Recurrent congestive heart failure in a child due to probable myocarditis.

Recurrent congestive heart failure (CHF) attributable to myocarditis is a seldom-discussed entity in the scientific literature. This report describes the case of an 8-year-old girl who had three clinically identical episodes of CHF, beginning at the age of 5 years, with each episode preceded by a viral prodrome. The clinical features and the echocardiography and electrocardiogram findings were most supportive of myocarditis. Symptoms and investigations completely normalized between episodes. The third episode, associated with influenza A (strain H1N1) infection, led to cardiac arrest and death on day 2 after admission. Autopsy showed mild cardiomegaly with microscopic foci of myocardial fibrosis and extensive contraction band necrosis. This report is the first to describe recurrent CHF due to probable myocarditis in a pediatric patient.

Factors in the choice of oral transmucosal fentanyl citrate dose for adult burns dressings.

Factors that influenced the choice of dose of oral transmucosal fentanyl at the time of burns dressing change were investigated in a prospective study. After Ethics committee approval, data was analysed from 29 consecutive patients who had been recruited and consented for a study of pain associated with burns dressings. Patients had completed an 11-point verbal pain intensity score (VRS) prior to and after the dressing change. Analgesic use during for this period was documented. Doses of 600 to 1200 mcg of transmucosal fentanyl (Actiq) were given based on individual assessment. The pre-dressing VRS (median [range]) in the 15 patients who received 600 mcg was 8 [3-10] and was higher than the VRS of 6 [2-9] in the 800-1200 mcg group. The time since the burn was longer in the low dose group at 7 [1-22] days compared with 5 [0-50] days in the higher dose group. In addition 73% of the low dose group was prescribed opioids regularly prior to the dressing compared with 57% of the high dose group. The choice of a lower transmucosal fentanyl dose was based on prior use of opioids and the age of the burn rather than on the patient's pain intensity.

Quantification of circulating cell-free plasma DNA and endothelial gene RNA in patients with burns and relation to acute thermal injury.

BACKGROUND: Major burn represents a multi-system insult to the human body. Despite improvements in mortality and morbidity, reliable predictors of outcome are lacking. Raised levels of cell-free nucleic acids have been detected in various pathological processes including burns. We quantified circulating nucleic acids as potential objective measures of burn severity with predictive and prognostic value. METHODS: Expression of endothelial specific cell-free mRNA and cell-free DNA were measured in plasma of 19 burn patients at days 1-3 and week 10 following acute thermal injury and in 19 healthy controls by real-time quantitative PCR. RESULTS: Expression of endothelial specific mRNA was higher in burn patients compared to controls (p<0.001). DNA levels were significantly higher in the burn population in the first 48 h following injury. Plasma RNA and DNA levels related to %TBSA burn in the first 24h and to the levels of circulating endothelial progenitor cells. CONCLUSIONS: We show that plasma levels of endothelial specific mRNA and DNA are elevated acutely following burns, and relate to severity in terms of %TBSA burnt.

Mobilization of endothelial progenitor cells into the circulation in burned patients.

BACKGROUND: Bone marrow-derived endothelial progenitor cells (EPCs) have been detected in the peripheral blood of patients following thermal injury. EPCs migrate to sites of active neovascularization in response to mediators released after trauma, contributing to wound healing. The aim was to characterize levels and kinetics of EPCs in burned patients, then relate these to key mobilizing factors, vascular endothelial growth factor (VEGF) and the chemokine (C-X-C motif) ligand 12 (CXCL 12), and compare them with those in healthy subjects. METHODS: The study included 19 adult patients with superficial or full-thickness burns and 50 blood donor volunteer controls. EPCs, identified by cell surface markers CD45(dim/-), CD133+, CD144+ and VEGF receptor 2, were quantified by four-colour flow cytometry. Plasma VEGF and CXCL12 were measured using enzyme-linked immunosorbent assay. RESULTS: Burned patients showed a rapid rise in EPC levels within 24 h, a ninefold increase compared with controls, returning to basal levels by 72 h. Body surface area burned correlated strongly with the degree of mobilization. EPC levels correlated significantly with rises in plasma VEGF and CXCL12. CONCLUSION: Thermal injury induced a rapid rise in EPCs that was proportional to the extent of the burn and significantly correlated with levels of angiogenic cytokines. Such cytokines may be used to stimulate EPCs as a future therapeutic target in burned patients.

