Alleviation of Trigeminal Nociception Using p75 Neurotrophin Receptor Targeted Lentiviral Interference Therapy.

Acute and chronic trigeminal (TG) neuropathies are the cause of considerable distress, with limited treatments available at present. Nociceptive neurons enriched with the vanilloid type 1 receptor (VR1) partake in pain sensation and sensitization in the TG system. While VR1 blockers with anti-nociceptive potential are of substantial medical interest, their use remains limited due to poor selectivity and lack of cell-targeting capabilities. This study describes a methodology for the alleviation of nociception via targeted depletion of VR1 in TG sensory neurons in rats. In cultured TG ganglion neurons, VR1 expression was virtually abolished by lentiviral short hairpin RNA (LV-VR1). By decorating GFP encoding LV (LV-GFP) and LV-VR1 with IgG192 for targeting TG sensory neurons enriched with the p75 neurotrophin receptor (p75NTR), transduction of a reporter GFP and VR1 depletion was achieved after injection of targeted vectors into the whisker pad. In IgG192/LV-VR1-injected rats, the behavioral response to capsaicin exposure as well as Erk 1/2 phosphorylation and VR1 current activation by capsaicin were significantly reduced. This pioneering investigation, thus, provides a proof of principle for a means of attenuating TG nociception, revealing therapeutic potential.

Increased levels of PD-1 expression on CD8 T cells in patients post-renal transplant irrespective of chronic high EBV viral load.

Studies have identified solid organ transplant recipients who remain asymptomatic despite maintaining CHL. Factors which determine the CHL state remain poorly understood but are likely to involve immunological control of the viral infection. We monitored expression of PD-1, a marker of T-cell exhaustion and viral persistence, on CD8 T cells in patients who resolved EBV infection as determined by undetectable EBV DNA (REI) and CHL patients. PD-1 expression on CD8 T cells was increased in the first year post-transplant irrespective of EBV outcome, and most CD8 T cells continued to express PD-1 for up to three yr post-transplant. Although all patient groups showed similar frequencies of EBV-specific CD8+ T cells, PD-1 expression on these cells increased in the post-transplant groups compared with the pretransplant patients. Functional studies of EBV-specific CD8+ T cells stimulated with BZLF or LMP2 peptide pools revealed monofunctional IFN-γ responses. Our results indicate that PD-1 expression on CD8 T cells post-transplant may result from factors other than antigenic stimulation.

Feasibility of correlation mapping optical coherence tomography (cmOCT) for anti-spoof sub-surface fingerprinting.

We propose the use of correlation mapping optical coherence tomography (cmOCT) to deliver additional biometrics associated with the finger that could complement existing fingerprint technology for law enforcement applications. The current study extends the existing fingerprint paradigm by measuring additional biometrics associated with sub-surface finger tissue such as sub-surface fingerprints, sweat glands, and the pattern of the capillary bed to yield a user-friendly cost effective and anti-spoof multi-mode biometric solution associated with the finger. To our knowledge no other method has been able to capture sub-surface fingerprint, papillary pattern and horizontal vessel pattern in a single scan or to show the correspondence between these patterns in live adult human fingertip. Unlike many current technologies this approach incorporates 'liveness' testing by default. The ultimate output is a biometric module which is difficult to defeat and complements fingerprint scanners that currently are used in border control and law enforcement applications.

Rapid quantification of histamine in human psoriatic plaques using microdialysis and ultra high performance liquid chromatography with fluorescence detection.

