The combined use of non-radioactive in situ hybridization and real-time RT-PCR to assess gene expression in cryosections

Gene expression changes in pathophysiological states can be spatiotemporally monitored by in situ hybridization and reliably quantified by real-time RT-PCR. Here we developed a new method whereby adjacent slides of frozen sections can be used for gene expression analysis by in situ hybridization and real-time RT-PCR. We applied this method to assess the mRNA expression of connexin 43 (Cx43), the major astrocytic connexin, after kainate-induced seizures in rat hippocampus. Gap junction-building connexins play a role in the pathogenesis of several diseases of the brain, including epilepsy. The number of Cx43 mRNA-positive cells in the hippocampus of kainate-treated and control rats was automatically quantified by computerized image analysis of brain sections hybridized with DIG-labeled RNA probes. In parallel, real-time RT-PCR was used to examine the relative Cx43 mRNA levels in hippocampal tissue from adjacent brain sections. Applying these two very sensitive methods we showed that kainate induced seizures do not affect hippocampal connexin 43 mRNA expression.     

Dental care and oral health in solid organ transplant recipients: a single center cross-sectional study and survey of German transplant centers

Aim of this study was to collect information about oral health of patients before and after SOT as well as information about center‐based recommendations for dental care. In a single center cross‐sectional study, the oral situation of 20 patients before and 20 after SOT were examined including dental (DMF‐T), periodontal (PSR^®/PSI), and oral hygiene findings (modified QHI). In a second project, a survey among 50 transplant centers in Germany was questioned regarding their recommendations for dental care of SOT recipients. Patients before and after SOT showed similar quality of dental findings (DMF‐T), but worse compared to the general population. In addition, most patients in both groups showed pronounced periodontal treatment need (PSR^®/PSI score 3 or 4). Oral hygiene findings (modified QHI) after SOT were significantly worse than in patients on the waiting list (P = 0.032). In a second project, the questionnaire was returned by 28 of 50 centers. Interpretation of data showed that 89% carry out a dental examination before SOT and 67% contacted the patients’ dentists. After SOT, 83% of the transplant centers recommend antibiotic cover before dental measures. The results of our study revealed lacks in the dental care of SOT recipients. Consistent recommendations regarding the dental care of patients before and after SOT should be determined.     

The release of systemic inflammatory mediators is independent of cardiopulmonary bypass temperature

OBJECTIVE: The aim of this study was to investigate the effect of systemic CPB temperature on the production of the key mediators of the systemic inflammatory response to coronary artery bypass graft (CABG) surgery. DESIGN: Randomized clinical study. SETTING: University hospital. PARTICIPANTS: Thirty patients undergoing first-time CABG surgery. INTERVENTIONS: The patients were randomized to hypothermic (32 degrees C, n = 15) or normothermic (36 degrees C, n = 15) CPB. MEASUREMENTS AND MAIN RESULTS: Plasma interleukin (IL)-6, IL-8, IL-10, C-reactive protein (CRP), cortisol, and neutrophils were measured the day before operation, at closure of the sternum, and 4, 16, and 44 hours later. The cytokine, CRP, cortisol, and neutrophil responses were independent of temperature during CPB with peak concentrations of IL-10 at closure of the sternum followed by IL-6, IL-8, cortisol, neutrophils, and finally CRP. A correlation between maximal plasma concentrations of IL-10 and cortisol was seen in both groups after surgery (p = 0.02). Drainage after surgery was lower after normothermic CPB (p=0.02), with no difference in the requirement for blood transfusion. All patients were discharged from the intensive care unit within 24 hours after surgery. CONCLUSIONS: The release of systemic inflammatory mediators after cardiac surgery was independent of mild hypothermia (32 degrees C) versus normothermia (36 degrees C) during CPB.     

Impact of clinical history on fracture detection with radiography

The effect of knowledge of localizing symptoms and signs in the detection of fractures was studied. Forty radiographs of the extremities were examined twice by seven radiologists; the sessions were separated by 4 months. In 26 cases, a subtle fracture was present; 14 cases were normal. In half of the cases at each session, the precise location of pain, tenderness, or swelling was provided. The observer was asked to determine if the case was normal or abnormal (provide the exact location of the fracture) and to indicate the degree of confidence in the diagnosis. Responses were converted to a numeric scale for analysis. Analysis of receiver operator characteristic parameters indicates that clues regarding location of trauma facilitate detection of fractures. The improvement is based largely on an increased true-positive rate without an increased false-positive rate, regardless of the decision criteria of the radiologist (overall willingness to "overread" or "underread"). This has direct clinical applicability and reinforces the plea of radiologists for precise clinical information.     

