Clinical and microbiological evaluation of epidural and regional anaesthesia catheters in injured UK military personnel.

INTRODUCTION: The adoption of regional and epidural analgesia in UK military personnel injured in action during Op HERRICK increased from 2008, in line with structural and environmental developments in the UK medical treatment facility. Historically, there have been concerns that invasive analgesic techniques could carry an increased risk of infection, due to the mechanism of injury and the environmental conditions in which the injuries were sustained. Consequently, the epidural and continuous peripheral nerve blockade (CPNB) catheters that were inserted in UK military personnel during a 33-month period of Op HERRICK were clinically and microbiologically examined, after subsequent admission to the University Hospitals Birmingham (UHB) NHS Trust. METHODS: Data on epidural and CPNB insertions were collected via the specialist pain service at UHB over the study period, including de novo and replacement insertions performed in both Afghanistan and the UK. Patients were regularly reviewed and relevant clinical concerns were documented in patients' case notes as necessary. The anatomical site, duration of placement and the results of microbiological culture of the epidural and CPNB catheter tips were all recorded. RESULTS: Overall, 236 catheters were assessed, of which 151 catheter tips (64%) were cultured (85 epidural, 66 CPNB). Of these, 48 grew bacteria (34% of cultured epidurals and 29% of cultured CPNB). There was no difference between the colonisation rates of epidurals inserted in Afghanistan and the UK. Only one infection related to a misplaced epidural catheter was confirmed. CONCLUSIONS: With the exception of the epidural (34%) and proximal sciatic (42%) catheters, these figures, in a military cohort characterised by significant injury scores, are consistent with those reported for civilian surgical patients. The results strongly support the expansion of regional analgesia during Op HERRICK from 2008 onwards. The outcomes suggest a possible translation into civilian major trauma practice.

Increased function of pronociceptive TRPV1 at the level of the joint in a rat model of osteoarthritis pain.

OBJECTIVES: Blockade of transient receptor potential vanilloid 1 (TRPV1) with systemic antagonists attenuates osteoarthritis (OA) pain behaviour in rat models, but on-target-mediated hyperthermia has halted clinical trials. The present study investigated the potential for targeting TRPV1 receptors within the OA joint in order to produce analgesia. METHODS: The presence of TRPV1 receptors in human synovium was detected using western blotting and immunohistochemistry. In a rat model of OA, joint levels of an endogenous ligand for TRPV1, 12-hydroxy-eicosatetraenoic acid (12-HETE), were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Effects of peripheral administration of the TRPV1 receptor antagonist JNJ-17203212 on afferent fibre activity, pain behaviour and core body temperature were investigated. Effects of a spinal administration of JNJ-17203212 on dorsal horn neuronal responses were studied. RESULTS: We demonstrate increased TRPV1 immunoreactivity in human OA synovium, confirming the diseased joint as a potential therapeutic target for TRPV1-mediated analgesia. In a model of OA pain, we report increased joint levels of 12-HETE, and the sensitisation of joint afferent neurones to mechanical stimulation of the knee. Local administration of JNJ-17203212 reversed this sensitisation of joint afferents and inhibited pain behaviour (weight-bearing asymmetry), to a comparable extent as systemic JNJ-17203212, in this model of OA pain, but did not alter core body temperature. There was no evidence for increased TRPV1 function in the spinal cord in this model of OA pain. CONCLUSIONS: Our data provide a clinical and mechanistic rationale for the future investigation of the therapeutic benefits of intra-articular administration of TRPV1 antagonists for the treatment of OA pain.

Calcitonin gene-related peptide receptor antagonists: beyond migraine pain--a possible analgesic strategy for osteoarthritis?

