Determination of a HIV protease inhibitor (DMP 450) in animal and human plasma by solid-phase extraction and high-performance liquid chromatography

Extraction of DMP 450 from plasma was performed with C₂ solid-phase extraction columns, using 0.1 M ammonium acetate in 90% methanol to elute DMP 450. The extraction recovery over the range of 10 to 10 000 ng/ml averaged 81.0, 96.2, 77.4, 95.2 and 68.0% from rat, dog, monkey, chimpanzee (25–10 000 ng/ml) and human plasma, respectively. HPLC analysis was carried out with a C₁₈ column and a mobile phase of acetonitrile, methanol and 30 mM potassium phosphate (pH 3), the composition dependent on the type of plasma being analyzed, and monitored at a wavelength of 229 nm. Intra-day and inter-day coefficients of variation were less than 9.9 and 12.9%, respectively. Absolute differences were less than 11.5%.     

Research letter: Predictive value of a self-reported history of varicella infection in determining immunity in adults

THE RECENT INTRODUCTION OF A VACCINE FOR VARICELLA has raised questions about whether, for adults, a patient''s history of varicella infection is useful in determining if vaccination is necessary. We report findings on 184 family medicine patients aged 18 to 65 years who were asked if they had a history of varicella infection and were subsequently tested for varicella antibodies. A history of infection was positive for 114 (62%) of the participants and negative or uncertain for 70 (38%). All 114 subjects who reported a varicella infection history were immune. All 4 subjects who were not immune reported an uncertain or negative infection history. Except for people who are at increased risk of varicella infection or complications from infection, serologic testing may not be required for adults in the general population who have a history of varicella infection. Varicella infection (chickenpox) is highly contagious¹ and is spread by respiratory droplets or direct contact. Varicella can occur in nonimmune adults, in whom severity of the infection increases with age,² often causing serious morbidity and loss of work time. In Canada, 70% of 53 reported deaths caused by varicella between 1987 and 1996 occurred in adolescents and adults.³ When acquired during pregnancy, varicella can cause significant maternal, perinatal and infant morbidity.³ After varicella infection, more than 95% of people develop antibodies,⁴ which are detected by serologic testing and indicate lifelong immunity.The recent introduction of a vaccine for varicella means that nonimmune adults may benefit from vaccination.⁵ This has led to studies to determine whether a self-reported history of varicella infection is an accurate indication of the presence of antibodies. Studies to date have yielded disparate conclusions and recommendations,^{6,7,8,9,10,11} and no study has yet investigated this question for adults in a primary care population. Our objective was to determine, in an adult primary care population, the accuracy of a self-reported history of varicella infection in determining immunity.St. Michael''s Hospital Family Practice Unit serves a population with a wide range of socioeconomic groups and countries of origin. The mean patient age is 44 years, and 60% of patients are female. On randomly selected clinic dates between October and December 2000 we enrolled patients aged 18 to 65 years who were having blood taken for reasons other than serologic testing for varicella antibodies. Using a structured interview, patients were asked about their history of varicella infection (Box 1), and their blood samples were analyzed for antibody titres using the VZVscan latex agglutination test (Becton Dickinson, Cockeysville, Md.). We excluded patients with psychiatric or medical conditions impairing memory, a history of varicella vaccination or active varicella infection. All patients gave informed consent, and the study was approved by the St. Michael''s Hospital Research Ethics Board.Open in a separate windowBox 1A sample size of 200 patients provides power to detect a 95% confidence interval (CI) of ± 3% if the baseline positive predictive value of a history of varicella is 95%.Of 204 participants enrolled, 184 had a serologic test for varicella antibodies. For unknown reasons, the serologic test was not performed in the remaining 20 cases.Of the 184 participants whose blood sample was tested for varicella antibodies, 101 (55%) were women, 117 (64%) were born in North America, and 107 (58%) had postsecondary education. The mean age was 43 (standard deviation 12.8) years. Participants for whom serologic testing was not done had similar characteristics.All 114 (62%) of the subjects who reported a history of varicella infection were immune, for a positive predictive value of 100% (95% CI 97%–100%) (Open in a separate windowIn this study, a self-reported history of varicella infection was a highly accurate indicator of immunity to the pathogen. The 98% (180/184) seroprevalence of varicella–zoster virus antibodies in our study population was slightly higher than other population estimates,² and consequently a large majority of those who were uncertain of or reported no past infection were also immune.Although we recruited participants from only 1 family medicine clinic in an urban teaching centre, the patient population of the clinic is diverse in terms of patient country of origin, socioeconomic status and demographic characteristics.Whether particular vaccination strategies are appropriate or cost-effective depends on the population examined and their circumstances. The costs of serologic testing and vaccination and the potential financial, social and medical consequences of infection should be considered.For people who are at increased risk of varicella infection or for whom it is crucial to establish immunity, such as health care workers,^9,10,12,13 pregnant women^7,11,14 and household contacts of immunocompromised people, it may be prudent to have them undergo routine serologic testing regardless of their self-reported infection history.In this primary care setting, a positive history of varicella infection was an accurate indicator of the presence of antibodies. Except for people at high risk of varicella infection, serologic testing may not be required for adults in the general population who have had the infection. Vaccination should be offered to nonimmune patients.     