Effect of the implementation of NICE guidelines for ultrasound guidance on the complication rates associated with central venous catheter placement in patients presenting for routine surgery in a tertiary referral centre.

BACKGROUND: The National Institute for Clinical Excellence (NICE) guidelines of 2002 recommended the use of ultrasound (US) for central venous catheterization in order to minimize complications associated with central line placement. An ongoing audit of line placement by anaesthetists in the theatre complex of a tertiary referral centre looked at the associated complication rates. The objective of the study was to compare complication rates pre- and post-implementation of NICE guidelines. METHODS: This prospective, single centre audit looked at all patients in whom a central venous catheter was placed for surgery. Complication rates were assessed for procedures that were performed pre- and post-implementation of NICE guidelines. In total, 438 patients were identified for the study, and the procedures were performed either by trainee or by consultant anaesthetists. RESULTS: The pre- and post-implementation complication rates were 10.5% (16/152) and 4.6% (13/284), respectively, representing an absolute risk reduction of 5.9% (95% CI 0.5-11.3%). Comparison of those procedures in which US was used when compared with the landmark technique after implementation found a reduction of 6.9% in complications (95% CI 1.4-12.4%). The reduction in complication rates was larger for specialist registrars than for consultants (11.2% vs 1.6%). CONCLUSIONS: The implementation of NICE guidelines has been associated with a significant reduction in complication rates in our tertiary referral centre. In the light of the cross-speciality evidence of US superiority and our results, it is imperative that routine use of US guidance becomes more widespread.

Biallelic mutation of MSH2 in primary human cells is associated with sensitivity to irradiation and altered RAD51 foci kinetics.

BACKGROUND: Reports of differential mutagen sensitivity conferred by a defect in the mismatch repair (MMR) pathway are inconsistent in their conclusions. Previous studies have investigated cells established from immortalised human colorectal tumour lines or cells from animal models. METHODS: We examined primary human MSH2-deficient neonatal cells, bearing a biallelic truncating mutation in MSH2, for viability and chromosomal damage after exposure to DNA-damaging agents. RESULTS: MSH2-deficient cells exhibit no response to interstrand DNA cross-linking agents but do show reduced viability in response to irradiation. They also show increased chromosome damage and exhibit altered RAD51 foci kinetics after irradiation exposure, indicating defective homologous recombinational repair. DISCUSSION: The cellular features and sensitivity of MSH2-deficient primary human cells are broadly in agreement with observations of primary murine cells lacking the same gene. The data therefore support the view that the murine model recapitulates early features of MMR deficiency in humans, and implies that the variable data reported for MMR-deficient immortalised human cells may be due to further genetic or epigenetic lesions. We suggest caution in the use of radiotherapy for treatment of malignancies in individuals with functional loss of MSH2.

Gestational stress induces post-partum depression-like behaviour and alters maternal care in rats.