Osteoarthritis (OA) pain is poorly understood and managed, as current analgesics have only limited efficacy and unwanted side effect profiles. A broader understanding of the pathological mechanisms driving OA joint pain is vital for the development of improved analgesics. Both clinical and preclinical data suggest an association between joint levels of the sensory neuropeptide calcitonin gene-related peptide (CGRP) and pain during OA. Whether a direct causative link exists remains an important unanswered question. Given the recent development of small molecule CGRP receptor antagonists with clinical efficacy against migraine pain, the interrogation of the role of CGRP in OA pain mechanisms is extremely timely. In this article, we provide the background to the importance of CGRP in pain mechanisms and review the emerging clinical and preclinical evidence implicating a role for CGRP in OA pain. We suggest that the CGRP receptor antagonists developed for migraine pain warrant further investigation in OA.

Parallel tagged amplicon sequencing reveals major lineages and phylogenetic structure in the North American tiger salamander (Ambystoma tigrinum) species complex.

Modern analytical methods for population genetics and phylogenetics are expected to provide more accurate results when data from multiple genome-wide loci are analysed. We present the results of an initial application of parallel tagged sequencing (PTS) on a next-generation platform to sequence thousands of barcoded PCR amplicons generated from 95 nuclear loci and 93 individuals sampled across the range of the tiger salamander (Ambystoma tigrinum) species complex. To manage the bioinformatic processing of this large data set (344 330 reads), we developed a pipeline that sorts PTS data by barcode and locus, identifies high-quality variable nucleotides and yields phased haplotype sequences for each individual at each locus. Our sequencing and bioinformatic strategy resulted in a genome-wide data set with relatively low levels of missing data and a wide range of nucleotide variation. structure analyses of these data in a genotypic format resulted in strongly supported assignments for the majority of individuals into nine geographically defined genetic clusters. Species tree analyses of the most variable loci using a multi-species coalescent model resulted in strong support for most branches in the species tree; however, analyses including more than 50 loci produced parameter sampling trends that indicated a lack of convergence on the posterior distribution. Overall, these results demonstrate the potential for amplicon-based PTS to rapidly generate large-scale data for population genetic and phylogenetic-based research.

Potential sources of bacteriological pollution for two bays with marinas in Trinidad

Welcome Bay and Chaguaramas Bay in the northwest peninsula of Trinidad contain large marinas and smaller sections of bathing beaches. Bacteriological surveys were conducted at both bays to assess water quality and to determine potential sources of pollution. These surveys were conducted during the wet season of 1996 and the dry season of 1997. Eleven sample stations were established at Welcome Bay and 12 at Chaguaramas Bay. Freshwater samples were collected from rivers and drains within the survey area. Marine water samples were collected from marinas, bathing beaches and inshore and outer areas at both bays. Five water samples were collected from each sampling station during the wet season of 1996 and six during the dry season of 1997. The membrane filter technique was used to determine faecal coliform and Escherichia coli levels in all samples. There was a seasonal effect on water quality, with significantly higher faecal coliform levels in the wet season, when water quality was not in compliance with international standards. This represents a potential health risk in bathing areas. Water quality was better at the outer area of both bays. Water quality at the inner bay areas was most likely adversely affected by land-based sources of pollution identified in this study. These sources include three drains and two rivers, which discharged into the bays. Yachts were apparently not a source of sewage pollution: there was no significant relationship between yacht number and faecal coliform levels

Sensitization of adenylate cyclase induced by a dopamine D2 receptor mutant: inverse agonism by D2 receptor antagonists.

Mutations of residues in the third intracellular loops of several G-protein coupled receptors have been shown to confer constitutive activation. The authors investigated the effects of one such mutation in the dopamine D2 receptor. Compared to the wild type D2, the mutant D2 receptor (D2T344K) showed a substantial increase in agonist affinity with affinity for antagonists unchanged. The increased agonist affinity was unaffected by pertussis toxin treatment, indicating it is an intrinsic property of the mutant receptor. The potency of dopamine for acute inhibition of forskolin-stimulated cAMP production in stably expressing Chinese Hamster Ovary (CHO) cells was higher for the mutant than the wild type receptor. CHO cells stably expressing D2T344K displayed enhanced responses to forskolin-stimulated adenylate cyclase activity compared with cells stably expressing the wild type D2 receptor. The increased forskolin responsiveness of adenylate cyclase is similar to the sensitization previously observed with wild type D2 receptor after agonist treatment. Adenylate cyclase responsiveness of CHO cells stably expressing D2T344K receptor was not further increased by agonist treatment. Sensitization was blocked by pertussis toxin and D2 receptor antagonists haloperidol, butaclamol, and clozapine, indicating inverse agonist activity of these compounds at D2T344K. Inverse agonist activity was further demonstrated by the finding that overnight treatment with these compounds drastically increased the density of the mutant receptor but had minimal effect on the density of the wild type receptor. Taken together, these results suggest the authors have generated a constitutively active dopamine D2 receptor capable of sensitizing adenylate cyclase in the absence of agonist activation.

