Transgene expression and effective gene silencing in vagal afferent neurons in vivo using recombinant adeno-associated virus vectors.

Vagal afferent fibres innervating thoracic structures such as the respiratory tract and oesophagus are diverse, comprising several subtypes of functionally distinct C-fibres and A-fibres. Both morphological and functional studies of these nerve subtypes would be advanced by selective, effective and long-term transduction of vagal afferent neurons with viral vectors. Here we addressed the hypothesis that vagal sensory neurons can be transduced with adeno-associated virus (AAV) vectors in vivo, in a manner that would be useful for morphological assessment of nerve terminals, using enhanced green fluorescent protein (eGFP), as well as for the selective knock-down of specific genes of interest in a tissue-selective manner. We found that a direct microinjection of AAV vectors into the vagal nodose ganglia in vivo leads to selective, effective and long-lasting transduction of the vast majority of primary sensory vagal neurons without transduction of parasympathetic efferent neurons. The transduction of vagal neurons by pseudoserotype AAV2/8 vectors in vivo is sufficiently efficient such that it can be used to functionally silence TRPV1 gene expression using short hairpin RNA (shRNA). The eGFP encoded by AAV vectors is robustly transported to both the central and peripheral terminals of transduced vagal afferent neurons allowing for bright imaging of the nerve endings in living tissues and suitable for structure-function studies of vagal afferent nerve endings. Finally, the AAV2/8 vectors are efficiently taken up by the vagal nerve terminals in the visceral tissue and retrogradely transported to the cell body, allowing for tissue-specific transduction.

Expression pattern of human P2Y receptor subtypes: a quantitative reverse transcription-polymerase chain reaction study.

The diverse biological actions of extracellular nucleotides in tissues and cells are mediated by two distinct classes of P2 receptor, P2X and P2Y. The G protein-coupled P2Y receptors comprise at least six mammalian subtypes (P2Y(1,2,4,6,11,12)), all of which have been cloned from human tissues, as well as other species. The P2Y receptor subtypes differ in their pharmacological selectivity for various adenosine and uridine nucleotides, which overlap in some cases. Data concerning the mRNA expression patterns of five P2Y receptors (P2Y(1,2,4,6,11)) in different human tissues and cells are currently quite limited, while P2Y mRNA distribution in the human brain has not previously been studied. In this study, we have addressed this deficiency in receptor expression data by using a quantitative reverse transcription-polymerase chain reaction approach to measure the precise mRNA expression pattern of each P2Y receptor subtype in a number of human peripheral tissues and brain regions, from multiple individuals, as well as numerous human cell lines and primary cells. All five P2Y receptors exhibited widespread yet subtype-selective mRNA expression profiles throughout the human tissues, brain regions and cells used. Our extensive expression data indicate the many potentially important roles of P2Y receptors throughout the human body, and will help in elucidating the physiological function of each receptor subtype in a wide variety of human systems.

Comparison of histidine-tryptophan ketoglutarate and University of Wisconsin solutions as primary preservation in renal allografts undergoing pulsatile perfusion.

INTRODUCTION: University of Wisconsin (UW) solution is the standard preservation solution for organ transplantation. Histidine-tryptophan ketogluatarate (HTK) solution has been used increasingly for kidney, pancreas, and liver transplantation. This study compared HTK and UW used during kidney procurement with subsequent pulsatile perfusion. METHODS: Between January and October 2003, 91 deceased renal and simultaneous kidney pancreas transplants were performed (UW, n = 41, and HTK, n = 50). There were no differences with regard to donor and recipient demographics or cold ischemia. RESULTS: Delayed graft function occurred in 3 (7%) of UW and 4 (8%) of HTK-preserved kidneys (P = NS). There were no significant differences between patient or graft survival. There was an anticipated difference between total preservative volumes used (HTK: 4.1 +/- 1.0 vs UW: 3.0 +/- 0.5; P < .005). CONCLUSION: UW and HTK appear to have similar efficacy in kidney preservation with pulsatile perfusion. HTK preservation solution can be used safely in conjunction with pulsatile preservation for cold storage of renal allografts.

Follow-up experience using histidine-tryptophan ketoglutarate solution in clinical pancreas transplantation.

