Polyethylene glycol treated allografts not tissue matched nor immunosuppressed rapidly repair sciatic nerve gaps, maintain neuromuscular functions, and restore voluntary behaviors in female rats.

Many publications report that ablations of segments of peripheral nerves produce the following unfortunate results: (1) Immediate loss of sensory signaling and motor control; (2) rapid Wallerian degeneration of severed distal axons within days; (3) muscle atrophy within weeks; (4) poor behavioral (functional) recovery after many months, if ever, by slowly-regenerating (∼1mm/d) axon outgrowths from surviving proximal nerve stumps; and (5) Nerve allografts to repair gap injuries are rejected, often even if tissue matched and immunosuppressed. In contrast, using a female rat sciatic nerve model system, we report that neurorrhaphy of allografts plus a well-specified-sequence of solutions (one containing polyethylene glycol: PEG) successfully addresses each of these problems by: (a) Reestablishing axonal continuity/signaling within minutes by nonspecific ally PEG-fusing (connecting) severed motor and sensory axons across each anastomosis; (b) preventing Wallerian degeneration by maintaining many distal segments of inappropriately-reconnected, PEG-fused axons that continuously activate nerve-muscle junctions; (c) maintaining innervation of muscle fibers that undergo much less atrophy than otherwise-denervated muscle fibers; (d) inducing remarkable behavioral recovery to near-unoperated levels within days to weeks, almost certainly by CNS and PNS plasticities well-beyond what most neuroscientists currently imagine; and (e) preventing rejection of PEG-fused donor nerve allografts with no tissue matching or immunosuppression. Similar behavioral results are produced by PEG-fused autografts. All results for Negative Control allografts agree with current neuroscience data 1-5 given above. Hence, PEG-fusion of allografts for repair of ablated peripheral nerve segments expand on previous observations in single-cut injuries, provoke reconsideration of some current neuroscience dogma, and further extend the potential of PEG-fusion in clinical practice.

Polyethylene glycol solutions rapidly restore and maintain axonal continuity, neuromuscular structures, and behaviors lost after sciatic nerve transections in female rats.

Complete severance of major peripheral mixed sensory-motor nerve proximally in a mammalian limb produces immediate loss of action potential conduction and voluntary behaviors mediated by the severed distal axonal segments. These severed distal segments undergo Wallerian degeneration within days. Denervated muscles atrophy within weeks. Slowly regenerating (∼1 mm/day) outgrowths from surviving proximal stumps that often nonspecifically reinnervate denervated targets produce poor, if any, restoration of lost voluntary behaviors. In contrast, in this study using completely transected female rat sciatic axons as a model system, we provide extensive morphometric, immunohistochemical, electrophysiological, and behavioral data to show that these adverse outcomes are avoided by microsuturing closely apposed axonal cut ends (neurorrhaphy) and applying a sequence of well-specified solutions, one of which contains polyethylene glycol (PEG). This "PEG-fusion" procedure within minutes reestablishes axoplasmic and axolemmal continuity and signaling by nonspecifically fusing (connecting) closely apposed open ends of severed motor and/or sensory axons at the lesion site. These PEG-fused axons continue to conduct action potentials and generate muscle action potentials and muscle twitches for months and do not undergo Wallerian degeneration. Continuously innervated muscle fibers undergo much less atrophy compared with denervated muscle fibers. Dramatic behavioral recovery to near-unoperated levels occurs within days to weeks, almost certainly by activating many central nervous system and peripheral nervous system synaptic and other plasticities, some perhaps to a greater extent than most neuroscientists would expect. Negative control transections in which neurorrhaphy and all solutions except the PEG-containing solution are applied produce none of these remarkably fortuitous outcomes observed for PEG-fusion.

Salivary Blockade Protects the Lower Respiratory Tract of Mice from Lethal Influenza Virus Infection.