Mu opioid receptor: a gateway to drug addiction

Mu opioid receptors mediate positive reinforcement following direct (morphine) or indirect (alcohol, cannabinoids, nicotine) activation, and our understanding of mu receptor function is central to the development of addiction therapies. Recent data obtained in native neurons confirm that mu receptor signaling and regulation are strongly agonist-dependent. Current functional mapping reveals morphine-activated neurons in the extended amygdala and early genomic approaches have identified novel mu receptor-associated proteins. A classification of about 30 genes either promoting or counteracting the addictive properties of morphine is proposed from the analysis of knockout mice data. The targeting of effectors or regulatory proteins, beyond the mu receptor itself, might provide valuable strategies to treat addictive disorders.     

Impaired NO signaling in small pulmonary arteries of chronically hypoxic newborn piglets

We performed studies to determine whether chronic hypoxia impairs nitric oxide (NO) signaling in resistance level pulmonary arteries (PAs) of newborn piglets. Piglets were maintained in room air (control) or hypoxia (11% O(2)) for either 3 (shorter exposure) or 10 (longer exposure) days. Responses of PAs to a nonselective NO synthase (NOS) antagonist, N(omega)-nitro-L-arginine methylester (L-NAME), a NOS-2-selective antagonist, aminoguanidine, and 7-nitroindazole, a NOS-1-selective antagonist, were measured. Levels of NOS isoforms and of two proteins involved in NOS signaling, heat shock protein (HSP) 90 and caveolin-1, were assessed in PA homogenates. PAs from all groups constricted to L-NAME but not to aminoguanidine or 7-nitroindazole. The magnitude of constriction to L-NAME was similar for PAs from control and hypoxic piglets of the shorter exposure period but was diminished for PAs from hypoxic compared with control piglets of the longer exposure period. NOS-3, HSP90, and caveolin-1 levels were similar in hypoxic and control PAs. These findings indicate that NOS-3, but not-NOS 2 or NOS-1, is involved with basal NO production in PAs from both control and hypoxic piglets. After 10 days of hypoxia, NO function is impaired in PAs despite preserved levels of NOS-3, HSP90, and caveolin-1. The development of NOS-3 dysfunction in resistance level PAs may contribute to the progression of chronic hypoxia-induced pulmonary hypertension in newborn piglets.     

Arachidonic acid metabolites and an early stage of pulmonary hypertension in chronically hypoxic newborn pigs

Our purpose was to determine whether production of arachidonic acid metabolites, particularly cyclooxygenase (COX) metabolites, is altered in 100-400-microm-diameter pulmonary arteries of piglets at an early stage of pulmonary hypertension. Piglets were raised in either room air (control) or hypoxia for 3 days. A cannulated artery technique was used to measure responses of 100-400-microm-diameter pulmonary arteries to arachidonic acid, a prostacyclin analog, or the thromboxane mimetic. Radioimmunoassay was used to determine pulmonary artery production of thromboxane B(2) (TxB(2)) and 6-keto-prostaglandin F(1alpha) (6-keto-PGF(1alpha)), the stable metabolites of thromboxane and prostacyclin, respectively. Assessment of abundances of COX pathway enzymes in pulmonary arteries was determined by immunoblot technique. Arachidonic acid induced less dilation in pulmonary arteries from hypoxic than in pulmonary arteries from control piglets. Pulmonary artery responses to prostacyclin and were similar for both groups. 6-Keto-PGF(1alpha) production was reduced, whereas TxB(2) production was increased in pulmonary arteries from hypoxic piglets. Abundances of both COX-1 and prostacyclin synthase were reduced, whereas abundances of both COX-2 and thromboxane synthase were unaltered in pulmonary arteries from hypoxic piglets. At least partly due to altered abundances of COX pathway enzymes, a shift in production of arachidonic acid metabolites, away from dilators toward constrictors, may contribute to the early phase of chronic hypoxia-induced pulmonary hypertension in newborn piglets.     