In May 2003, at Indiana University, the standard cold preservation solution University of Wisconsin (UW) solution was replaced by histidine-tryptophan ketogluatarate (HTK) solution. Earlier, we presented our initial experience with HTK in pancreas preservation with an analysis of the first 10 pancreas transplants. Here we report updated results with HTK in pancreas transplantation over the past 18 months. Between May 2003 and March 2005, a total of 87 pancreas transplants were performed with 78 of these organs utilizing HTK. Seventy five patients received 78 organ transplants. Surgical procedures performed were: simultaneous kidney pancreas transplantation (n = 50, 64%), pancreas after kidney transplantation (n = 19, 24%), solitary pancreas transplantation (n = 9, 12%). Donor and recipient data were collected with primary outcomes as primary nonfunction and 30-day graft and patient survivals, and compared to the UW cohort from our original report. Donor and recipient demographics were similar. Mean follow-up time is 12 +/- 6 months. The mean cold ischemia time was 9 +/- 3 hours. There were no cases of primary graft nonfunction. Thirty-day and 1-year patient survivals were 99% and 93%. The 30-day and 1-year graft survivals were 96% and 93%. There were five grafts lost, including three within the first month (two venous and one arterial thrombosis). There was one case of chronic rejection and one noncompliance. All other patients were insulin-independent by discharge. Serum fasting blood glucose and serial amylase remained comparable at all intervals posttransplantation. Within this range of cold ischemia time, HTK appears to provide effective pancreas preservation.

Human Ntera-2/D1 neuronal progenitor cells endogenously express a functional P2Y1 receptor.

We report here that human Ntera-2/D1 (NT-2) cells, an undifferentiated committed neuronal progenitor cell line, endogenously express a functional P2Y(1) receptor, while other P2Y subtypes, except perhaps P2Y(4), are not functionally expressed. Quantitative RT-PCR analysis showed that NT-2 cells abundantly express mRNA for P2Y(1) and P2Y(11) receptors, while P2Y(2) and P2Y(4) receptors were detected at considerably lower levels. Western blot analysis also demonstrated expression of P2Y(1) receptors and Galpha(q/11) subunits. Various nucleotides induced intracellular Ca(2+) mobilisation in NT-2 cells in a concentration-dependent manner with a rank order potency of 2-MeSADP > 2-MeSATP > ADP > ATP > UTP > ATPgammaS, a profile resembling that of human P2Y(1) receptors. Furthermore, P2Y(1) receptor-specific (A3P5P) and P2Y-selective (PPADS, suramin) antagonists inhibited adenine nucleotide-induced Ca(2+) responses in a concentration-dependent manner, consistent with expression of a P2Y(1) receptor. Moreover, of seven adenine nucleotides tested, only Bz-ATP and ATPgammaS elicited small increases in cAMP formation suggesting that few, if any, functional P2Y(11) receptors were expressed. P2Y(1) receptor-selective adenine nucleotides, including 2-MeSADP and ADP, also induced concentration-dependent phosphorylation and hence, activation of the extracellular-signal regulated protein kinases (ERK1/2). NT-2 cells, therefore, provide a useful neuronal-like cellular model for studying the precise signalling pathways and physiological responses mediated by a native P2Y(1) receptor.

von Willebrand factor activity detected in a monoclonal antibody-based ELISA: an alternative to the ristocetin cofactor platelet agglutination assay for diagnostic use.

The monoclonal antibody RFF-VIII:R/1 recognises an epitope on von Willebrand factor involved in its interaction with GPIb alpha. A two-site, solid phase ELISA has been established using RFF-VIII:R/1 as the solid-phase, capture antibody and an enzyme-conjugated, polyclonal antibody to human VWF, which provides an assay for VWF functional activity with a detection limit of 0.5 U/dl VWF and an interassay %CV < 10. Plasma from 192 VWD patients (48 studied retrospectively; 144 prospectively) showed VWF levels of < 50 U/dl in type 1 patients (n = 156), < 25 U/dl in type 2A (n = 26) and < 35 U/dl in type 2B (n = 8) which, in type 1 and 2A patients, correlated with RiCoF activity (r > or = 0.82). In plasma from patients with type 1 VWD values of VWF in the Mab-based ELISA were similar to levels of VWF:Ag measured in a polyclonal antibody-based ELISA (r > or = 0.87) but were significantly lower than VWF:Ag in type 2A and 2B plasmas (p < or = 0.0005), allowing discrimination of variant VWD. The Mab-based ELISA has advantages of sensitivity and reproducibility over the RiCoF assay to measure VWF activity and can be used to analyse stored samples. In conjunction with an ELISA for VWF:Ag and VWF multimer analysis, it provides a reliable method for the laboratory diagnosis of VWD.

