Vimentin Regulates Chemokine Expression and NOD2 Activation in Brain Endothelium during Group B Streptococcal Infection.

Streptococcus agalactiae (group B Streptococcus, or GBS) is an opportunistic pathogen capable of causing invasive disease in susceptible individuals, including the newborn. Currently, GBS is the leading cause of meningitis in the neonatal period. We have recently shown that GBS interacts directly with host type III intermediate filament vimentin to gain access to the central nervous system. This results in characteristic meningeal inflammation and disease progression; however, the specific role of vimentin in the inflammatory process is unknown. Here, we investigate the contribution of vimentin to the pathogenesis of GBS meningitis. We show that a CRISPR-targeted deletion of vimentin in human cerebral microvascular endothelial cells (hCMEC) reduced GBS induction of neutrophil attractants interleukin-8 (IL-8) and CXCL-1 as well as NF-κB activation. We further show that inhibition of vimentin localization also prevented similar chemokine activation by GBS. One known chemokine regulator is the nucleotide-binding oligomerization domain containing protein 2 (NOD2), which is known to interact directly with vimentin. Thus, we hypothesized that NOD2 would also promote GBS chemokine induction. We show that GBS infection induced NOD2 transcription in hCMEC comparably to the muramyl dipeptide (MDP) NOD2 agonist, and the chemokine induction was reduced in the presence of a NOD2 inhibitor. Using a mouse model of GBS meningitis, we also observed increased NOD2 transcript and NOD2 activation in brain tissue of infected mice. Lastly, we show that NOD2-mediated IL-8 and CXCL1 induction required vimentin, further indicating the importance of vimentin in mediating inflammatory responses in brain endothelium.

Parental And Clinician Views Of Consent In Neonatal Research.

AIM: To determine parental and clinician views of the informed consent process in neonatal research. METHODS: A questionnaire-based study on the informed consent process. Two questionnaires were developed and distributed to parents and clinicians over a four-month period. RESULTS: Thirty-four parents (79%) surveyed had consented their baby to a research study. The majority of clinicians (72%) had a preference for antenatal provision of information. A desire to help future babies (97%, n=32) and a belief that their baby's healthcare would directly benefit (72%, n=28) were primary reasons for consenting. The majority (76% n=28) of parents were not in favour of a waiver of consent. However twenty clinicians (56%) agreed that a waiver of consent may be appropriate in neonatal research. Thirty-one (86%) clinicians rated GCP training as important. DISCUSSION: Parents are generally supportive of neonatal research. Good clinical practice training is essential for clinicians involved in neonatal research.

Doctors' attitudes towards the introduction and clinical operation of do not resuscitate orders (DNRs) in Ireland.

BACKGROUND: Do not resuscitate orders (DNRs) are documents which state that should a patient suffer from cardiopulmonary failure, resuscitation should not be attempted. Internationally, DNRs are often misunderstood and used inappropriately in a clinical setting. AIMS: The aim of this paper was to determine the current understanding of DNRs and their clinical operation among hospital doctors in Ireland. METHODS: A cross-sectional, questionnaire-based study was conducted involving doctors from the Cork teaching hospitals. The questionnaire sought information regarding understanding of DNRs and their clinical operation, as well as attitudes regarding the current absence of relevant Irish guidelines. The questionnaire also collected information regarding demographics, clinical specialty, and level of experience. RESULTS: 45.9% (47/103) of all doctors stated that their clinical knowledge was sufficient to draft a DNR, but 48.7% of this group (n = 23) chose the incorrect definition for a DNR when provided with three separate options. Thirty-five percent (n = 36) of all doctors surveyed demonstrated an incorrect understanding of a DNR. Neither specialty nor experience level had any effect on level of understanding of DNRs (p > 0.05). 93.2% (n = 96) agreed that there is a need for introduction of domestic guidelines regarding DNRs. 57.6% (n = 59) would draft more DNRs in the event that such domestic guidelines were in place. CONCLUSIONS: A substantial proportion of hospital doctors surveyed demonstrated an incomplete understanding of DNRs and their clinical operation. However, the overwhelming majority of the present sample believe that domestic guidelines are needed on the matter.