Gestational stress (GS) produces profound behavioural impairments in the offspring and may permanently programme hypothalamic-pituitary-adrenal (HPA) axis function. We investigated whether or not GS produced changes in the maternal behaviour of rat dams, and measured depression-like behaviour in the dam, which might contribute to effects in the progeny. We used the Porsolt test, which measures immobility in a forced-swim task, and models depression in rodents, while monitoring maternal care (arched-back nursing, licking/grooming, nesting/grouping pups). Pregnant rats underwent daily restraint stress (1 h/day, days 10-20 of gestation), or were left undisturbed (control). On post-parturition days 3 and 4, dams were placed into a swim tank, and time spent immobile was measured. GS significantly elevated immobility scores by approximately 25% above control values on the second test day. Maternal behaviours, in particular arched-back nursing and nesting/grouping pups, were reduced in GS dams over post-natal days 1-10. Adult offspring showed increased immobility in the Porsolt test, and also hypersecreted ACTH and CORT in response to an acute stress challenge. These data show that GS can alter maternal behaviour in mothers, and this might contribute to alterations in the offspring. GS may be an important factor in maternal post-natal depression, which may in turn detrimentally effect the offspring because depressed mothers do not sufficiently care for their offspring.

Reputation, public information, and physician adoption of an innovation.

A dynamic expected utility framework is developed to explore the timing and extent of a physician's adoption of an innovation. The benefits to adoption are uncertain, although an informative public information signal is released with some delay and possibly some inaccuracy. Improvements to the accuracy of the signal do not necessarily cause risk-averse physicians to scale back adoption prior to the signal's release - if physicians are confident enough in the innovation's worth, they may accelerate early adoption in order to capture an earlymover advantage in reputation building.

Coexpression of band 3 mutants and Rh polypeptides: differential effects of band 3 on the expression of the Rh complex containing D polypeptide and the Rh complex containing CcEe polypeptide.

K562 cells were stably transfected with cDNAs encoding the band 3 found in Southeast Asian ovalocytosis (B3SAO, deletion of residues 400-408), band 3 with a transport-inactivating E681Q point mutation (B3EQ), or normal band 3 (B3). Flow cytometric analysis and quantitative immunoblotting revealed that B3SAO expressed alone was translocated to the plasma membrane, at levels similar to B3 or B3EQ. Nine monoclonal antibodies that reacted with extracellular loops of B3 also reacted with B3SAO, although the affinity of most antibodies for the mutant protein was reduced. Both known Wr(b) epitopes were expressed on K562/B3SAO cells, demonstrating that B3SAO interacts with glycophorin A. The growth rates of K562 clones expressing equivalent amounts of B3 and B3EQ were the same, suggesting that the potentially toxic transport function of band 3 may be regulated in K562 cells. The band 3-mediated enhancement of Rh antigen reactivity and the depression of Rh epitopes on SAO erythrocytes were investigated by comparing the coexpression of B3, B3SAO, or B3EQ in K562 clones expressing exogenous RhcE or RhD polypeptides. The results are consistent with an interaction between band 3 and the Rh polypeptide-Rh glycoprotein (RhAG) complex, which may enhance translocation of the complex or affect its conformation in the plasma membrane. The data suggest that the interaction between band 3 and the RhD-RhAG complex is weaker than it is between band 3 and the RhCcEe-RhAG complex.

Expression of C antigen in transduced K562 cells.

BACKGROUND: The Rh blood group system is involved in HDN and transfusion reactions. A retrovirus-expression system was previously used to show that polypeptides carrying the Rh blood group antigens are encoded by the RHD and RHCE genes. This study investigated the structure of the C antigen. STUDY DESIGN AND METHODS: K562 cells were transduced with full-length cDNA encoding Ce and CE antigens, and the expression of C, e, and E antigens was examined by flow cytometry using MoAbs. The importance of Cys16 in C antigen expression was examined by utilizing site-directed mutagenesis to convert Cys16 to Trp in cDNA encoding Ce and CE before expression in K562 cells. RESULTS: When K562 cells were transduced with cDNA that was predicted to encode Ce antigens, clear reactivity with anti-e and anti-C was obtained. In contrast, K562 cells transduced with cDNA that was predicted to encode CE antigens gave strong reactivity with anti-E but failed to react with two examples of anti-C. A third example of anti-C gave weak reactivity. When cDNA encoding Ce antigens was mutated to encode Trp16, one example of anti-C had the same reactivity with the mutated polypeptide as with the wild-type molecule, but reactivity with two other anti-C examples was reduced by 50 percent. CONCLUSIONS: The nature of polymorphic residue 226 (proline when E is expressed, alanine when e is expressed) has a marked effect on the epitopes recognized by the three C MoAbs studied. The presence of Cys16 in Ce polypeptides influences the presentation of the C epitope recognized by two of the three MoAbs. These experiments provide the first direct demonstration that C and E/e antigens can be expressed on the same polypeptide.

