Adeno-associated virus vector gene transfer and sarcolemmal expression of a 144 kDa micro-dystrophin effectively restores the dystrophin-associated protein complex and inhibits myofibre degeneration in nude/mdx mice

Duchenne muscular dystrophy is a severe life-threatening X-linked recessive disorder, caused by mutations in the dystrophin gene, for which currently there is no effective treatment. Because of the large size of the dystrophin cDNA (14 kb) this precluded it from being used in early adenovirus- or retrovirus-based gene therapy vectors. However, some therapeutic success has been achieved in mdx mice using adenovirus- and retrovirus-mediated transfer of a 6.3 kb recombinant mini-dystrophin cDNA. Despite this, problems with immunogenicity and inefficient transduction of mature myofibres make these vectors less than ideal for gene transfer to skeletal muscle. Adeno-associated viral (AAV) vectors overcome many of the problems associated with other vector systems. However, AAV vectors can only accommodate <5 kb of foreign DNA. For this reason we have produced a micro-dystrophin cDNA gene construct that is <3.8 kb. This construct, driven by a CMV promoter, was introduced into the skeletal muscle of 12-day-old nude/mdx mice using an AAV vector, resulting in specific sarcolemmal expression of micro-dystrophin in >50% of myofibres up to 20 weeks of age, and effective restoration of the dystrophin-associated protein (DAP) complex components. Additionally, evaluation of central nucleation indicated a significant inhibition of degenerative dystrophic muscle pathology. We have therefore shown that the current micro-dystrophin gene delivered in vivo using an AAV vector is not only capable of restoring sarcolemmal DAP complexes, but can also ameliorate dystrophic pathology at the cellular level.     

Impact of depressive symptoms on adult asthma outcomes.

BACKGROUND: Psychological disorders, including depression, are common in adults with asthma. Although depression is treatable, its impact on longitudinal asthma outcomes is not clear. OBJECTIVE: To elucidate the impact of depressive symptoms on patient-centered outcomes and emergency health care use in adults with asthma. METHODS: We conducted a prospective cohort study of 743 adults with asthma who were recruited after hospitalization for asthma. Depressive symptoms were defined as having a score of 16 or more on the Center for Epidemiologic Studies Depression Scale. We examined the impact of depressive symptoms on patient-centered outcomes (validated severity-of-asthma score, Marks Asthma Quality of Life Questionnaire, and 12-Item Short-Form Health Survey physical component summary score) and on future emergency health care use for asthma ascertained from computerized databases. RESULTS: The prevalence of depressive symptoms was 18% (95% confidence interval [CI], 15%-21%) among adults with asthma. Depressive symptoms were associated with greater severity-of-asthma scores after controlling for age, sex, race/ ethnicity, educational attainment, and cigarette smoking (mean score increment, 2.6 points; 95% CI, 1.8-3.4 points). Furthermore, depressive symptoms were associated with poorer asthma-specific quality of life (mean score increment, 19.9 points; 95% CI, 17.7-22.1 points) and poorer physical health status (mean score decrement, 3.7 points; 95% CI, 1.5-5.8 points). Depressive symptoms were associated with a greater longitudinal risk of hospitalization for asthma (hazard ratio, 1.34; 95% CI, 0.98-1.84). After controlling for differences in preventive care for asthma, the relationship was stronger (hazard ratio, 1.45; 95% CI, 1.05-2.0). CONCLUSION: Depressive symptoms are common in adults with asthma and are associated with poorer health outcomes, including greater asthma severity and risk of hospitalization for asthma.     