Localization and quantification of 5-hydroxytryptophan and serotonin in the central nervous systems of Tritonia and Aplysia.

Serotonin (5-hydroxytryptamine, 5-HT) plays a central role in several behaviors in marine molluscs and other species. In an effort to better understand the regulation of 5-HT synthesis, we used high performance liquid chromatography (HPLC) with electrochemical detection and immunohistochemistry to measure and map the distribution of the immediate precursor of 5-HT, 5-hydroxytryptophan (5-HTP), in two model opisthobranch molluscs, the nudibranch Tritonia diomedea and the anaspid Aplysia californica. HPLC measurements showed that 5-HTP is present at approximately the same level as the 5-HT metabolite, 5-hydroxyindolacetic acid (5-HIAA) but is more than 100 times lower in concentration than either 5-HT or dopamine in the same tissue. Specific 5-HTP immunoreactivity was colocalized with serotonin in both species. The overall intensity of 5-HTP immunoreactivity in individual ganglia agreed with HPLC measurements for those ganglia. The intensity of 5-HTP immunolabeling varied between cell types and was correlated with the intensity of 5-HT immunolabeling. In particular, differences in staining intensity were consistently seen among the three dorsal swim interneurons of the Tritonia swim central pattern generator circuit. Some nonserotonergic neurons also displayed low levels of 5-HTP immunolabeling that were above background levels. Together, these results support the notion that production of 5-HTP is a rate-limiting step in serotonin synthesis and suggest that there may be additional regulation that allows 5-HTP to accumulate to varying levels.

A case-control study of differences between regular and causal adult attenders in general dental practice.

OBJECTIVES: To assess whether adults attending a dental practice for regular dental care have better oral health than adults attending casually in response to a dental problem, and to explore the barriers to asymptomatic attendance. METHODS: An observational case-control study comparing the characteristics of 100 regular attenders with 100 causal attenders in one general dental practice in North Staffordshire. All study subjects were aged 18 years or over. Sociodemographic characteristics of the two groups were collated, including age, gender, social class, marital status, employment status and smoking status. The primary outcome measure was the observed number of teeth with dentinal caries diagnosed using bitewing radiographs. RESULTS: Regular attenders were observed to have better oral health with respect to dental caries and tooth mobility (p < 0.05). This was not explained by the observation that causal attenders were more likely than regular attenders to be male, aged 18-44 years, in social class III or IV. Secondary outcomes, including number of subjects with mobile teeth and teeth with > 30% bone-loss, were also significantly worse in the casual attenders. However, the median number of teeth present in both groups was 27. In regular attenders, the most common reason for attending was to 'keep the teeth' (96%). In casual attenders, 'fear/dislike of dental treatment' was the most frequent indicator of non-attendance (56%). CONCLUSIONS: In our study, adults who regularly attended general dental practice were shown to have better oral health, including less overall tooth decay, mobility and bone-loss, compared with adults who did not attend on a regular basis. Assuming this result to be externally valid, a challenge for the dental profession in the future will be to develop effective oral health promotion initiatives.

Regulation of transport of the dopamine D1 receptor by a new membrane-associated ER protein.