Psoriasis is a chronic skin disease resulting from abnormal immune function and is characterized by the presence of scaly psoriatic plaques which are areas of inflammation and excessive skin production. The psoriatic plaques contain mast cells which are increased in number in the uppermost dermis of the psoriatic lesion and which may play a role in the initiation and maintenance of the lesion. These processes are thought to be mediated via the local release of histamine along with other mediators from the mast cells; however their precise role still remains a mystery. Our study involved the development of a rapid and ultra-sensitive liquid chromatographic method for the separation and detection of histamine. To this end a state-of-the-art ultra high pressure liquid chromatography (UHPLC) system incorporating the latest technology in fluorescence detection system was employed which allowed for the rapid and reliable trace level detection of histamine in human derived microdialysate samples. This new reverse phase method utilized a sub-two-micron packed C(18) stationary phase (50 mm × 4.6 mm, 1.8 µm particle size) and a polar mobile phase of ACN:H(2)O:acetic acid (70:30:0.05) (v/v). The column temperature was maintained at (30±2°C), the injection volume was (8 µl), with a flow rate of (1.1 ml/min). Dermal microdialysis was used to collect (20 µl) samples from healthy, peri-lesional and lesional skin regions, in the forearms of a small cohort of subjects (n=6), and the ultra sensitive liquid chromatographic method allowed for nanomolar quantitation of histamine in 6.7 min. To date this represents one of the fastest reported separations of histamine using fluorescence detection with very high chromatographic efficiency (258,000/m) and peak symmetry of (0.88). Prior to sample analysis being performed method linearity, precision and limit of detection (LOD) were investigated. The results showed that intracutaneous histamine measured at 70 min after catheter implantation was (3.44±.52 nmol) (mean±SEM) in non-lesional (control) skin and was not dissimilar to that observed in either lesional (3.10±.76 nmol) or peri-lesional skin (2.24±.20 nmol). A second fraction collected 190 min after implantation also revealed similar levels with no difference in intracutaneous histamine observed between control (2.41±.56 nmol), lesional (2.69±.54 nmol), or peri-lesional skin (2.25±.50 nmol).

Neurotherapeutics to inhibit exocytosis from sensory neurons for the control of chronic pain.

There is a pressing unmet need for long-acting and effective therapeutics to alleviate symptoms of the varied forms of chronic pain. As many sufferers do not respond satisfactorily to non-addictive anti-nociceptives, a new treatment has emerged using inhibitors for the release of pain mediators from peripheral sensory nerves to give prolonged benefit. This strategy relies on proteolytically inactivating intra-neuronal SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptors) proteins which are essential for regulated exocytosis of transmitters, peptides and other pain signalling molecules. Success has been achieved with botulinum neurotoxin A (BoNT/A) which targets neuronal acceptors via its heavy chain, becomes endocytosed and translocated into the cytosol where the long-lived protease of its light chain potently and specifically cleaves SNAP-25 (synaptosomal-associated protease of Mr=25k). Encouragingly, clinical trials have shown that local injections of BOTOX(®) (BoNT/A complex) reduce chronic migraine symptoms including frequency and intensity for many months. Several serotypes of the neurotoxin moiety alone have been prepared recombinantly using Escherichia coli, which exhibit optimal neuroparalysis. Moreover, an engineered chimera of BoNT/E in which its binding domain was replaced with that from /A efficaciously inhibits the TRPV1 (transient receptor potential vanilloid type 1)-triggered release of CGRP (calcitonin gene-related peptide) from cultured sensory neurons, and suppresses the resultant excitatory effects in brain slices. A longer acting composite toxin, containing the protease of type E attached to BoNT/A, displays prolonged amelioration of pain symptoms in an animal model of inflammatory pain. This provides proof of principle that therapeutically advantageous features of /E (most robust inhibitor of CGRP release) and /A (targeting to sensory neurons and dramatic extension of the longevity of E protease) can be incorporated into a single synergistically active anti-nociceptive.

Qualitative analysis using Raman spectroscopy and chemometrics: a comprehensive model system for narcotics analysis.

The rapid, on-site identification of illicit narcotics, such as cocaine, is hindered by the diverse nature of the samples, which can contain a large variety of materials in a wide concentration range. This sample variance has a very strong influence on the analytical methodologies that can be utilized and in general prevents the widespread use of quantitative analysis of illicit narcotics on a routine basis. Raman spectroscopy, coupled with chemometric methods, can be used for in situ qualitative and quantitative analysis of illicit narcotics; however, careful consideration must be given to dealing with the extensive variety of sample types. To assess the efficacy of combining Raman spectroscopy and chemometrics for the identification of a target analyte under real-world conditions, a large-scale model sample system (633 samples) using a target (acetaminophen) mixed with a wide variety of excipients was created. Materials that exhibit problematic factors such as fluorescence, variable Raman scattering intensities, and extensive peak overlap were included to challenge the efficacy of chemometric data preprocessing and classification methods. In contrast to spectral matching analyte identification approaches, we have taken a chemometric classification model-based approach to account for the wide variances in spectral data. The first derivative of the Raman spectra from the fingerprint region (750-1900 cm(-1)) yielded the best classifications. Using a robust segmented cross-validation method, correct classification rates of better than ∼90% could be attained with regression-based classification, compared to ∼35% for SIMCA. This study demonstrates that even with very high degrees of sample variance, as evidenced by dramatic changes in Raman spectra, it is possible to obtain reasonably reliable identification using a combination of Raman spectroscopy and chemometrics. The model sample set can now be used to validate more advanced chemometric or machine learning algorithms being developed for the identification of analytes such as illicit narcotics.

