. Lavages harvested from the oral cavity and lower genital tracts were equally infectious at both cutaneous and mucosal sites, supporting the broad tissue tropism of this papillomavirus. Importantly, two secondary skin lesions on the forearms of two mice displayed a malignant phenotype at about 9.5 months post-primary infection. Therefore, MmuPV1 induces not only dysplasia at mucosal sites such as the vagina, anus and oral cavity but also skin carcinoma at cutaneous sites. These findings demonstrate that MmuPV1 mucosal infection can be spread to cutaneous sites and suggest that the model could serve a useful role in the study of the viral life cycle and pathogenesis of papillomavirus.

Induction and Purification of C. difficile Phage Tail-Like Particles

Due to the inherent limitations of conventional antibiotics for the treatment of C. difficile infection (CDI), there is a growing interest in the development of alternative treatment strategies. Both bacteriophages and R-type bacteriocins, also known as phage tail-like particles (PTLPs), show promise as potential antibacterial alternatives for treating CDI. Similar to bacteriophages, but lacking a viral capsid and genome, PTLPs remain capable of killing target bacteria. Here we describe our experience in the induction and purification of C. difficile PTLPs. These methods have been optimized to allow production of concentrated, non-contractile, and non-aggregated samples for both sensitivity testing and structural electron microscopy studies.