Orphan G-protein-coupled receptors: the next generation of drug targets?

The pharmaceutical industry has readily embraced genomics to provide it with new targets for drug discovery. Large scale DNA sequencing has allowed the identification of a plethora of DNA sequences distantly related to known G protein-coupled receptors (GPCRs), a superfamily of receptors that have a proven history of being excellent therapeutic targets. In most cases the extent of sequence homology is insufficient to assign these 'orphan' receptors to a particular receptor subfamily. Consequently, reverse molecular pharmacological and functional genomic strategies are being employed to identify the activating ligands of the cloned receptors. Briefly, the reverse molecular pharmacological methodology includes cloning and expression of orphan GPCRs in mammalian cells and screening these cells for a functional response to cognate or surrogate agonists present in biological extract preparations, peptide libraries, and complex compound collections. The functional genomics approach involves the use of 'humanized yeast cells, where the yeast GPCR transduction system is engineered to permit functional expression and coupling of human GPCRs to the endogenous signalling machinery. Both systems provide an excellent platform for identifying novel receptor ligands. Once activating ligands are identified they can be used as pharmacological tools to explore receptor function and relationship to disease.

Characterization of recombinant human orexin receptor pharmacology in a Chinese hamster ovary cell-line using FLIPR.

The cellular mechanisms underlying the physiological effects of the orexins are poorly understood. Therefore, the pharmacology of the recombinant human orexin receptors was studied using FLIPR. Intracellular calcium ([Ca2+]i) was monitored in Chinese hamster ovary (CHO) cells stably expressing orexin-1 (OX1) or orexin-2 (OX2) receptors using Fluo-3AM. Orexin-A and orexin-B increased [Ca2+]i in a concentration dependent manner in CHO-OX1 (pEC50=8.03+/-0.08 and 7. 30+/-0.08 respectively, n=5) and CHO-OX2 (pEC50=8.18+/-0.10 and 8. 43+/-0.09 respectively, n=5) cells. This response was typified as a rapid peak in [Ca2+]i (maximal at 6 - 8 s), followed by a gradually declining secondary phase. Thapsigargin (3 microM) or U73122 (3 microM) abolished the response. In calcium-free conditions the peak response was unaffected but the secondary phase was shortened, returning to basal values within 90 s. Calcium (1.5 mM) replacement restored the secondary phase. In conclusion, orexins cause a phospholipase C-mediated release of calcium from intracellular stores, with subsequent calcium influx.

Endocrine responses to tryptophan infusion in schizophrenic patients treated with neuroleptics.

The growth hormone (GH) and prolactin (PRL) responses to intravenous L-tryptophan (LTP) were measured in 20 schizophrenics receiving long-term treatment with neuroleptics and 20 unmedicated control subjects. In the patients, the PRL response was significantly enhanced, and it correlated with PRL baseline concentration. In contrast, the patients' GH response was markedly reduced. These opposite changes in PRL and GH responses to LTP are unlikely to be accounted for by the effect of neuroleptics on serotonin receptors, but they may have been due to the blockade of DA receptors, which is known to disinhibit PRL release and to suppress that of GH.

Characterisation of gene expression changes following permanent MCAO in the rat using subtractive hybridisation.