Adeno-associated virus and lentivirus vectors mediate efficient and sustained transduction of cultured mouse and human dorsal root ganglia sensory neurons.

Peripheral nervous system (PNS) sensory neurons are directly involved in the pathophysiology of numerous inherited and acquired neurological conditions. Therefore, efficient and stable gene delivery to these postmitotic cells has significant therapeutic potential. Among contemporary vector systems capable of neuronal transduction, only those based on herpes simplex virus have been extensively evaluated in PNS neurons. We therefore investigated the transduction performance of recombinant adeno-associated virus type 2 (AAV) and VSV-G-pseudotyped lentivirus vectors derived from human immunodeficiency virus (HIV-1) in newborn mouse and fetal human dorsal root ganglia (DRG) sensory neurons. In dissociated mouse DRG cultures both vectors achieved efficient transduction of sensory neurons at low multiplicities of infection (MOIs) and sustained transgene expression within a 28-day culture period. Interestingly, the lentivirus vector selectively transduced neurons in murine cultures, in contrast to human cultures, in which Schwann and fibroblast-like cells were also transduced. Recombinant AAV transduced all three cell types in both mouse and human cultures. After direct microinjection of murine DRG explants, maximal transduction efficiencies of 20 and 200 transducing units per neuronal transductant were achieved with AAV and lentivirus vectors, respectively. Most importantly, both vectors achieved efficient and sustained transduction of human sensory neurons in dissociated cultures, thereby directly demonstrating the exciting potential of these vectors for gene therapy applications in the PNS.

Use of teleoptometry to evaluate acceptability of rigid gas-permeable contact lens fits.

BACKGROUND: Teleoptometry involves the transmission of digitized optometric information from a remote site for analysis by an expert. This project assessed computer compression of video data showing contact lens fitting relationships and the transmission of these data to a specialist for evaluation METHODS: Fifty-five sets of video clips showing dynamic fluorescein patterns of rigid gas-permeable lens fits, topographic corneal maps, and basic information on lens parameters were evaluated-live and again after digitization and electronic compression-by a contact lens specialist. The evaluator was asked to determine whether lens fit was acceptable and, if not, how the lens parameters should be changed. RESULTS: Comparison of lens evaluations made live versus compressed showed agreement on fit acceptability for approximately 80% of the subject/lens combinations. When the evaluator concluded for both presentations that the lens was unacceptable, the same change in lens parameters was recommended 67% of the time. DISCUSSION: Agreement for the majority of live and compressed video observations suggests that teleoptometric consultation on contact lens fitting is feasible. When technology advances to the point at which large files can be sent quickly via the Internet, it is likely the practice of transmitting video clips and other information to obtain fitting assistance will become commonplace.

Adeno-associated virus vectors show variable dependence on divalent cations for thermostability: implications for purification and handling.

