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.

Tracking vaginal, anal and oral infection in a mouse papillomavirus infection model.

Noninvasive and practical techniques to longitudinally track viral infection are sought after in clinical practice. We report a proof-of-principle study to monitor the viral DNA copy number using a newly established mouse papillomavirus (MmuPV1) mucosal infection model. We hypothesized that viral presence could be identified and quantified by collecting lavage samples from cervicovaginal, anal and oral sites. Nude mice infected at these sites with infectious MmuPV1 were tracked for up to 23 weeks starting at 6 weeks post-infection. Viral DNA copy number was determined by SYBR Green Q-PCR analysis. In addition, we tracked viral DNA load through three complete oestrous cycles to pinpoint whether there was a correlation between the DNA load and the four stages of the oestrous cycle. Our results showed that high viral DNA copy number was reproducibly detected from both anal and cervicovaginal lavage samples. The infection and disease progression were further confirmed by histology, cytology, in situ hybridization, immunohistochemistry and transmission electron microscopy. Interestingly, the viral copy number fluctuated over the oestrous cycle, with the highest level at the oestrus stage, implying that multiple sampling might be necessary to provide a reliable diagnosis. Virus DNA was detected in oral lavage samples at a later time after infection. Lower viral DNA load was found in oral samples when compared with those in anal and vaginal tracts. To our knowledge, our study is the first in vivo study to sequentially monitor papillomavirus infection from mucosal anal, oral and vaginal tracts in a preclinical model.

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

Neurologic disorders such as Alzheimer's, Parkinson's 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.

Demonstrating collagen tendon fibril segments involvement in intrinsic tendon repair.

Severed tendons can undergo regenerative healing, intrinsic tendon repair. Fibrillogenesis of chick tendon involves "collagen fibril segments" (CFS), which are the building blocks of collagen fibers that make up tendon fascicles. The CFS are 10.5 micron in length, composed of tropocollagen monomers arranged in parallel arrays. Rather than incorporating single tropocollagen molecules into growing collagen fibers, incorporating large CFS units is the mechanism for generating collagen fibers. Is intrinsic tendon repair through the reestablishment of tendon embryogenesis? Gentamicin treated 10-day-old chick embryo tendons released CFS were fluorescently tagged with Rhodamine (Rh). Organ cultured severed 14-day-old embryo tendon explants received Rh tagged CFS. At day 4 auto fluorescent tagged CFS were identified at the severed tendon ends by fluorescent microscopy. Accumulation of fluorescent tagged CFS was exclusively localized to the severed ends of tendon explants. Parallels between collagen fiber growth during embryonic fibrillogenesis and tendon repair reveal CFS incorporation is responsible for collagen fibers growth. CFS incorporation into frayed collagen fibers from severed tendons is the proposed mechanism for intrinsic tendon repair, which is an example of regenerative repair.

Mitochondria impairment correlates with increased sensitivity of aging RPE cells to oxidative stress.

Impairment of mitochondria function and cellular antioxidant systems are linked to aging and neurodegenerative diseases. In the eye, the retinal pigment epithelium (RPE) is exposed to a highly oxidative environment that contributes to age-related visual dysfunction. Here, we examined changes in mitochondrial function in human RPE cells and sensitivity to oxidative stress with increased chronological age. Primary RPE cells from young (9-20)-, mid-age (48-60)-, and >60 (62-76)-year-old donors were grown to confluency and examined by electron microscopy and flow cytometry using several mitochondrial functional assessment tools. Susceptibility of RPE cells to H(2)O(2) toxicity was determined by lactate dehydrogenase and cytochrome c release, as well as propidium iodide staining. Reactive oxygen species, cytoplasmic Ca(2+) [Ca(2+)](c), and mitochondrial Ca(2+) [Ca(2+)](m) levels were measured using 2',7'-dichlorodihydrofluorescein diacetate, fluo-3/AM, and Rhod-2/AM, respectively, adenosine triphosphate (ATP) levels were measured by a luciferin/luciferase-based assay and mitochondrial membrane potential (ΔΨm) estimated using 5,5',6,6'-tetrachloro 1,1'3,3'-tetraethylbenzimid azolocarbocyanine iodide. Expression of mitochondrial and antioxidant genes was determined by real-time polymerase chain reaction. RPE cells show greater sensitivity to oxidative stress, reduction in expression of mitochondrial heat shock protein 70, uncoupling protein 2, and superoxide dismutase 3, and greater expression of superoxide dismutase 2 levels with increased chronological age. Changes in mitochondrial number, size, shape, matrix density, cristae architecture, and membrane integrity were more prominent in samples obtained from >60 years old compared to mid-age and younger donors. These mitochondria abnormalities correlated with lower ATP levels, reduced ΔΨm, decreased [Ca(2+)](c), and increased sequestration of [Ca(2+)](m) in cells with advanced aging. Our study provides evidence for mitochondrial decay, bioenergetic deficiency, weakened antioxidant defenses, and increased sensitivity of RPE cells to oxidative stress with advanced aging. Our findings suggest that with increased severity of mitochondrial decay and oxidative stress, RPE function may be altered in some individuals in a way that makes the retina more susceptible to age-related injury.