Failure of several putative neuroprotectants in large multicentred clinical trials has re-focussed attention on the predictability of pre-clinical animal models of stroke. Model characterisation and relationship to heterogeneous patient sub-groups remains of paramount importance. Information gained from magnetic resonance imaging (MRI) signatures indicates that the Zea Longa model of rat middle cerebral artery occlusion may be more representative of slowly evolving infarcts. Understanding the molecular changes over several hours following cerebral ischaemia will allow detailed characterisation of the adaptive response to brain injury. Using a fully characterised model of Zea Longa middle cerebral artery occlusion we have used the representational difference analysis (RDA) subtractive hybridisation method to identify transcripts that accumulate in the ischaemic cortex. Along with a number of established ischaemia-induced gene products (including MCP-1, TIMP-1, hsp 70) we were also able to identify nine genes which have not previously been shown to accumulate following focal ischaemia (including SOCS-3, GADD45gamma, Xin).

Cloning, localisation and functional expression of a novel human, cerebellum specific, two pore domain potassium channel.

We have isolated, by degenerate PCR, a complementary DNA encoding a novel two pore domain potassium channel. This is the 7th functional member of the human tandem pore domain potassium channel family to be reported. It has an open reading frame of 1.125 kb and encodes a 374 amino acid protein which shows 62% identity to the human TASK-1 gene: identity to other human members of the family is 31-35% at the amino acid level. We believe this gene to be human TASK-3, the ortholog of the recently reported rat TASK-3 gene: amino acid identity between the two is 74%. 'Taqman' mRNA analysis demonstrated a very specific tissue distribution pattern, showing human TASK-3 mRNA to be localised largely in the cerebellum, in contrast rat TASK-3 was reported to be widely distributed. We have shown by radiation hybrid mapping that human TASK-3 can be assigned to chromosome 8q24.3. Human TASK-3 was demonstrated to endow Xenopus oocytes with a negative resting membrane potential through the presence of a large K(+) selective conductance. TASK-3 is inhibited by extracellular acidosis with a mid-point of inhibition around pH 6. 5, supporting the predictions from the sequence data that this is a third human TASK (TWIK-related acid sensitive K(+) channel) gene.

The neuroprotective agent sipatrigine (BW619C89) potently inhibits the human tandem pore-domain K(+) channels TREK-1 and TRAAK.

We have cloned and functionally expressed the human orthologue of the mouse TRAAK gene. When cDNA for hTRAAK is expressed in either Xenopus oocytes or HEK293 cells it forms a K(+)-selective conductance and hyperpolarises the resting membrane potential. Quantitative mRNA expression analysis using Taqman revealed that hTRAAK mRNA is predominantly present in the central nervous system where it exhibits a regionally diverse pattern of expression. Like the related channel TREK-1, the activity of TRAAK was potentiated by arachidonic acid. The neuroprotective agent sipatrigine (10 microM) inhibited both hTREK-1 (73.3+/-4.4%) and hTRAAK (45.1+/-11.2%) in a reversible, voltage-independent manner. Inhibition of both channels was dose-dependent and for TREK-1 occurred with an IC(50) of 4 microM. The related compound lamotrigine, which is a better anticonvulsant but weaker neuroprotective agent than sipatrigine, was a far less effective antagonist of both channels, producing <10% inhibition at a concentration of 10 microM.

Development and evaluation of ELISAs for factor XIIIA and XIIIB subunits in plasma.

Severe, congenital deficiency of factor XIII is extremely rare. However, a moderate reduction in the plasma level of the functional subunit (factor XIIIA) and also to a lesser extent of the carrier subunit (factor XIIIB), and a decrease in the XIIIA:B subunit ratio, have recently been reported in patients with the inflammatory bowel disorder Crohn's disease, particularly during clinical relapse. In order to accurately monitor patients, sensitive, reliable assays for the two subunits of factor XIII are required. We report here the development and validation of ELISAs for these components. The assays are identical except in respect of the specificity of the polyclonal antiserum used as starting material, both of which are commercially available. The antisera are purified by n-octanoic acid precipitation and portions of these purified immunoglobulins are used as coating antibodies. The remaining portions are biotinylated and used with streptavidin and horse-radish peroxidase as tracer antibodies. A normal range (n = 24) was established for factor XIIIA (mean 95 range 60-130 U/dl) and for factor XIIIB (mean 99 range 60-130 U/dl). There were no significant differences between the ELISA and electroimmunodiffusion assays either for factor XIIIA (means +/- 1 standard deviation 95 +/- 15.9 and 89 +/- 22.7 respectively) or for factor XIIIB (99 +/- 18.3 and 106 +/- 23.4 respectively). These assays have been in routine use for six months, during which time two further antisera purifications and biotinylations have been carried out without significant problems of reproducibility or stability.