Recombinant adeno-associated virus (rAAV) shows significant promise as a vector for gene transfer in pre-clinical models of human disease, and is currently being evaluated in human clinical trials. As a consequence, increasing attention is being turned to the important tasks of optimizing rAAV titer, purity, and stability. We have observed dramatic variation in divalent cation dependence for thermostability of different rAAV vectors. To further investigate this observation, the thermostability of eight different vector constructs ranging in size from 73 to 107% of wild-type genome size (4.68 kilobases) was determined in the presence and absence of divalent cations. Virions containing smaller genomes (i.e., <85% wild type) were relatively divalent cation independent for thermostability. In contrast, virions containing recombinant genomes close to, or exceeding, wild-type size (i.e., >95% wild type) were dependent on divalent cations for thermostability. Genome sequence also appeared to be a factor in the thermostability of the larger rAAV vectors. These observations are of both practical and theoretical significance. Divalent cations should be included in all buffer solutions used during rAAV purification and storage, and unnecessary heat exposure avoided. These data also demonstrate that different recombinants of a particular virus should not be assumed to possess the same thermostability profile.

Human PBMC-derived dendritic cells transduced with an adenovirus vectorinduce cytotoxic T-lymphocyte responses against a vector-encoded antigen in vitro.

Dendritic cells (DC) are among the most potent antigen-presenting cells known and play an important role in the initiation of antigen-specific T-lymphocyte responses. Several recent studies have demonstrated that DC expressing vector-encoded tumor-associated antigens can induce protective and therapeutic immunity in murine cancer models. In the current study we set out to examine in vitro the utility of adenovirus vectors in the transduction of human DC for the induction of antigen-specific T-lymphocyte responses against a defined vector-encoded antigen. DC were derived from the adherent fraction of PBMC by culture in defined medium containing GM-CSF and IL-4. A replication-defective E1/E3-deleted type 5 adenovirus vector encoding bacterial beta-galactosidase (beta-gal) under the transcriptional control of a CMV promoter was used to transduce DC at multiplicities of infection (MOI) up to 1000. While high MOI were required to achieve efficient transduction there was no significant effect on DC morphology, immunophenotype or potency in allogeneic lymphocyte proliferation assays. Furthermore, transduced DC-induced antigen-specific CTL activity against adenoviral proteins and more significantly, the vector-encoded antigen beta-gal. These data clearly demonstrate the potential of adenovirus vectors in anticancer DC vaccine strategies and provide an important link between existing animal data and human clinical application.

Human fibroblasts transduced with CD80 or CD86 efficiently trans-costimulate CD4+ and CD8+ T lymphocytes in HLA-restricted reactions: implications for immune augmentation cancer therapy and autoimmunity.

Augmenting immunogenicity by genetically modifying tumor cells to express costimulatory molecules has proven to be a promising therapeutic strategy in murine tumor models and is currently under investigation in human clinical trials for metastatic cancer. However, there are significant technical and logistic problems associated with implementing strategies requiring direct gene modification of primary tumor cells. In an effort to circumvent these problems, we are developing a strategy in which the costimulatory signal required for tumor-specific T lymphocyte activation is provided by a genetically modified human fibroblast (trans-costimulation). We have evaluated the efficiency of CD80- and CD86-mediated trans-costimulation in the activation of human CD8+ and CD4+ T lymphocytes in MHC class I- and class II-restricted lymphoproliferation reactions. Our studies demonstrate that the efficiency of CD80- or CD86-mediated trans-costimulation of purified human CD8+ and CD4+ T lymphocytes is comparable to cis-costimulation under defined conditions. Moreover, a dose-response relationship consistent with the predicted two-hit kinetics of the reaction was evident in trans-costimulation reactions in which the ratio of target cells expressing either signal 1 or signal 2 was varied incrementally from 1:10 to 10:1. Importantly, the level of cell-surface CD86 required for trans-costimulation is equivalent to that constitutively expressed by human peripheral blood monocytes. These results may have significant implications for the clinical implementation of this type of cancer immunotherapy and also raise questions about the possibility of trans-costimulating autoreactive T lymphocytes in vivo.

Changes in hippocampal theta following intrahippocampal corticotropin-releasing hormone (CRH) infusions in the rat.

