Rapid detection and identification of human adenovirus species by adenoplex, a multiplex PCR-enzyme hybridization assay

Human adenoviruses (AdV) have been implicated in a wide variety of diseases and are ubiquitous in populations worldwide. These agents are of concern particularly in immunocompromised patients, children, and military recruits, resulting in severe disease or death. Clinical diagnosis of AdV is usually achieved through routine viral cell culture, which can take weeks for results. Immunofluorescence and enzyme-linked immunosorbent assay-based techniques are more timely but lack sensitivity. The ability to distinguish between the six different AdV species (A to F) is diagnostically relevant, as infections with specific AdV species are often associated with unique clinical outcomes and epidemiological features. Therefore, we developed a multiplex PCR-enzyme hybridization assay, the Adenoplex, using primers to the fiber gene that can simultaneously detect all six AdV species A through F in a single test. The limit of detection (LOD) based on the viral 50% tissue culture infective dose/ml for AdV A, B, C, D, E, and F was 10(-2), 10(-1), 10(-1), 10(-2), 10(-1), and 10(-2), respectively. Similarly, the LOD for the six DNA controls ranged from 10(2) to 10(3) copies/ml. Twelve common respiratory pathogens were tested with the Adenoplex, and no cross-reactivity was observed. We also validated our assay using clinical specimens spiked with different concentrations of AdV strains of each species type and tested by multiplex PCR and culture. The results demonstrated an overall sensitivity and specificity of Adenoplex of 100%. This assay can be completed in as few as 5 h and provides a rapid, specific, and sensitive method to detect and subtype AdV species A through F.     

A chronic mouse model of Parkinson's disease has a reduced gait pattern certainty

The purpose of this investigation was to determine if a chronic Parkinson's disease mouse model will display less certainty in its gait pattern due to basal ganglia dysfunction. A chronic Parkinson's disease mouse model was induced by injecting male C57/BL mice with 10 doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (25 mg/kg) (MPTP) and probenecid (250 mg/kg) (P) over 5 weeks. This chronic model produces a severe and persistent loss of nigrostriatal neurons resulting in dopamine depletion and locomotor impairment. The control mice were treated with probenecid alone. Fifteen weeks after the last MPTP/P treatment, the mice were videotaped in the sagittal plane with a digital camera (60 Hz) as they ran on a motorized treadmill at a speed of 10 m/min. The indices of gait and gait variability were calculated. Stride length was significantly (p = 0.016) more variable in the chronic MPTP/P mice. Additionally, the chronic MPTP/P mice had a statistically less certain gait pattern when compared to the control mice (p = 0.02). These results suggest that variability in the gait pattern can be used to evaluate changes in neural function. Additionally, our results imply that disorder of the basal ganglia results in less certainty in modulating the descending motor command that controls the gait pattern.     

A randomised controlled trial to test the feasibility of a collaborative care model for the management of depression in older people

BACKGROUND: Depression is the most common mental health disorder in people aged over 65 years. Late-life depression is associated with chronic illness and disability. AIM: To investigate the feasibility of a collaborative care model for depression in older people in a primary care setting. DESIGN OF STUDY: Randomised controlled trial with 16-weeks follow up. SETTING: A primary care trust in Manchester. METHOD: Participants were 105 people aged 60 years or older who scored 5 or more on the Geriatric Depression Scale; 53 were randomly allocated to an intervention group and 52 to a usual care group. The intervention group received care managed by a community psychiatric nurse who delivered an intervention comprising a facilitated self-help programme with close liaison with primary care professionals and old-age psychiatry according to a defined protocol. The usual care group received usual GP care. A nested qualitative study explored the views of the health professionals and patients regarding the acceptability and effectiveness of the intervention. RESULTS: The main outcome measure was recovery from depression. Patients in the intervention group were less likely to suffer from major depressive disorder at follow up compared with usual care (0.32, 95% confidence = interval = 0.11 to 0.93, P = 0.036). The qualitative component of the study demonstrated the acceptability of the intervention to patients. CONCLUSION: A model of collaborative care for older people with depression, used in a primary care setting with a facilitated self-help intervention is more effective than usual GP care. This study demonstrates that the implementation of a collaborative care model is feasible in UK primary care and that the intervention is effective and acceptable to patients.     

Engineering porcine arteries: effects of scaffold modification

Techniques have been developed to culture bovine or porcine vascular cells on polyglycolic acid (PGA) scaffolds to form engineered vessels. Previously, it was shown that smooth muscle cells (SMCs) that were in close proximity to PGA remnants after 8 weeks of culture had lower expression of SMC markers of differentiation and were more mitotic compared with SMCs that were distant from polymer residuals. Modifications of PGA were explored as a means to minimize residual polymer fragments after culture. To hasten degradation, polymer was treated with heat, NaOH, or gamma-irradiation. Differential scanning calorimetry, mass and tensile strength degradation, and inherent viscosity were used to assess polymer characteristics. When polymer was maintained in aqueous conditions, tensile strength of treated PGA degraded to zero within 3 weeks for each treatment. Engineered vessel constructs cultured on NaOH and gamma-treated polymer displayed smooth muscle alpha-actin throughout the vessel wall. Scaffold treatment impacted graft morphology, cellular differentiation, and mechanical integrity.     