Characterization of host immunity during persistent vaginal colonization by Group B Streptococcus.

Streptococcus agalactiae (Group B Streptococcus, GBS) is a Gram-positive bacterium, which colonizes the vaginal tract in 10-30% of women. Colonization is transient in nature, and little is known about the host and bacterial factors controlling GBS persistence. Gaining insight into these factors is essential for developing therapeutics to limit maternal GBS carriage and prevent transmission to the susceptible newborn. In this work, we have used human cervical and vaginal epithelial cells, and our established mouse model of GBS vaginal colonization, to characterize key host factors that respond during GBS colonization. We identify a GBS strain that persists beyond a month in the murine vagina, whereas other strains are more readily cleared. Correspondingly, we have detected differential cytokine production in human cell lines after challenge with the persistent strain vs. other GBS strains. We also demonstrate that the persistent strain more readily invades cervical cells compared with vaginal cells, suggesting that GBS may potentially use the cervix as a reservoir to establish long-term colonization. Furthermore, we have identified interleukin-17 production in response to long-term colonization, which is associated with eventual clearance of GBS. We conclude that both GBS strain differences and concurrent host immune responses are crucial in modulating vaginal colonization.

The at-risk medical student--what more can we do?

Securing a place in medical school is extremely difficult-students who are successful all have similar high levels of academic achievement. So why do some students, and not others, have difficulty with the course, and in some cases, leave the programme? Studies on medical school attrition offer valuable insight into why medical students under-perform. Identification of the 'at-risk' student can trigger additional support and early remediation, helping some students remain in their chosen profession.

Host-dependent requirement for specific DnaA boxes for plasmid RK2 replication.

The replication origin of the broad-host-range plasmid RK2, oriV, contains four DnaA boxes, which bind the DnaA protein isolated from Escherichia coli. Using a transformation assay, mutational analysis of these boxes showed a differential requirement for replication in different Gram-negative bacteria. DnaA boxes 3 and 4 were required in E. coli and Pseudomonas putidabut not as strictly in Azotobacter vinelandii and not at all in P. aeruginosa. In vitro replication results using an extract prepared from E. coli demonstrated that the activity of origin derivatives containing mutations in boxes 3 or 4 or a deletion of all four DnaA boxes could be restored by the addition of increasing amounts of purified DnaA protein. High levels of DnaA protein in the presence of the TrfA protein also resulted in the stimulation of open complex formation and DnaB helicase loading on oriV, even in the absence of the four DnaA boxes. These observations at least raise the possibility that an alternative mechanism of initiation of oriV is being used in the absence of the four DnaA boxes and that this mechanism may be similar to that used in P. aeruginosa, which does not require these four DnaA boxes for replication.

A critical DnaA box directs the cooperative binding of the Escherichia coli DnaA protein to the plasmid RK2 replication origin.

The requirement of DnaA protein binding for plasmid RK2 replication initiation the Escherichia coli was investigated by constructing mutations in the plasmid replication origin that scrambled or deleted each of the four upstream DnaA boxes. Altered origins were analyzed for replication activity in vivo and in vitro and for binding to the E. coli DnaA protein using a gel mobility shift assay and DNase I footprinting. Most strikingly, a mutation in one of the boxes, box 4, abolished replication activity and eliminated stable DnaA protein binding to all four boxes. Unlike DnaA binding to the E. coli origin, oriC, DnaA binding to two of the boxes (boxes 4 and 3) in the RK2 origin, oriV, is cooperative with box 4 acting as the "organizer" for the formation of the DnaA-oriV nucleoprotein complex. Interestingly, the inversion of box 4 also abolished replication activity, but did not result in a loss of binding to the other boxes. However, DnaA binding to this mutant origin was no longer cooperative. These results demonstrate that the sequence, position, and orientation of box 4 are crucial for cooperative DnaA binding and the formation of a nucleoprotein structure that is functional for the initiation of replication.

Controlled trial of pharmacist intervention in general practice: the effect on prescribing costs.