Phase Variation of Campylobacter jejuni 81-176 Lipooligosaccharide Affects Ganglioside Mimicry and Invasiveness In Vitro

The outer cores of the lipooligosaccharides (LOS) of many strains of Campylobacter jejuni mimic human gangliosides in structure. A population of cells of C. jejuni strain 81-176 produced a mixture of LOS cores which consisted primarily of structures mimicking GM(2) and GM(3) gangliosides, with minor amounts of structures mimicking GD(1b) and GD(2). Genetic analyses of genes involved in the biosynthesis of the outer core of C. jejuni 81-176 revealed the presence of a homopolymeric tract of G residues within a gene encoding CgtA, an N-acetylgalactosaminyltransferase. Variation in the number of G residues within cgtA affected the length of the open reading frame, and these changes in cgtA corresponded to a change in LOS structure from GM(2) to GM(3) ganglioside mimicry. Site-specific mutation of cgtA in 81-176 resulted in a major LOS core structure that lacked GalNAc and resembled GM(3) ganglioside. Compared to wild-type 81-176, the cgtA mutant showed a significant increase in invasion of INT407 cells. In comparison, a site-specific mutation of the neuC1 gene resulted in the loss of sialic acid in the LOS core and reduced resistance to normal human serum but had no affect on invasion of INT407 cells.     

Population-based case control study of seroprevalence of Mycobacterium paratuberculosis in patients with Crohn's disease and ulcerative colitis

There is renewed enthusiasm for exploring the possibility that Mycobacterium paratuberculosis may be causative in Crohn's disease (CD). We aimed to determine whether CD subjects are more likely to be M. paratuberculosis seropositive than controls. Using our population-based University of Manitoba Inflammatory Bowel Disease Research Registry, we recruited CD and ulcerative colitis (UC) subjects between 18 and 50 years of age for a study involving detailed questionnaires and venipuncture. We accessed the population-based databases of Manitoba Health (single provincial health insurer) to get age-, gender-, and geography-matched controls to our inflammatory bowel disease (IBD) population. We asked enrolling IBD subjects for potential nonaffected sibling controls. We used an enzyme-linked immunosorbent assay (ELISA) for serum antibodies to M. paratuberculosis initially developed for cattle but adapted for human use. The rate of positive ELISA results, based on previously published interpretation criteria, was significantly higher for all study groups. There was no difference in M. paratuberculosis seropositivity rate among CD patients (37.8%; n = 283), UC patients (34.7%; n = 144), healthy controls (33.6%; n = 402), and nonaffected siblings (34.1%; n = 138). For siblings, there was no correlation between M. paratuberculosis serological status and that of the corresponding IBD affected sibling. None of the demographic or questionnaire variables studied were predictive of M. paratuberculosis status. Subjects with CD and UC were less likely to have ingested unpasteurized milk and less likely to have had a non-tap water source as a primary water source. In conclusion, in this population-based case control study, the M. paratuberculosis seropositivity rate was approximately 35% for all groups and there was no difference in rates between CD patients, UC patients, healthy controls, or nonaffected siblings. The much higher rate of seropositivity for subjects from Manitoba, Canada, than for those from Denmark or Wisconsin cannot be obviously explained. While these data seem to refute any association of CD with M. paratuberculosis, the high seroprevalence in Manitobans raises the possibility that the high rates of CD in Manitoba could be related to high exposure rates for M. paratuberculosis. Hence, the possibility of an association between M. paratuberculosis and CD remains inconclusive.     

Video analysis of communication in paediatric consultations in primary care.

BACKGROUND: There is a paucity of research evidence concerning communication in paediatric consultations between GPs, adults, and child patients. AIM: This study was carried out to identify features of the interaction between a doctor, a child patient aged 6-12 years, and their carer in the consultation associated with the child's participation. DESIGN OF STUDY: A qualitative analysis of video recordings of 31 primary care paediatric consultations was undertaken, using strategies from the methodology of conversation analysis. SETTING: Primary care, Suffolk, UK. METHOD: NHS GPs from three primary care trusts (PCTs), were invited to participate in this study. Sixteen volunteers from this sample took part. RESULTS: Analysis of the interaction in the consultations revealed that the children had little involvement. Children participated when invited to do so, and took more time than adults to answer a doctor's question. An adult carer was less likely to answer on behalf of a child, when they were in a position to see that the doctor's gaze was directed at the child, and the doctor addressed the child by name. Adult carers, who had not voiced their own concerns first, were seen to interrupt doctor-child talk. In consultations where the participants sat in a triangular arrangement, all parties being an equal distance apart, triadic talk was noted. CONCLUSION: Child involvement in the primary care consultation is associated with adult carers being able to voice their own concerns early in the consultation, and children being invited to speak with the appropriate recipient design.     