Many structural determinants for G protein-coupled receptor (GPCR) functions have been defined, but little is known concerning the regulation of their transport from the endoplasmic reticulum (ER) to the cell surface. Here we show that a carboxy-terminal hydrophobic motif, FxxxFxxxF, which is highly conserved among GPCRs, functions independently as an ER-export signal for the dopamine D1 receptor. A newly identified ER-membrane-associated protein, DRiP78, binds to this motif. Overexpression or sequestration of DRiP78 leads to retention of D1 receptors in the ER, reduced ligand binding, and a slowdown in the kinetics of receptor glycosylation. Our results indicate that DRiP78 may regulate the transport of a GPCR by binding to a specific ER-export signal.

Urban-rural differences in the quality of care for medicare patients with acute myocardial infarction.

BACKGROUND: There are urban-rural differences in health care utilization in Kansas. This study was conducted to determine if similar differences exist in the quality of inpatient care provided for patients with acute myocardial infarction (AMI). METHODS: All acute care hospitals in the state were stratified into 12 urban, 31 semirural, and 76 rural hospitals according to their location. Data from medical records of 2521 Medicare patients 65 years and older who had survived AMI and were discharged alive from hospitals during an 8-month period in 1994/1995 were abstracted. The measures of the quality of care (quality indicators [QIs]) were the use of aspirin (during hospital stay and at discharge) and the administration of beta-blockers, intravenous (IV) nitroglycerin, heparin, and reperfusion by thrombolytic therapy or primary angioplasty. RESULTS: A significantly higher proportion of ideal candidates for the use of aspirin during hospital stay and at discharge, heparin, and IV nitroglycerin received these medications in urban hospitals, and a lower proportion of similar patients received these medications in rural hospitals compared with the patients in semirural hospitals (P<.001). Similar trends in each of the 6 QIs were observed for less than ideal patients (P<.05). Patient age was associated with a relatively poor quality of care in terms of the 6 QIs. Except for the administration of IV nitroglycerine to less than ideal patients, age adjustments did not change the observed urban-rural differences in the QI measures. CONCLUSION: Relatively poor quality of care for patients with AMI was provided by rural hospitals where greater opportunity for improvement exists.

Identification of two prokineticin cDNAs: recombinant proteins potently contract gastrointestinal smooth muscle.

The motility of gastrointestinal tract is regulated by classical neurotransmitters, neuropeptides, and humoral agents. Two novel human cDNAs have been cloned based on their sequence similarity to a frog skin secretion protein, Bv8, and a nontoxic protein of mamba snake venom. These human cDNAs encode two secreted proteins of 86 and 81 amino acids. Northern blot hybridization has revealed that these cDNAs are expressed in gastrointestinal tract, particularly the stomach. Recombinant proteins with authentic N-terminal sequences have been produced in Escherichia coli and refolded into functional proteins by careful control of protein aggregation. Mass spectrometry has confirmed the formation of five pairs of disulfide bonds. The refolded recombinant proteins potently contract gastrointestinal smooth muscle with EC(50) values in the subnanomolar range. The contractile effects of the recombinant proteins are specific for gastrointestinal smooth muscle, because they have no effect on vascular or respiratory smooth muscle. To reflect their potent and specific effects on gastrointestinal smooth muscle cells, we have named these recombinant proteins prokineticins. Ligand binding studies with iodinated prokineticin revealed the presence of a high-affinity site in ileal smooth muscle. The displacement of specific binding by GTP gamma S suggests that the prokineticin receptor may belong to the family of G protein-coupled receptors. Experiments with verapamil and nifedipine revealed that calcium influx is essential for the contractile activity of prokineticins on gastrointestinal smooth muscle. In summary, we have identified two novel endogenous regulators of gastrointestinal motility. The availability of recombinant prokineticins should provide novel therapeutic agents for disorders involving impaired gastrointestinal motility.

Site-specific gene targeting for gene expression in eukaryotes.

