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A Wakefield

Publications and source records attributed to A Wakefield.

18 recordsLinked to original sources
PubMed2000

Detection and sequencing of measles virus from peripheral mononuclear cells from patients with inflammatory bowel disease and autism.

It has been reported that measles virus may be present in the intestine of patients with Crohn's disease. Additionally, a new syndrome has been reported in children with autism who exhibited developmental regression and gastrointestinal symptoms (autistic enterocolitis), in some cases soon after MMR vaccine. It is not known whether the virus, if confirmed to be present in these patients, derives from either wild strains or vaccine strains. In order to characterize the strains that may be present, we have carried out the detection of measles genomic RNA in peripheral mononuclear cells (PBMC) in eight patients with Crohn's disease, three patients with ulcerative colitis, and nine children with autistic enterocolitis. As controls, we examined healthy children and patients with SSPE, SLE, HIV-1 (a total of eight cases). RNA was purified from PBMC by Ficoll-paque, followed by reverse transcription using AMV; cDNAs were subjected to nested PCR for detection of specific regions of the hemagglutinin (H) and fusion (F) gene regions. Positive samples were sequenced directly, in nucleotides 8393-8676 (H region) or 5325-5465 (from noncoding F to coding F region). One of eight patients with Crohn disease, one of three patients with ulcerative colitis, and three of nine children with autism, were positive. Controls were all negative. The sequences obtained from the patients with Crohn's disease shared the characteristics with wild-strain virus. The sequences obtained from the patients with ulcerative colitis and children with autism were consistent with being vaccine strains. The results were concordant with the exposure history of the patients. Persistence of measles virus was confirmed in PBMC in some patients with chronic intestinal inflammation.

Adult↗
PubMed1998

A sensitive and robust method for measles RNA detection.

The aim of this study was to compare measles RNA amplification methods and to develop and select the most rapid, sensitive and robust procedure. The use of hybrid capture for measles RNA isolation was evaluated, and three RNA amplification detection techniques were compared. These were: (a) reverse transcription followed by nested polymerase chain reaction (RT-PCR) with MMLV reverse transcriptase and Taq polymerase; (b) a combined RT-PCR reaction using rTth polymerase; and (c) NASBA. An internal positive control was also developed. The sensitivities of the detection methods were quantified by using a dilution series of a known amount of total RNA from measles-infected Vero cells or by calculation of the number of transcript molecules (produced from a recombinant plasmid containing an insert measles nucleoprotein DNA) present in each amplification reaction, respectively. The results indicated that hybrid capture followed by combined RT-PCR with rTth polymerase was the most reproducibly robust and sensitive protocol and could detect as few as 10(4) synthetic measles RNA transcripts added to tissue homogenates. However, NASBA proved to be the most sensitive method for measles RNA detection in water.

Animals↗
PubMed1998

Sign reading: making sense of what is going on in the ward.

When entering a ward the environment observed can be perceived in one of two ways: either from the perspective of 'background', this being the backdrop against which events take place, or as the main focus of attention, thereby alerting the observer to what is going on in the setting. Whilst undertaking their work nurses emit signs which patients, relatives and other staff interpret and through which they gain an understanding of the type of work about to be undertaken at the clinical interface. The themes of private work, showdown work and tragic plight work, which were generated from an observational study in a surgical ward, are discussed in this article. Findings show that, despite nurses attempts to hide the gravity of a message from patients, they in fact give more away than they hide.

Attitude of Health Personnel↗
PubMed1997

The affinity of lipid-coated microbubbles for maturing brain injury sites.

The availability of a vehicle to deliver lipid soluble agents to a brain injury site may be of potential value in management of brain injury. This work describes the aggregation of intravenously administered Lipid-Coated Microbubbles (LCM) in the injury site following an experimental radiofrequency rat brain lesion. The bubbles can be identified around the region of the injury after the lesion has matured at least 48 h. The greatest bubbles density is evident after the lesion has matured for 10 days. This bubble density, reflecting "affinity," decreases to a plateau level from the second to the third week after injury. In order to investigate the potential relationship of bubble influx to posttraumatic astrocytosis and to cell turnover in the region, we utilized dual-channel laser-scanning confocal microscopy to track both bubble influx into the region and concomitant Glial Fibrillary Acidic Protein (GFAP) expressing astroctyte cell distribution. Cell turnover was assayed in separate sections using immunohistochemical staining of Proliferating Cell Nuclear Antigen (PCNA). We suggest a relationship between the LCM affinity and reactive astrocytes, but found no affinity of LCM for cells which stained positive with PCNA.

Animals↗
PubMed1995

In situ hybridization detection of short viral amplicon sequences within cultured cells and body fluids after the in situ polymerase chain reaction.