Membrane-associated sickle hemoglobin: a major determinant of sickle erythrocyte rigidity

Micropipette aspiration tests on single erythrocytes have previously shown that the static rigidity (membrane shear modulus) of oxygenated sickle cells increased with increasing hemoglobin concentration, whereas the rigidity of normal cells was independent of hemoglobin concentration. Moreover, it was observed that after mechanical extension, sickle cells exhibited persistent deformation more frequently and to a greater extent than normal cells. To ascertain if differences in association of normal and sickle hemoglobin with the membrane could account for these observations, we measured rheologic properties of normal membranes reconstituted with sickle hemoglobin and sickle membranes reconstituted with normal hemoglobin. The static rigidity of normal ghosts reloaded with sickle hemoglobin was higher than those of either normal ghosts reloaded with normal hemoglobin or native normal cells. On the other hand, the increased rigidity of native sickle cells decreased to near-normal values following reconstitution with normal hemoglobin. Furthermore, we observed that normal ghosts reconstituted with sickle hemoglobin exhibited persistent bumps after mechanical extension, but no bumps formed on normal ghosts reconstituted with normal hemoglobin. Moreover residual bumps were not produced on sickle cells reloaded with normal hemoglobin. Since mechanical characteristics peculiar to sickle cells could be induced in normal cells by incorporation of sickle hemoglobin, and since normal characteristics could be restored to sickle cells by incorporation of normal hemoglobin, we suggest that the interaction of sickle hemoglobin with the cell membrane is responsible for augmented static rigidity of oxygenated sickle erythrocytes.     

Deferred consent in a minimal-risk study involving critically ill subarachnoid hemorrhage patients

INTRODUCTION:

Alterations from first-party and surrogate decision-maker consent can enhance the feasibility of research involving critically ill patients.

OBJECTIVE:

To describe the use of a deferred-consent model to enable participation of critically ill patients in a minimal-risk biomarker study.

METHODS:

A prospective observational study was conducted in which serum biomarker samples were collected three times daily over the first 14 days following aneurysmal subarachnoid hemorrhage. Sample collection was initiated on intensive care unit admission and consent was obtained when research personnel could approach the patient or the patient’s surrogate decision maker.

RESULTS:

Twenty-seven patients were eligible for the study, of whom only five were capable of providing informed consent. Full consent was obtained for 21 (78%) patients through self- (n=4) and surrogate (n=17) consent. Partial consent or refusal (only permitting the collection of blood samples as a part of routine care or use of data) occurred in three patients. Among the 22 consents sought from surrogates, three (11%) refused participation. The refusals included the sickest patients in the cohort. Once consent was provided, no patient or surrogate withdrew consent before study completion.

DISCUSSION:

Use of a deferred consent model enabled participation of critically ill patients in a minimal-risk biomarker study with no withdrawals.

CONCLUSIONS:

Further research and enhanced awareness of the potential utility of hybrid models, including deferred consent in addition to patient or surrogate consent, in the conduct of low-risk and minimally interventional time-sensitive studies of critically ill patients are required.     

Does anxiety predict the use of urgent care by people with long term conditions? A systematic review with meta-analysis

Objective

The role of anxiety in the use of urgent care in people with long term conditions is not fully understood. A systematic review was conducted with meta-analysis to examine the relationship between anxiety and future use of urgent healthcare among individuals with one of four long term conditions: diabetes; coronary heart disease, chronic obstructive pulmonary disease and asthma.

Methods

Electronic searches of MEDLINE, EMBASE, PSYCINFO, CINAHL, the British Nursing Library and the Cochrane Library were conducted These searches were supplemented by hand-searching bibliographies, citation tracing eligible studies and asking experts within the field about relevant studies. Studies were eligible for inclusion if they: a) used a standardised measure of anxiety, b) used prospective cohort design, c) included adult patients diagnosed with coronary heart disease (CHD), asthma, diabetes or chronic obstructive pulmonary disease (COPD), d) assessed urgent healthcare use prospectively. Data regarding participants, methodology, and association between anxiety and urgent care use was extracted from studies eligible for inclusion. Odds ratios were calculated for each study and pooled using random effects models.

Results

8 independent studies were identified for inclusion in the meta-analysis, with a total of 28,823 individual patients. Pooled effects indicate that anxiety is not associated with an increase in the use of urgent care (OR = 1.078, p = 0.476), regardless of the type of service, or type of medical condition.

Conclusions

Anxiety is not associated with increased use of urgent care. This finding is in contrast to similar studies which have investigated the role of depression as a risk factor for use of urgent care.