In-vivo dynamic characterization of microneedle skin penetration using optical coherence tomography.

The use of microneedles as a method of circumventing the barrier properties of the stratum corneum is receiving much attention. Although skin disruption technologies and subsequent transdermal diffusion rates are being extensively studied, no accurate data on depth and closure kinetics of microneedle-induced skin pores are available, primarily due to the cumbersome techniques currently required for skin analysis. We report on the first use of optical coherence tomography technology to image microneedle penetration in real time and in vivo. We show that optical coherence tomography (OCT) can be used to painlessly measure stratum corneum and epidermis thickness, as well as microneedle penetration depth after microneedle insertion. Since OCT is a real-time, in-vivo, nondestructive technique, we also analyze skin healing characteristics and present quantitative data on micropore closure rate. Two locations (the volar forearm and dorsal aspect of the fingertip) have been assessed as suitable candidates for microneedle administration. The results illustrate the applicability of OCT analysis as a tool for microneedle-related skin characterization.

Tissue viability (TiVi) imaging: temporal effects of local occlusion studies in the volar forearm.

Tissue Viability (TiVi) imaging is a promising new technology for the assessment of microcirculation in the upper human dermis. Although the technique is easily implemented and develops large amounts of observational data, its role in the clinical workplace awaits the development of standardised protocols required for routine clinical practice. The present study investigates the use of TiVi technology in a human, in vivo, localized, skin blood flow occlusion protocol. In this feasibility study, the response of the cutaneous microcirculation after provocation on the volar surface of the forearm was evaluated using a high temporal-low spatial resolution TiVi camera. 19 healthy subjects - 10 female and 9 male - were studied after a localized pressure was applied for 5 different time periods ranging from 5 to 25 seconds. Areas corresponding to 100 x 100 pixels (2.89 cm(2)) were monitored for 60 seconds prior to, during and after each occlusion period. Our results demonstrated the removal of blood from the local area and a hyperaemic response supporting the suitability of TiVi imaging for the generation of detailed provocation response data of relevance for the physiological function of the skin microcirculation in health and disease.

Basiliximab induced non-cardiogenic pulmonary edema in two pediatric renal transplant recipients.

We report two cases of non-cardiogenic pulmonary edema as a complication of basiliximab induction therapy in young pediatric renal transplant patients identified following a retrospective review of all pediatric renal transplant cases performed in the National Paediatric Transplant Centre, Childrens University Hospital, Temple Street, Dublin, Ireland. Twenty-eight renal transplantations, of which five were living-related (LRD) and 23 were from deceased donors (DD), were performed in 28 children between 2003 and 2006. In six cases, transplantations were pre-emptive. Immunosuppression was induced pre-operatively using a combination of basiliximab, tacrolimus and methylprednisolone in all patients. Basiliximab induction was initiated 2 h prior to surgery in all cases and, in 26 patients, basiliximab was re-administered on post-operative day 4. Two patients, one LRD and one DD, aged 6 and 11 years, respectively, developed acute non-cardiogenic pulmonary edema within 36 h of surgery. Renal dysplasia was identified as the primary etiological factor for renal failure in both cases. Both children required assisted ventilation for between 4 and 6 days. While both grafts had primary function, the DD transplant patient subsequently developed acute tubular necrosis and was eventually lost within 3 weeks due to thrombotic microangiopathy and severe acute antibody-mediated rejection despite adequate immunosuppression. Non-cardiogenic pulmonary edema is a potentially devastating post-operative complication of basiliximab induction therapy in young pediatric patients following renal transplantation. Early recognition and appropriate supportive therapy is vital for patient and, where possible, graft survival.