Comparison of detection methods for liquid chromatographic determination of 3-nitro-l-tyrosine

A liquid chromatographic method has been developed for the determination of 3-nitro-l-tyrosine. Different detection methods, including UV, oxidative and redox electrochemistry, and postcolumn photolysis followed by electrochemical detection, have been optimized and compared in terms of analysis time, detection limit and dynamic range. It was demonstrated that liquid chromatography with postcolumn photolysis followed by electrochemical detection is the most effective method, with an analysis time of 5 min, detection limit of 0.01 pmol, and a linear dynamic range from 2 nM to 100 μM.     

Differential Gene Expression Profiling of Dystrophic Dog Muscle after MuStem Cell Transplantation

BackgroundSeveral adult stem cell populations exhibit myogenic regenerative potential, thus representing attractive candidates for therapeutic approaches of neuromuscular diseases such as Duchenne Muscular Dystrophy (DMD). We have recently shown that systemic delivery of MuStem cells, skeletal muscle-resident stem cells isolated in healthy dog, generates the remodelling of muscle tissue and gives rise to striking clinical benefits in Golden Retriever Muscular Dystrophy (GRMD) dog. This global effect, which is observed in the clinically relevant DMD animal model, leads us to question here the molecular pathways that are impacted by MuStem cell transplantation. To address this issue, we compare the global gene expression profile between healthy, GRMD and MuStem cell treated GRMD dog muscle, four months after allogenic MuStem cell transplantation.ResultsIn the dystrophic context of the GRMD dog, disease-related deregulation is observed in the case of 282 genes related to various processes such as inflammatory response, regeneration, calcium ion binding, extracellular matrix organization, metabolism and apoptosis regulation. Importantly, we reveal the impact of MuStem cell transplantation on several molecular and cellular pathways based on a selection of 31 genes displaying signals specifically modulated by the treatment. Concomitant with a diffuse dystrophin expression, a histological remodelling and a stabilization of GRMD dog clinical status, we show that cell delivery is associated with an up-regulation of genes reflecting a sustained enhancement of muscle regeneration. We also identify a decreased mRNA expression of a set of genes having metabolic functions associated with lipid homeostasis and energy. Interestingly, ubiquitin-mediated protein degradation is highly enhanced in GRMD dog muscle after systemic delivery of MuStem cells.ConclusionsOverall, our results provide the first high-throughput characterization of GRMD dog muscle and throw new light on the complex molecular/cellular effects associated with muscle repair and the clinical efficacy of MuStem cell-based therapy.     

Developing the Bias Blind Spot: Increasing Skepticism towards Others

Two experiments with eighty-eight 7- to 10-year-olds examined the bias blind spot in children. Both younger and older children rated themselves as less likely than a specific other (Experiment 1) or an average child (Experiment 2) to commit various biases. These self-other differences were also more extreme for biased behaviors than for other behaviors. At times, older children demonstrated stronger self-other differences than younger children, which seemed primarily driven by older children’s judgments about bias in others. These findings suggest that, although the bias blind spot exists as soon as children recognize other-committed biases, what changes over development is how skeptical children are towards others.     

Impact of Community Based Peer Support in Type 2 Diabetes: A Cluster Randomised Controlled Trial of Individual and/or Group Approaches

Background

Diabetes peer support, where one person with diabetes helps guide and support others, has been proposed as a way to improve diabetes management. We have tested whether different diabetes peer support strategies can improve metabolic and/or psychological outcomes.

Methods

People with type 2 diabetes (n = 1,299) were invited to participate as either ‘peer’ or ‘peer support facilitator’ (PSF) in a 2x2 factorial randomised cluster controlled trial across rural communities (130 clusters) in England. Peer support was delivered over 8–12 months by trained PSFs, supported by monthly meetings with a diabetes educator. Primary end point was HbA1c. Secondary outcomes included quality of life, diabetes distress, blood pressure, waist, total cholesterol and weight. Outcome assessors and investigators were masked to arm allocation. Main factors were 1:1 or group intervention. Analysis was by intention-to-treat adjusting for baseline.

Results

The 4 arms were well matched (Group n = 330, 1:1(individual) n = 325, combined n = 322, control n = 322); 1035 (79•7%) completed the mid-point postal questionnaire and 1064 (81•9%) had a final HbA1c. A limitation was that although 92.6% PSFs and peers were in telephone contact, only 61.4% of intervention participants attended a face to face session. Mean baseline HbA1c was 57 mmol/mol (7•4%), with no significant change across arms. Follow up systolic blood pressure was 2•3mm Hg (0.6 to 4.0) lower among those allocated group peer-support and 3•0mm Hg (1.1 to 5.0) lower if the group support was attended at least once. There was no impact on other outcomes by intention to treat or significant differences between arms in self-reported adherence or medication.

Conclusions

Group diabetes peer support over 8–12 months was associated with a small improvement in blood pressure but no other significant outcomes. Long term benefits should be investigated.

Trial Registration

ISRCTN.com ISRCTN6696362166963621