BACKGROUND: It has been suggested that the employment of pharmacists in general practice might moderate the growth in prescribing costs. However, empirical evidence for this proposition has been lacking. We report the results of a controlled trial of pharmacist intervention in United Kingdom general practice. AIM: To determine whether intervention practices made savings relative to controls. METHOD: An evaluation of an initiative set up by Doncaster Health Authority. Eight practices agreed to take part and received intensive input from five pharmacists for one year (September 1996 to August 1997) at a cost of 163,000 Pounds. Changes in prescribing patterns were investigated by comparing these practices with eight individually matched controls for both the year of the intervention and the previous year. Prescribing data (PACTLINE) were used to assess these changes. The measures used to take account of differences in the populations of the practices included the ASTRO-PU for overall prescribing and the STAR-PU for prescribing in specific therapeutic areas. Differences between intervention and control practices were subjected to Wilcoxon matched-pairs, signed-ranks tests. RESULTS: The median (minimum to maximum) rise in prescribing costs per ASTRO-PU was 0.85 Pound (-1.95 Pounds to 2.05 Pounds) in the intervention practices compared with 2.55 Pounds (1.74 Pounds to 4.65 Pounds) in controls (P = 0.025). Had the cost growth of the intervention group been as high as that of the controls, their total prescribing expenditure would have been around 347,000 Pounds higher. CONCLUSION: This study suggests that the use of pharmacists did control prescribing expenditure sufficiently to offset their employment costs.

Health needs of coronary artery bypass graft surgery patients at discharge.

Shortened hospital stays could potentially lead to unmet patient needs. This descriptive study utilizes critical pathway data and the Omaha System to identify the health needs of coronary artery bypass surgery patients at time of discharge. The most common health needs were education, sleep/rest and rehabilitation. Nursing implications relating to these health needs are also identified.

Replication origin of the broad host range plasmid RK2. Positioning of various motifs is critical for initiation of replication.

The 393-base pair minimal origin, oriV, of plasmid RK2 contains three iterated motifs essential for initiation of replication: consensus sequences for binding the bacterial DnaA protein, DnaA boxes, which have recently been shown to bind the DnaA protein; 17-base pair direct repeats, iterons, which bind the plasmid encoded replication protein, TrfA; and A + T-rich repeated sequences, 13-mers, which serve as the initial site of helix destabilization. To investigate how the organization of the RK2 origin contributes to the mechanism of replication initiation, mutations were introduced into the minimal origin which altered the sequence and/or spacing of each particular region relative to the rest of the origin. These altered origins were analyzed for replication activity in vivo and in vitro, for localized strand opening and for DnaB helicase mediated unwinding. Mutations in the region between the iterons and the 13-mers which altered the helical phase or the intrinsic DNA curvature prevented strand opening of the origin and consequently abolished replication activity. Insertions of more or less than one helical turn between the DnaA boxes and the iterons also inactivated the replication origin. In these mutants, however, strand opening appeared normal but the levels of DnaB helicase activity were substantially reduced. These results demonstrate that correct helical phasing and intrinsic DNA curvature are critical for the formation of an open complex and that the DnaA boxes must be on the correct side of the helix to load DnaB helicase.

Interregional healthcare: patient stories and chart reviews.

The literature and healthcare provider experiences leave questions about which interventions might best assist patients during interregional healthcare. The research was conducted to gain information on the current reality of interregional healthcare for patients needing tertiary cardiovascular care distant from their home. A purposive sample of patients having a cardiovascular diagnosis who were transferred for procedures or surveillance to a tertiary site were interviewed (n = 17), and their charts were reviewed (n = 27). Six broad themes were extracted from the interviews and chart reviews: healthcare provider behaviors, healthcare system issues, patient education or information, discharge from the hospital, overall reflections on the healthcare experience, and healthcare communication issues. A redesign of interregional healthcare is needed to address the areas of care and expert behaviors by providers, documentation, continuity, communication, education, and rehabilitation/adaption. The advanced practice nurse is well suited to lead these practice changes.