Major advances in the use of site-specific recombinases to facilitate sustained gene expression via chromosomal targeting have been made during the past year. New tools for genomic manipulations using this technology include the discovery of epitopes in recombinases that confer nuclear localization, crystal structures that show the precise topology of recombinase-DNA-substrate synaptic complexes, manipulations of the DNA recognition sequences that select for integration over excision of DNA, and manipulations that make changes in gene expression inducible by drug administration. In addition, endogenous eukaryotic and mammalian DNA sequences have been discovered that can support site-specific recombinase-mediated manipulations.

The role of the Advanced Casualty Management Team in St John Ambulance Australia (New South Wales District).

St John Ambulance is a household name synonymous with the teaching and provision of first aid. Recently the organisation has developed pre-hospital emergency care services through the introduction of the St John Ambulance Australia Advanced Casualty Management Team in New South Wales. The Advanced Casualty Management Team represents a move away from the practice of first aid by lay personnel and is a natural extension of the traditional work and principles of St John Ambulance. This article provides an overview of the Advanced Casualty Management Team and discusses its contribution to pre-hospital trauma care delivery.

Vascular inflammation inhibits gene transfer to the pulmonary circulation in vivo.

We report gene transfer to the normal and injured murine pulmonary circulation via systemic (intravascular) and airway (intratracheal) delivery of novel polycationic liposomes (imidazolium chloride, imidazolinium chloride-cholesterol, and ethyl phosphocholine). With use of the reporter genes chloramphenicol acetyltransferase (CAT) or human placental alkaline phosphatase (hpAP), intravascular injection of lipid-DNA complexes resulted in gene expression primarily in the lung, with lesser expression in the heart (11% of lung, P < 0.05) and spleen (8% of lung, P < 0.05). Histochemical staining for the hpAP reporter gene showed localized transgene expression in the microvascular endothelium. Monocrotaline (80 mg/kg body wt sc) treatment produced endovascular inflammation and reduced lung CAT activity (2 days postintravascular transfection) by 75 +/- 8 and 86 +/- 6% at 7 and 21 days, respectively, after monocrotaline (P < 0. 05). Despite the apparent decrease in functional CAT protein, Southern blot analysis suggested maintained plasmid delivery, whereas quantitative PCR (TaqMan) showed decreased CAT mRNA levels in monocrotaline mice. In contrast, intratracheal delivery of lipid-DNA complexes showed enhanced CAT expression in monocrotaline mice. Transfection in alternate pulmonary vascular disorders was studied by inducing hypoxic pulmonary hypertension (4 wk at barometric pressure of 410 mmHg). Efficiency and duration of gene transfer, assessed by CAT activity, were similar in pulmonary hypertensive and normal lungs. We conclude that imidazolinium-derived polycationic liposomes provide a means of relatively selective and efficient gene transfer to the normal and injured murine microvascular circulation, although translation of transgene mRNA may be reduced by preexisting endothelial injury.

A humanized anti-CD3 antibody, HuM291, with low mitogenic activity, mediates complete and reversible T-cell depletion in chimpanzees.

BACKGROUND: An anti-CD3 antibody that reduces cytokine release syndrome (CRS) while maintaining immunosuppression would be a major advance in the treatment of acute allograft rejection. A humanized (Hu) anti-CD3 IgG2 Ab, HuM291 gamma2 M3 (HuM291; Protein Design Labs, Inc., Mountain View, CA), was engineered with mutations in the upper CH2 region of the Fc domain. The mutations were intended to reduce affinity for Fcgamma receptors, thought to be relevant to CRS. METHODS: In vitro studies using chimpanzee peripheral blood mononuclear cells (PBMCs) were conducted to characterize HuM291 and to establish an animal model. A multidose study was conducted in chimpanzees to evaluate the safety, pharmacokinetics, immunomodulatory activity, and immunogenicity of HuM291, when administered at doses ranging from 0.1 to 10 mg. RESULTS: HuM291 bound to and effectively downmodulated CD3 from chimpanzee PBMCs and stimulated substantially less cytokine secretion and proliferation of chimpanzee PBMCs compared with OKT3 (Orthoclone OKT3; Ortho Pharmaceutical Corp., Raritan, NJ). Multiple doses of HuM291 (0.1, 1.0, or 10 mg/dose) were not associated with adverse events, signs of toxicity, or CRS, despite cytokine release. HuM291 exhibited a long elimination t1/2 (81.5 hr) and, after three 10-mg doses, sustained serum concentrations > 1000 ng/ml were maintained for 1 week. Multiple 10-mg doses induced complete depletion of circulating CD2+CD3+ T cells for up to 10 days after the last dose; T cells recovered by Day 28. Anti-HuM291 Abs were observed in only 4 of 12 animals and were transient in 2 of those animals. CONCLUSIONS: In vitro, HuM291 is substantially less mitogenic than OKT3. In chimpanzees, HuM291 effectively depleted peripheral T cells without eliciting clinical signs of CRS, and recovered T cells were functionally normal.