Hippocampal theta activity is a large amplitude, sinusoidal wave that occurs during attentive immobility and exploratory behaviour in the rat, and it is thought to be involved in memory formation. Recent reports suggest that corticotropin-releasing hormone (CRH) has pro-mnemonic effects in rodents. Because memory-enhancing substances/manipulations generally alter either theta frequencies or amplitudes, these variables were monitored in urethane-anaesthetised rats following intrahippocampal infusions of CRH. Adult male, Lister hooded rats were implanted with a hippocampal recording electrode and a guide cannula, both aimed at the dentate gyrus. When CRH was infused into the hippocampus, the main change in the hippocampal EEG was a slow onset increase in the amplitude of spontaneous theta and, paradoxically, a significant decrease in the amount of time spent displaying theta. These data suggest that CRH has the ability to modulate ongoing hippocampal theta, but, considering the slow effect, the involvement of hippocampal CRH receptors is suspect. Regardless of locus, the described electrophysiological changes suggest that hippocampal cholinergic systems may play a role in the memory-enhancing effects of CRH.

Inhibition of human immunodeficiency virus type 1 replication by the K10-K42 peptide of GAP31 is due to induction of rapid but nonspecific precipitation of viral and nonviral proteins.

The 33-amino acid peptide K10-K42 has previously been described as having potent anti-HIV-1 activity, and antiviral efficacy against hepatitis B and human cytomegalovirus in vitro. Although the exact mechanism of antiviral activity was unknown, it was hypothesised that the K10-K42 peptide inhibited HIV-1 by interfering with one or more of the intracellular processes of reverse transcription, integration, and/or viral gene expression. We performed a series of experiments to identify and characterize the inhibitory mechanism, and to determine whether intracellular expression of the K10-K42 peptide would potentiate its antiviral efficacy in vitro. Surprisingly, our results revealed that the antiviral activity of the K10-K42 peptide could be explained without implicating intracellular inhibition of HIV-1 replication. The activity appeared to be due to an extraordinary capacity of the K10-K42 peptide to precipitate viral and nonviral proteins in vitro. The protein-precipitating capacity of the K10-K42 peptide was sequence specific and a scrambled version of the 33-amino acid peptide did not retain the activity. Although the unusual biochemical properties of the K10-K42 peptide probably negate a number of potential therapeutic applications, they do merit further investigation. Moreover, these findings provide a plausible explanation of the mechanism by which the K10-K42 peptide can inhibit replication of viruses from families as genetically and functionally diverse as Retroviridae, Hepadnaviridae, and Herpesviridae.

Effects of SCH23390 and raclopride on anxiety-like behavior in rats tested in the black-white box.

Dopamine (DA) systems are activated by stress, and this response has as a corollary the induction of stress-related behaviors such as anxiety. In mice, D2 receptor blockade produces an apparent anxiogenic effect, although locomotor impairments might have been present. We investigated the effects of D1 and D2 antagonists on a variety of anxiety-like behaviors induced by the black-white box in rats and carefully screened for any locomotor deficits. Adult male Lister hooded rats were injected with either the D1 antagonist SCH23390 (0. 0.1. or 0.25 mg/kg i.p.) or the D2 antagonist raclopride (0, 0.05, or 0.10 mg/kg i.p.) 20 min prior to being placed into the white chamber of the black-white box (n = 8-10/group). Rats were videotaped and the tapes were scored for latency to exit the white chamber, latency to reenter the white chamber, time spent in the white chamber, intercompartmental crossing, and locomotor activity. ANOVA revealed no effect of the D1 antagonist SCH23390 on any behavioral measure. However, the raclopride-treated rats left the white area sooner than control rats (p < 0.01). Raclopride-treated rats also exhibited delayed reentry times to the white chamber compared to control rats (p < 0.01) and spent significantly less time in the white chamber (p < 0.05). Neither SCH23390 nor raclopride affected locomotor activity in a manner that confounded these behaviors. These results confirm that D2 receptor blockade enhances anxiety in rats tested in the black-white box.