It is possible to model the progression of influenza virus from the upper respiratory tract to the lower respiratory tract in the mouse using viral inoculum delivered in a restricted manner to the nose. In this model, infection with the A/Udorn/307/72 (Udorn) strain of virus results ultimately in high viral titers in both the trachea and lungs. In contrast, the A/Puerto Rico/8/34 (PR8) strain causes an infection that is almost entirely limited to the nasal passages. The factors that govern the progression of virus down the respiratory tract are not well understood. Here, we show that, while PR8 virus grows to high titers in the nose, an inhibitor present in the saliva blocks further progression of infection to the trachea and lungs and renders an otherwise lethal dose of virus completely asymptomatic. In vitro, the salivary inhibitor was capable of potent neutralization of PR8 virus and an additional 20 strains of type A virus and two type B strains that were tested. The exceptions were Udorn virus and the closely related H3N2 strains A/Port Chalmers/1/73 and A/Victoria/3/75. Characterization of the salivary inhibitor showed it to be independent of sialic acid and other carbohydrates for its function. This and other biochemical properties, together with its virus strain specificity and in vivo function, indicate that the mouse salivary inhibitor is a previously undescribed innate inhibitory molecule that may have evolved to provide pulmonary protection of the species from fatal influenza virus infection.IMPORTANCE Influenza A virus occasionally jumps from aquatic birds, its natural host, into mammals to cause outbreaks of varying severity, including pandemics in humans. Despite the laboratory mouse being used as a model to study influenza virus pathogenesis, natural outbreaks of influenza have not been reported in the species. Here, we shed light on one mechanism that might allow mice to be protected from influenza in the wild. We show that virus deposited in the mouse upper respiratory tract will not progress to the lower respiratory tract due to the presence of a potent inhibitor of the virus in saliva. Containing inhibitor-sensitive virus to the upper respiratory tract renders an otherwise lethal infection subclinical. This knowledge sheds light on how natural inhibitors may have evolved to improve survival in this species.

Reducing the impact of influenza-associated secondary pneumococcal infections.

When administered prophylactically, we show that the Toll-like receptor-2 (TLR-2) agonist PEG-Pam2Cys (pegylated-S-(2,3-bis(palmitoyloxy)propyl)cysteine) not only mediates potent anti-viral activity against influenza virus but also reduces the impact of secondary infections with Streptococcus pneumoniae (the pneumococcus) by reducing (i) pulmonary viral and bacterial burdens, (ii) the levels of proinflammatory cytokines that normally accompany influenza and S. pneumoniae secondary infections and (iii) the vascular permeability of the pulmonary tract that can allow bacterial invasion of the blood in mice. We also show that an inactivated detergent-disrupted influenza virus vaccine formulated with the Pam2Cys-based adjuvant R4-Pam2Cys provides the host with both immediate and long-term protection against secondary pneumococcal infections following influenza virus infection through innate and specific immune mechanisms, respectively. Vaccinated animals generated influenza virus-specific immune responses that provided the host with long-term protection against influenza virus and its sequelae. This vaccine, which generates an immediate response, provides an additional countermeasure, which is ideal for use even in the midst of an influenza outbreak.

Neurokinin-1 antagonist orvepitant for EGFRI-induced pruritus in patients with cancer: a randomised, placebo-controlled phase II trial.

OBJECTIVE: To evaluate the efficacy of orvepitant (10 or 30 mg given once daily, orally for 4 weeks), a neurokinin-1 receptor antagonist, compared with placebo in reducing the intensity of epidermal growth factor receptor inhibitor (EGFRI)-induced intense pruritus. DESIGN: Randomised, double-blind, placebo-controlled clinical trial. SETTING: 15 hospitals in Italy and five hospitals in the UK. PARTICIPANTS: 44 patients aged ≥18 years receiving an EGFRI for a histologically confirmed malignant solid tumour and experiencing moderate or intense pruritus after EGFRI treatment. INTERVENTION: 30 or 10 mg orvepitant or placebo tablets once daily for 4 weeks (randomised 1:1:1). PRIMARY AND SECONDARY OUTCOME MEASURES: The primary endpoint was change from baseline in mean patient-recorded numerical rating scale (NRS) score (over the last three recordings) at week 4. Secondary outcome measures were NRS score, verbal rating scale score, Skindex-16 and Leeds Sleep Evaluation Questionnaire at each study visit (baseline, weeks 1, 4, 8); rescue medication use; EGFRI dose reduction; and study withdrawal because of intense uncontrolled pruritus. RESULTS: The trial was terminated early because of recruitment challenges; only 44 of the planned 90 patients were randomised. All patients were analysed for efficacy and safety. Mean NRS score change from baseline to week 4 was -2.78 (SD: 2.64) points in the 30 mg group, -3.04 (SD: 3.06) points in the 10 mg group and -3.21 (SD: 1.77) points in the placebo group; the difference between orvepitant and placebo was not statistically significant. No safety signal was detected. Adverse events related to orvepitant (asthenia, dizziness, dry mouth, hyperhidrosis) were all of mild or moderate severity. CONCLUSIONS: Orvepitant was safe and well tolerated. No difference in NRS score between the orvepitant and placebo groups was observed at the week 4 primary endpoint. A number of explanations for this outcome are possible. TRIAL REGISTRATION NUMBER: EudraCT2013-002763-25.