The effect of diazepam on indices of 5-HT function in man.

The effects of acute and chronic diazepam administration on L-tryptophan induced prolactin release was studied in seven male volunteers. Acute diazepam diminished the prolactin neuroendocrine response to L-tryptophan. On chronic administration this effect was lost, suggesting tolerance had developed. The sedative effects of L-tryptophan were unaltered by either acute or chronic diazepam administration. A possible explanation for the tolerance development to the neuroendocrine effects may be the observed reduction in platelet 3H-imipramine binding that was observed.

Army nurse readiness instrument: psychometric evaluation and field administration.

The purpose of this study was to construct and evaluate the psychometric properties of an instrument to estimate the level of individual readiness among U.S. Army nurses. This study constitutes phase II of congressionally sponsored research to establish the degree to which Army nurses are prepared for the expectations of deployment. An expert panel established the validity of the initial readiness questionnaire. Changes were then incorporated into the first Readiness Estimate and Deployability Index (READI) questionnaire. Internal consistency and test-retest techniques assessed multiple reliabilities from pilot administrations. The READI was refined based on the results. Analysis of field administrations of the revised READI to three separate groups of nurses replicated earlier reliability results. Principle component analyses appear to support the hypothesized dimensional structure underlying questionnaire attitude items. The READI produced psychometrically stable ratings and results with great utility for the Army and potential adaptation for other military services.

The fate of failed bone graft surgery for scaphoid nonunions.

Twenty patients with scaphoid nonunions had bone grafting procedures that failed to achieve union. Nineteen had persistent wrist pain. Electrical stimulation after bone grafting proved useless in obtaining union in five patients. Sixteen patients had additional surgery. Ten had repeat bone grafting. Six scaphoids united after a second grafting and one united after a third graft. However, at follow-up only three of these seven patients had no pain in their wrists. The rate of union was not affected by fracture location, the presence of proximal pole avascular necrosis, or instability. The three patients with nonunion after two bone grafts remain symptomatic. Six patients had salvage procedures; silicone replacement arthroplasty (3), wrist fusion (1), proximal pole excision (1), intercarpal fusion (1). Four were asymptomatic after one of these procedures and two (silicone arthroplasty and intercarpal fusion) became asymptomatic after wrist fusions. Five fractures, believed to be united on the basis of plain radiographs, later demonstrated persistent nonunions. We recommend adequate radiologic follow-up, including tomography, to determine whether or not fracture union has occurred.

Thrombotic risk factors associated with transurethral prostatectomy.

OBJECTIVE: To ascertain the potential thrombotic risk associated with transurethral prostatectomy (TURP). PATIENTS AND METHODS: The changes in coagulation variables were assessed in a prospective study of 40 patients undergoing TURP. RESULTS: There was a significant increase in thrombin-antithrombin complexes 6 h after TURP (anova, P=0.01) combined with a significant decrease in activated partial thromboplastin time (anova, P=0.006), suggesting a postoperative hypercoagulable state. The significant increase in d-dimer 24 h after TURP (anova, P=0.015) in the absence of any significant rise in tissue plasminogen activator antigen levels perioperatively (anova, P=0.737) suggests a physiological fibrinolytic response to the developing procoagulant state. The absence of any significant increase in plasminogen activator inhibitor-1 antigen perioperatively (anova, P=0.348) suggests the observed hypercoagulability is not due to a 'fibrinolytic shutdown' reported in other forms of surgery. CONCLUSION: TURP is associated with a hypercoagulable prothrombotic state; aspirin withdrawal perioperatively may be hazardous, and low-dose heparin prophylaxis for venous thrombosis should be considered.

Conducting population behavioral health surveillance by using automated diagnostic and pharmacy data systems.