In vivo studies of gene expression via transient transgenesis using lipid-DNA delivery.

As the sequencing of the human genome proceeds, the need for a new screen for in vivo function is becoming apparent. Many investigators are turning to various transgenic models as a means of studying function. However, these approaches are very time consuming, with a transgene-expressing mouse model often taking months to establish. We have developed an efficient system for delivering genes in vivo, which allows the gene product to be studied as early as 24 h after introduction into the mouse model. The delivery system employs a novel cationic lipid, 1-[2-(9-(Z)-octadecenoyloxy)ethyl]-2-(8-(Z)-heptadecenyl)-3- (hydroxyethyl)imidazolinium chloride (DOTIM), and a neutral lipid, cholesterol, complexed with an expression vector containing the reporter gene chloramphenicol acetyl transferase (CAT). After a single intravenous injection of these complexes, several tissues were seen to express the transgene. High, persistent expression in the vascular endothelial cells in the mouse lung was obtained. Delivery of DNA in vivo has been evaluated by quantitative polymerase chain reaction and protein expression by CAT activity assays. In vivo studies showed reproducible expression in more than 500 mice injected via the tail vein. An early peak of expression was followed by lower, but sustained, expression for > 50 days. Transgene expression of CAT could also be identified by immunohistochemistry staining in mouse lung and appeared to be located within the capillaries. The pattern of in vivo expression could be modulated and targeted to specific organs by altering the lipid-DNA formulation. New expression vectors with altered introns and polyadenylation sites further improved expression. The expression reported here may be sufficient in magnitude, duration, and flexibility to be an attractive alternative, in some cases, to establishing transgenic animals by stable gene transfer.

Urban-rural differences in Medicare in Missouri.

Health Care Financing Administration's (HCFA) administrative data were analyzed to examine urban-rural differences in the characteristics of Medicare beneficiaries, their utilization of medical care, the providers of services, and selected outcomes in Missouri. As compared to the residents of urban areas, the residents of the rural counties were a little poorer, had equal access to health care, and utilized more health care services; but the cost of major services was lower.

Pulse oximetry in infants of < 1500 gm birth weight on supplemental oxygen: a national survey.

OBJECTIVE: To determine practices related to the use of pulse oximetry in monitoring infants of < 1500 gm birth weight on supplemental oxygen. STUDY DESIGN: A mailing list of all neonatal intensive care units with accredited Neonatal-Perinatal Fellowship programs was prepared. A questionnaire was prepared and mailed to collect information on the following: Method used for noninvasive monitoring of oxygen therapy, acceptable maximum and minimum arterial pulse oxygen saturation levels, high and low alarm settings, and whether oxygen was administered at a fixed or variable rate. RESULTS: A response rate of 70% to 85% was achieved for different items of the questionnaire. A wide variation exists regarding acceptable arterial pulse oxygen saturation levels and alarm settings. Many units accepted an arterial pulse oxygen saturation level of 100% or set the high alarm at 100%. CONCLUSION: There is a need for greater awareness of the potential for hyperoxemia that may result from accepting an arterial pulse oxygen saturation level of 100% or setting high alarms at 100%. We urge stricter adherence to published recommendations.