The profile enhancer (reverse-pull headgear).

The purpose of this article is to re-acquaint the reader with the etiology of Class III malocclusions, review the use of the Reverse-Pull Headgear (Profile Enhancer) in Class III patients, understand the limitations in older patients, and provide alternative treatment to finish the case. Often orthodontic treatment of certain patients requires combinations of extra-oral as well as intra-oral appliances. The patient presented in this article was beyond the ideal envelope of skeletal changes with this appliance, however, as an alternative to surgical intervention the results were pleasing.

Polyethylene glycol (PEG) and other bioactive solutions with neurorrhaphy for rapid and dramatic repair of peripheral nerve lesions by PEG-fusion.

BACKGROUND: Nervous system injuries in mammals often involve transection or segmental loss of peripheral nerves. Such injuries result in functional (behavioral) deficits poorly restored by naturally occurring 1-2 mm/d axonal outgrowths aided by primary repair or reconstruction. "Neurorrhaphy" or nerve repair joins severed connective tissues, but not severed cytoplasmic/plasmalemmal extensions (axons) within the tissue. NEW METHOD: PEG-fusion consists of neurorrhaphy combined with a well-defined sequence of four pharmaceutical agents in solution, one containing polyethylene glycol (PEG), applied directly to closely apposed viable ends of severed axons. RESULTS: PEG-fusion of rat sciatic nerves: (1) restores axonal continuity across coaptation site(s) within minutes, (2) prevents Wallerian degeneration of many distal severed axons, (3) preserves neuromuscular junctions, (4) prevents target muscle atrophy, (5) produces rapid and improved recovery of voluntary behaviors compared with neurorrhaphy alone, and (6) PEG-fused allografts are not rejected, despite no tissue-matching nor immunosuppression. COMPARISON WITH EXISTING METHODS: If PEG-fusion protocols are not correctly executed, the results are similar to that of neurorrhaphy alone: (1) axonal continuity across coaptation site(s) is not re-established, (2) Wallerian degeneration of all distal severed axons rapidly occurs, (3) neuromuscular junctions are non-functional, (4) target muscle atrophy begins within weeks, (5) recovery of voluntary behavior occurs, if ever, after months to levels well-below that observed in unoperated animals, and (6) allografts are either rejected or not well-accepted. CONCLUSION: PEG-fusion produces rapid and dramatic recovery of function following rat peripheral nerve injuries.

Creating a successful school-based mobile dental program.

BACKGROUND: Dental disease is one of the leading causes of school absenteeism for children. This article describes the creation and evolution of the St. David's Dental Program, a mobile school-based dental program for children. METHODS: The dental program is a collaboration of community partners in Central Texas that provides free dental care to low-income children in schools without relying on reimbursements or government funding. RESULTS: Since 1998, the program has provided 132,791 screenings for oral health treatment needs and 38,634 encounters for sealants or treatment. In 2005, the program provided $2.1 million worth of services at a cost of $1.2 million (not including donated services). Factors important to the program's success included sustained funding for general operating costs; well-compensated clinicians to deliver care and experienced human service workers to manage program operations; the devotion of resources to maximize consent form return rates; and the development of strong relationships with school district and individual school staff. CONCLUSIONS: By removing cost, time, transportation, and bureaucratic barriers, the program was able to reach more children than fixed-site clinics. The program was a merging of private and public health dentistries. This model can be useful to other communities in light of the unmet need for dental care and tighter federal, state, and local government budgets.

PEGylation of a TLR2-agonist-based vaccine delivery system improves antigen trafficking and the magnitude of ensuing antibody and CD8+ T cell responses.