Mode of delivery and risk of developing allergic disease

The aim of this study was to quantify the relationship between mode of delivery and subsequent incidence of allergic disease. The analysis is based on data derived from a birth cohort of 24,690 children who contributed data to the West Midlands General Practice Research Database. We found no convincing evidence to suggest that babies born by caesarean, forceps, or breech delivery had an increased risk of developing allergic disease.     

Frequency of cytokine polymorphisms in populations from western Europe,Africa, Asia,the Middle East and South America

PCR-SSOP identification procedures for IL-2, IL-6, IL-10, TNF-alpha and TNF-beta cytokine polymorphisms have been developed. Application of the procedures to a range of diverse geographically distributed populations has identified ethnic differences within the groups studied. Five populations were investigated, Northern Ireland, South African Zulu, Omani, Singapore Chinese and Mexican Mestizos.     

Knowledge about asthma and COPD: associations with sick leave, health complaints, functional limitations, adaptation, and perceived control

We sought to investigate associations between knowledge about the disease and sick leave, health complaints, functional limitations, adaptation and perceived control. Patients with asthma (n = 101) and COPD (n = 64) underwent lung function tests and completed questionnaires. In addition, all were asked the question: ‘what is the diagnosis of your disease?’, with the response categories: ‘asthma’ and ‘COPD (chronic bronchitis or emphysema)’. Thirty-five percent of the asthma patients and 30% of the COPD patients did not know their correct diagnosis. Sick leave was not associated with knowledge about the disease in asthma and COPD. In asthma, much knowledge about management of the disease was associated with better adaptation (P = 0.01) and less perceived control over health by external factors (P = 0.02). Knowing the correct diagnosis was associated with less control over health by powerful others (P = 0.02). For COPD, more knowledge about management of the disease was associated with better adaptation (P = 0.02) and less control over health by internal factors (P = 0.01). Knowing the correct diagnosis was associated with less control over dyspnea at work (P = 0.01).     

Foxl2 disruption causes mouse ovarian failure by pervasive blockage of follicle development

FOXL2 mutations cause gonadal dysgenesis or premature ovarian failure (POF) in women, as well as eyelid/forehead dysmorphology in both sexes (the 'blepharophimosis-ptosis-epicanthus inversus syndrome', BPES). Here we report that mice lacking Foxl2 recapitulate relevant features of human BPES: males and females are small and show distinctive craniofacial morphology with upper eyelids absent. Furthermore, in mice as in humans, sterility is confined to females. Features of Foxl2 null animals point toward a new mechanism of POF, with all major somatic cell lineages failing to develop around growing oocytes from the time of primordial follicle formation. Foxl2 disruption thus provides a model for histogenesis and reproductive competence of the ovary.     

Monkeypox virus detection in rodents using real-time 3'-minor groove binder TaqMan assays on the Roche LightCycler

During the summer of 2003, an outbreak of human monkeypox occurred in the Midwest region of the United States. In all, 52 rodents suspected of being infected with monkeypox virus were collected from an exotic pet dealer and from private homes. The rodents were euthanized and submitted for testing to the United States Army Medical Research Institute of Infectious Diseases by the Galesburg Animal Disease Laboratory, Illinois Department of Agriculture. The rodent tissue samples were appropriately processed and then tested by using an integrated approach involving real-time polymerase chain reaction (PCR) assays, an antigen-detection immunoassay, and virus culture. We designed and extensively tested two specific real-time PCR assays for rapidly detecting monkeypox virus DNA using the Vaccinia virus F3L and N3R genes as targets. The assays were validated against panels of orthopox viral and miscellaneous bacterial DNAs. A pan-orthopox electrochemiluminescence (ECL) assay was used to further confirm the presence of Orthopoxvirus infection of the rodents. Seven of 12 (58%) animals (seven of 52 (15%) of all animals) tested positive in both monkeypox-specific PCR assays and two additional pan-orthopox PCR assays (in at least one tissue). The ECL results showed varying degrees of agreement with PCR. One hamster and three gerbils were positive by both PCR and ECL for all tissues tested. In addition, we attempted to verify the presence of monkeypox virus by culture on multiple cell lines, by immunohistology, and by electron microscopy, with negative results. Sequencing the PCR products from the samples indicated 100% identity with monkeypox virus strain Zaire-96-I-16 (a human isolate from the Congo). These real-time PCR and ECL assays represent a significant addition to the battery of tests for the detection of various orthopoxviruses. In light of the recent monkeypox virus transmissions, early detection of the virus is crucial for both natural outbreaks and potential acts of bioterrorism.     

Structure and genomic sequence of the myotonic dystrophy (DM kinase) gene

The mutation causing myotonic dystrophy (DM) has recently beenidentified as an unstable CTG trinucleotide repeat located inthe 3' untranslated region of a gene encoding for a proteinwith putative serine-threonine protein kinase activity. In thisreport we present the genomic sequences of the human and murineDM kinase gene. A comparison of these sequences with each otherand with known cDNA sequences from both species, led us to predicta translation initiation codon, as well as determine the organizationof the DM kinase gene. Several polymorphisms within the humanDM kinase gene have been identified, and PCR assays to detecttwo of these are described. The complete sequence and characterizationof the structure of the DM kinase gene, as well as the identificationof novel polymorphisms within the gene, represent an importantstep in a further understanding of the genetics of myotonicdystrophy and the molecular biology of the gene.