The antigenicity of sequential polypeptides: II. The antigenicity of some sequential polymers including several related to collagen

The antigenicity in guinea-pigs of a series of sequential polymers has been studied. Almost all the polymers had a repeating sequence of three amino acids and with few exceptions glycine was one of the three residues and proline or a proline homologue one of the others. As a result the majority of the peptides used probably possessed at least in part, the collagen type of triple helix, although with the exception of (Gly-Pro-Gly)_n all these polymers were antigenic inducing both a humoral and cell-mediated response.With (D-Ala-Gly-D-Pro)_n no humoral response was obtained but there were excellent cell-mediated reactions. The mutual cross-reactivity, both humoral and cell-mediated, between (Gly-Pro-Ala)_n and (Pro-Ala-Gly)_n emphasized the importance of the sequence in the body of the polymers rather than the sequence at the free ends in determining specificity. This is confirmed by the absence of cross-reactivity of both of these polymers with (Ala-Pro-Gly)_n.With a series of samples of (Ala-Gly-Pro)_n of increasing single chain weight average molecular weight ranging from ca 2,000 to ca 15,000 the most immunogenic for cell-mediated reactions was that of lowest molecular weight, but for humoral responses immunogenicity ran parallel with increasing molecular weight with the exception of the polymer of highest molecular weight, which was almost insoluble.     

Pathogenesis and diagnosis of human meningococcal disease using immunohistochemical and PCR assays

Neisseria meningitidis remains the leading cause of fatal sepsis. Cultures may not be available in fulminant fatal cases. An immunohistochemical assay for N meningitidis was applied to formalin-fixed samples from 14 patients with meningococcal disease. Histopathologic findings in 12 fatal cases included interstitial pneumonitis, hemorrhagic adrenal glands, myocarditis, meningitis, and thrombi in the glomeruli and choroid plexus. Meningeal inflammation was observed in 6 patients. Skin biopsies of 2 surviving patients showed leukocytoclastic vasculitis and cellulitis. By using immunohistochemical analysis, meningococci and granular meningococcal antigens were observed inside monocytes, neutrophils, and endothelial cells or extracellularly. By using real-time polymerase chain reaction (PCR) on formalin-fixed tissue samples, meningococcal serogroup determination was possible in 11 of 14 cases (8 serogroup C, 2 Y, and 1 B). Diagnosis and serogrouping of N meningitidis can be performed using immunohistochemical analysis and PCR on formalin-fixed tissue samples. Immunohistochemical analysis determined the distribution of meningococci and meningococcal antigens in tissue samples, allowing better insights into N meningitidis pathogenesis.     

Surgery-attached psychogeriatric nurses: an evaluation of psychiatric nurses in the primary care team

Aspects of the work of five community psychiatric nurses are examined by means of a questionnaire completed by the nurses for a sample of 50 patients. Information sharing and a close working relationship with the general practitioner, skills relating to assessment and psychological support of patients, organization of care by other agencies, a detailed knowledge of community agencies, and a capacity for interdisciplinary working were all found to be important. Tasks relating to body care were reported in only a small number of cases. The nurses saw themselves as advising on the planning of patient management and the selection of medication in a substantial minority of cases. The original brief had been to care for psychogeriatric patients but a third of the patients fell into a younger age group. These findings are discussed in connection with their implications for the future training of nurses and the relationship between primary care teams and local psychiatric services.