Differentiation of xenografted human fetal lung parenchyma.

The goal of this study was to characterize xenografted human fetal lung tissue with respect to developmental stage-specific cytodifferentiation. Human fetal lung tissue (pseudoglandular stage) was grafted either beneath the renal capsule or the skin of athymic mice (NCr-nu). Tissues were analyzed from 3 to 42 days post-engraftment for morphological alterations by light and electron microscopy (EM), and for surfactant protein mRNA and protein by reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry (ICC), respectively. The changes observed resemble those seen in human lung development in utero in many respects, including the differentiation of epithelium to the saccular stage. Each stage occurred over approximately one week in the graft in contrast to the eight weeks of normal in utero development. At all time points examined, all four surfactant proteins (SP-A, SP-B, SP-C, and SP-D) were detected in the epithelium by ICC. Lamellar bodies were first identified by EM in 14 day xenografts. By day 21, a significant increase in lamellar body expression was observed. Cellular proliferation, as marked by PCNA ICC and elastic fiber deposition resembled those of canalicular and saccular in utero development. This model in which xenografted lung tissue in different stages of development is available may facilitate the study of human fetal lung development and the impact of various pharmacological agents on this process.

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.

Persistent vascular defects in lung allografts attributed to defective endogenous endothelial progenitors.

BACKGROUND: A major pathological finding in human newborns with pulmonary hypoplasia and congenital diaphragmatic hernia is the presence of vascular abnormalities in lungs. Vasculogenesis/angiogenesis are crucial to lung development. To study lung alveolar development, including microvascular formation in fetal lung implants, Schwarz et al. [1] developed a subcutaneous allograft model. We adopted their model to assess the influence of neovascularization or the "host-graft vascular development" on hypoplastic lung structure and growth. MATERIALS AND METHODS: Normal and hypoplastic lungs at pseudoglandular stage were implanted subcutaneously under the dorsolateral fold of immunocompromised nude mice (athymic, nu/nu). Lung allografts were removed and assessed at 2, 4, 6, and 8 weeks postimplantation. RESULTS: Neovascularization of implanted lungs from subcutaneous vasculature of nude mice resulted in varying degrees of maturation of implanted normal and hypoplastic lungs. By 4 weeks, implanted normal lungs contained Type 2-like cells and by 7 to 8 weeks, Type 2 and Type 1-like cells, air spaces had enlarged, and surfactant secretion was observed. Despite some differentiation and maturation of hypoplastic lungs, there was more mesenchymal tissue, no secondary septa, and smaller air spaces compared to normal lungs. CONCLUSIONS: (a) Neovascularization or host-graft vascular development occurs in both normal and hypoplastic lung allografts. (b) Development and maturation of implanted normal and hypoplastic lungs follow the establishment of the vascular connections between the host and grafts. (c) The host-graft vascular connections do not improve the growth of normal or hypoplastic lungs. (d) Neovascularization failed to overcome the embryonic defects in vascular formation and the pulmonary vasculogenesis remained defective in hypoplastic lung allografts, which may be attributed to the defective endogenous endothelial progenitor cells.

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.

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 alpha-smooth muscle actin within stress fibers, were absent in vanadate-treated granulation tissue. The expression of alpha-smooth muscle actin was restricted to smooth muscle cells of blood vessels. Controls had densely packed alpha-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.