INTRODUCTION: The Walter Reed Army Institute of Research used the Electronic Surveillance System for the Early Notification of Community-Based Epidemics (ESSENCE) to conduct population-based behavioral health surveillance among military-health-system beneficiaries. The study analyzed the effectiveness of using prescribing patterns of psychotropic medications to monitor changes in a community's behavioral health status. OBJECTIVES: The objectives of this study were to 1) determine the feasibility of tracking psychiatric illnesses by monitoring prescriptions for psychiatric medications; 2) assess how often psychiatric medications are prescribed for patients with no record of psychiatric illness; 3) determine at what types of clinics these medications are prescribed most often and what other diagnoses are attributed to these patients; and 4) analyze data for potential changes in the population's mental health after high-stress events. METHODS: Correlation analysis and calculations of sensitivity and specificity were used to determine how well prescription medications correlate with outpatient diagnoses and how well they serve as proxies for outpatient diagnoses. A descriptive analysis was conducted of the types of clinics (e.g., primary care, behavioral health, or other specialty clinics) treating patients and the associated percentage of concurrence between prescriptions and diagnostic codes. RESULTS: In military treatment facilities, a diagnosis of depression or anxiety correlated significantly (r = 0.82) with antidepressant or anxiolytic prescriptions. Sensitivity of prescriptions when compared with outpatient visits was 0.76, and specificity was 0.94. Among those patients who visited a primary care clinic either the day before or the same day as an antidepressant or anxiolytic prescription was filled, 60.1% did not receive a diagnosis of any mental health disorder. Behavioral health clinics had the highest correlation between diagnoses and prescriptions; specialty clinics had the lowest. CONCLUSIONS: Behavioral health trends in a population can be monitored by automated analysis of prescribing patterns alone. This method might be a rapid indicator of needed mental health interventions after acute stress-inducing events and be more sensitive than tracking diagnoses alone.

A simple monoclonal antibody based ELISA for free protein S. Comparison with PEG precipitation.

A simple monoclonal antibody based ELISA for free Protein S, compatible with out existing ELISA for total Protein S has been developed, and its performance compared with the conventional PEG precipitation method of free Protein S assay. The normal range (mean +/- 2 SD) was 0.19-0.54 iu/ml free Protein S. The mean intra assay variation was 5.24% and the mean inter assay variation was 5.50%. A total of 102 routine diagnostic samples from patients referred for prothrombotic investigation (six assays for each method) were assayed by PEG precipitation (mean 0.32 iu/ml, SD 10.60), and the monoclonal ELISA (mean 0.34, SD 0.9). Paired t-test analysis of the two data sets indicated no significant difference between them (P < 0.001). In this sample population, there was no significant difference in free Protein S values when assayed by monoclonal based ELISA or by PEG precipitation. The monoclonal assay has proved to be reliable, accurate and precise. The monoclonal ELISA is simpler, quicker and easier to perform in routine use. Data generated is directly comparable to that generated by PEG precipitation. This methodology would be suitable for laboratories currently measuring Protein S by ELISA.

Thrombelastographic evaluation of coagulation in transurethral prostatectomy.

OBJECTIVE: To assess the changes in overall coagulation status and define the degree of systemic fibrinolysis occurring in patients undergoing transurethral prostatectomy (TURP). PATIENTS AND METHODS: Thirty patients undergoing TURP, 23 for benign prostatic hyperplasia and seven for prostatic carcinoma, were studied prospectively. Serial venous blood samples were taken using the two-syringe technique. Samples were taken before, during and at intervals up to 72 h and 10-14 days after surgery. Thrombelastography (TEG) was performed on native whole blood samples. Peri-operative blood loss was assessed, until the catheter was removed, by photometric estimation of the haemoglobin content of the irrigant fluid and the measurement of clot volume. RESULTS: There was no evidence of fibrinolysis (TEG Percentage Clot Lysis Ly60 > 15%) in any patient over the whole peri-operative period. There was a significant change in the mean TEG variables towards hypercoagulation from 3 h until 10-14 days postoperatively, compared with the pre-operative values (P < 0.05). There was a significant correlation between blood loss and clot volume. CONCLUSION: These results question the role of systemic fibrinolysis in primary and secondary haemorrhage following TURP and thus the rationale of using antifibrinolytics in these patients. The persistent hypercoagulable state post-operatively indicates a possible role of hypercoagulability in clot retention.