The lipopeptide R4Pam2Cys is an agonist for toll-like receptor-2 (TLR2), a key pathogen-associated molecular pattern receptor expressed on many antigen-presenting cells such as dendritic cells (DCs). Electrostatic association of R4Pam2Cys with soluble protein antigens significantly enhances their immunogenicity and there is evidence to suggest that reducing the size of suitably adjuvanted-antigen complexes in solution may further improve their immunostimulatory capabilities. In this study, we investigated how incorporation of polyethylene glycol (PEG) into R4Pam2Cys affects the size, activity and efficacy of formed antigen-lipopeptide complexes. The presence of PEG was shown to increase solubility with a concomitant reduction in the particle size of vaccine formulations that was dependent on the length of PEG used. When compared to non-PEGylated R4Pam2Cys, vaccination of animals with antigen-complexed PEGylated R4Pam2Cys resulted not only in improvements in antibody production but significantly higher antigen-specific CD8+ T cell responses. Both lipopeptides exhibited similar in vitro capabilities to induce DC maturation, facilitate antigen uptake and presentation to T cells. Moreover, analyses of the transcriptomes obtained from DCs treated with either lipopeptide revealed a large number of commonly induced genes with similar transcript expression levels, suggesting that common signalling pathways and processes were engaged following activation by either lipopeptide. In vivo analysis however revealed that vaccination with antigen-complexed PEGylated R4Pam2Cys resulted in improved antigen presentation to T cells. These heightened responses were not attributed to prolonged antigen persistence but rather due to more rapid transportation of antigen from the injection site into the draining lymph nodes over a short period of time. Our results indicate that reducing the size of formed antigen-TLR2-agonist complexes by PEGylation does not compromise the activity of the agonist but in fact enhances its trafficking in vivo ultimately leading to improved humoral and cell-mediated immune responses.

A single dose biodegradable vaccine depot that induces persistently high levels of antibody over a year.

In this study, we describe a biodegradable vaccine depot which persists in vivo for at least 4-months, provides synergistic adjuvant effects and also allows dose sparing of both antigen and adjuvant. A single administration results in immediate release of a priming dose of vaccine, by a process of syneresis, which is then followed by release of remaining vaccine which maintains robust antibody levels that last for more than a year. The platform technology comprises two aqueous components; one contains chitosan and hydroxyapatite, in which the vaccine is incorporated, and the other consists of a crosslinking agent, tripolyphosphate (TPP) and chondroitin sulphate. When co-injected into tissue, they spontaneously crosslink forming a firm yet compliant vaccine-containing depot. Whole body imaging of animals inoculated with the material show that the depot persists in situ for up to 19 weeks. Vaccination of mice with depot formulations containing ovalbumin (OVA) emulsified in Montanide ISA 61 adjuvant results in the induction of robust antibody responses using doses of adjuvant 40-fold less than those recommended by the manufacturer. Dose sparing effects were also apparent with antigen when delivered in the depot. Similar dose sparing effects were observed with Montanide ISA 50, complete and incomplete Freund's adjuvants but not with aluminium hydroxide nor Quil A. Antibody titres, induced by a single dose of antigen/adjuvant formulation incorporated in the depot, persisted at high levels for at least 55 weeks following a single dose of vaccine.

Correlation of quantitative light-induced fluorescence and optical coherence tomography applied for detection and quantification of early dental caries.

Fluorescence loss in enamel following demineralization has been correlated with the amount of mineral lost during the demineralization. The correlation between fluorescence loss measured by quantitative light-induced fluorescence (QLF) and the reflectivity loss measured by a versatile en face optical coherence tomography (OCT) system was investigated in a demineralization process to produce artificial dental caries. We used an OCT system that can collect A-scans (reflectivity versus depth), B-scans (longitudinal images), and C-scans (en face images). The power to the sample was 250 microW, the wavelength lambda = 850 nm, and the depth resolution in air 16 microm. A-scans, which show the profile of the reflectivity versus the depth of penetration into the tooth tissue, were used for quantitative analysis of the reflectivity loss. The results have shown that both the fluorescence radiance and reflectivity of the enamel decrease with increasing de- mineralization time. A linear correlation was observed between the percentage of fluorescence loss measured by QLF and the percentage of reflectivity loss measured by OCT. It was concluded that the decrease in reflectivity of the enamel during demineralization, measured by OCT, could be related to the amount of mineral lost during the de- mineralization process.

Quantification of root caries using optical coherence tomography and microradiography: a correlational study.

PURPOSE: The use of transverse microradiography (TMR) to quantify the amount of mineral lost during demineralization of tooth tissue has long been established. In the present study, the use of an en-face Optical Coherence Tomography (OCT) technology to detect and quantitatively monitor the mineral changes in root caries was investigated and correlated with TMR. MATERIALS AND METHODS: We used an OCT system, developed initially for retina imaging, and which can collect A-scans, B-scans (longitudinal images) and C-scans (en-face images) to quantitatively assess the development of root caries. The power to the sample was 250 microW, wavelength lambda = 850 nm and the optical source linewidth was 16 microm. RESULTS: Both the transversal and longitudinal images showed the caries lesion as volumes of reduced reflectivity. Quantitative analysis using the A-scan (reflectivity versus depth curve) showed that the tissue reflectivity decreased with increasing demineralization time. A linear correlation (r = 0.957) was observed between the mineral loss measured by TMR and the percentage reflectivity loss in demineralized tissue measured by OCT. CONCLUSION: We concluded that OCT could be used to detect incipient root caries, and that the reflectivity loss in root tissue during demineralization, measured by OCT, could be related to the amount of mineral lost during the demineralization.

Zirconia enriched dental adhesive: a solution for OCT contrast enhancement. Demonstrative study by synchrotron radiation microtomography.

OBJECTIVE: The major aim of this study was to prove the capability of the optical coherence tomography (OCT) method in visualizing the integrity of the adhesive fillings and of the interfaces between the adhesive, tooth structures and composite resin. As zirconium dioxide was added to the composition of the adhesive layer in order to strengthen the backscattered light in the OCT investigation, for a better visualization of the interfaces, the determination of a proper zirconia concentration was another aim of our study. METHOD: Several class II cavities were prepared in human premolars and were filled with dental adhesive containing different zirconia concentrations and light-curing composite resin. Both OCT and synchrotron radiation microtomography (micro-CT) were used to analyse the morphology of the tooth-adhesive-composite interfaces and to investigate the adhesive layer. RESULTS: The pore distribution, both at the interfaces level and in the resin, and the analysis of the adhesive layer integrity were obtained. A good agreement between OCT and micro-CT analyses was observed in terms of detecting discontinuities in the adhesive layer. Furthermore, micro-CT showed that zirconia percentages in the adhesive higher than 20 vol.% lead to conglomerates formation, which can negatively influence mechanical properties. Meanwhile, OCT confirmed a factor of 3 for the contrast enhancement when 20% of zirconia was included in the adhesive composition. SIGNIFICANCE: The present study proved the capability of the OCT method in visualizing the morphology and integrity of zirconia doped tooth adhesive fillings, to be used for a further in vivo tool development.

Effects of sorghum (Sorghum bicolor (L.) Moench) tannins on α-amylase activity and in vitro digestibility of starch in raw and processed flours.

The purpose of this study was to investigate the effects of tannins on starch digestion in tannin-containing sorghum extracts and wholegrain flours from 12 sorghum varieties. Extracts reduced amylase activity in a tannin concentration-dependent manner when the extract was mixed with the enzyme before substrate (amylopectin) addition, with higher molecular weight tannins showing greater reduction. Conversely, when the extract and substrate were combined before enzyme addition an enhancement in amylase activity was experienced. In uncooked, cooked, and cooked and stored wholegrain sorghum flours, rapidly digestible, slowly digestible, and resistant starches were not correlated with tannin content or molecular weight distribution. Resistant starch increased from 6.5% to 22-26% when tannins were added to starch up to 50% (starch weight). Tannin extracts both reduced and enhanced amylase activity depending on conditions, and, while these trends were clear in extracts, the effects on starch digestion in wholegrain flours was more complex.

Control of size dispersity of chitosan biopolymer microparticles and nanoparticles to influence vaccine trafficking and cell uptake.

Structurally related surfactant molecules were exploited to generate chitosan emulsions to provide systematic variation in micelle radii of curvature and size. These compositions provide precise control of chitosan particle dispersity, that is, size distribution according to three quantitative distribution parameters as well as shape distribution. This resulted in a suite of particle size distributions spanning 71 nm to 3.7 µm and a very high degree of particle sphericity, allowing the influence of particle size to be isolated in two in vivo studies relating biopolymer particle size to cellular uptake and trafficking to lymph nodes. Flow cytometry and fluorescence microscopy indicated that the three cell lines examined preferentially internalized chitosan microparticles to a greater extent than nanoparticles over a 24 h period. In an in vivo mouse model, nanoparticles initially trafficked rapidly to lymph nodes draining the site of inoculation followed by further slower uptake. Microparticles trafficked to the lymph nodes with a similar pattern except that the initial discharge was ∼50-fold less than that observed with nanoparticles indicating a profound difference in the physiological transport properties of the two particle types.