Using single primer pairs, intracellular gene sequences of cytomegalovirus (CMV-Towne's strain) and alpha-tubulin were amplified (in situ PCR) from cells in human body fluids and in suspensions. Visualization of CMV amplificants was carried out by in situ hybridization (ISH), using both a biotinylated double-stranded DNA probe and a radiolabelled oligonucleotide probe. Visualization of alpha-tubulin amplificants was achieved using both radiolabelled single-stranded cRNA and oligonucleotide probes. Liberated amplificants were also identified by bands of expected size by gel electrophoresis. The specificity of the PCR products was confirmed by Southern blot analysis. Intracellular amplification was identified both in unfixed cells and, optimally, after brief alcohol fixation, whilst maintaining relative isotonicity in all working solutions. For CMV, enhanced signal was observed in cells (cultured fibroblasts or urine sediment) undergoing in situ PCR using either biotinylated or radiolabelled probes compared with controls undergoing ISH alone. For alpha-tubulin, radiolabelled riboprobes and oligoprobes only produced signals within cells (human peripheral lymphocytes, ascitic fluid and bladder washings from routine cytological specimens) after in situ PCR, but not after ISH alone. Morphological evaluation was superior with biotinylated probes, and minimal back-diffusion effect was found compared with radiolabelled probes. Up to 80% of cells survived thermal cycling. In situ PCR detected short sequence (100 bp) foreign DNA and low copy number genomic DNA, and was superior to ISH alone. In contrast to radiolabelled probes, very small CMV amplificants could be detected without a significant 'back-diffusion' effect when using the large biotinylated probe in this model system.

Animals↗
PubMed1990

Fulminant hepatitis.

Explore the source record for details and available documents.

Blood Coagulation Disorders↗
PubMed1990

Lipid-coated ultrastable microbubbles as a contrast agent in neurosonography.

Lipid-coated microbubbles can be synthesized from selected lipid monolayer systems for use as ultrasonic contrast. These microbubbles have the property of longevity of weeks in vitro (ultrastability) and longevity of hours in vivo. The bubbles can be manufactured with a mean diameter of approximately 2 microns in a tight diameter distribution; all are less than 6 microns and 99% are smaller than 4.5 microns. The current study compared the in vivo survival characteristics of these lipid-coated microbubbles with microbubbles produced by saline. The comparison was made in the rat brain using direct intraparenchymal injections and injections into a previously created cyst/coagulum. The echogenic enhancement by the lipid-coated microbubbles persisted in vivo for over 24 hours in both the intraparenchymal environment and in the cyst/coagulum. The saline bubble echos were not detectable by 3 hours in a cyst/coagulum, and not detectable in the parenchyma after 2 hours. The sonographic characteristics and longevity of lipid-coated microbubbles make this agent a potentially useful clinical contrast material for neurosonography.

Animals↗
PubMed1989

Thrombogenicity of total parenteral nutrition solutions: I. Effect on induction of monocyte/macrophage procoagulant activity.

Although thrombosis is a frequent complication of total parenteral nutrition (TPN), its pathogenesis has received little scientific attention. We have studied, in vitro, the effects of the component solutions of TPN on the induction and modulation of human monocyte procoagulant activity, an initiator of coagulation. Human peripheral blood mononuclear cells were cultured with (a) 200 microliters of dextrose solution (10%, 15%, 20%, 25%, and 50%), (b) 200 microliters of amino acid solution (full, one-half, and one-quarter strength), and (c) 200 microliters of isosmolar 10% lipid emulsion (LE). Cocultures of LE and 20% dextrose, LE and full-strength amino acid solution, and LE and bacterial lipopolysaccharide were also studied. Cells cultured with lipopolysaccharide or medium alone constituted positive and negative controls, respectively. In addition, cocultures of LE and 20% dextrose, LE and full-strength amino acid solution, and LE and lipopolysaccharide were also studied. Cells were incubated for intervals of 12-72 h, washed, frozen, and assayed for monocyte procoagulant activity (MPCA). Milliunits of MPCA were derived from a standard thromboplastin curve. In addition, spontaneous MPCA levels were measured in healthy volunteers (n = 4) and "home" total parenteral nutrition patients (n = 4) before and after a 2-h infusion of 500 ml of LE. Our results show that, in vitro, hypertonic dextrose and full-strength amino acid solutions induce significant levels of MPCA. Induction of MPCA by dextrose was lymphocyte-independent. Although a significant increase in MPCA by full-strength amino acid solution was seen in cultures of isolated monocytes, a lymphocyte requirement was demonstrated for full MPCA. In contrast, LE significantly inhibited the induction of MPCA by 20% dextrose and full-strength amino acid solution. This inhibitory activity was at the monocyte level. Subfractionation of the LE into triglyceride and phospholipid phases showed the inhibitory capacity to reside in the former. In vivo, patients on home total parenteral nutrition expressed higher spontaneous MPCA levels than normal controls. Ten percent lipid emulsion infusion abolished MPCA expression in both groups. These corroborative in vitro and in vivo data suggest a mechanism for the thrombogenicity of total parenteral nutrition solutions and that the inhibitory properties of LE may be of practical advantage in preventing thrombosis.

Adult↗
PubMed1989

Thrombogenicity of total parenteral nutrition solutions: II. Effect on induction of endothelial cell procoagulant activity.

Thrombosis is a common sequela of total parenteral nutrition. We have recently demonstrated in vitro that hypertonic total parenteral nutrition solutions are potent inducers of a tissue factor monocyte procoagulant activity, the initiating cofactor of the extrinsic clotting cascade. We have further studied, in vitro, the effects of the component solutions of total parenteral nutrition on the induction and modulation of endothelial cell procoagulant activity. Cultured porcine aortic endothelial cells were incubated with (a) 200 microliters of dextrose solution (5%, 10%, 20%, 25%, and 50%), (b) 200 microliters of amino acid solution [full strength (N), one-fourth strength, and one-half strength], and (c) 200 microliters of 10% lipid emulsion. Cocultures of lipid emulsion and 20% dextrose, lipid emulsion and full-strength 10% amino acid solution (N-amino acid), and lipid emulsion and bacterial lipopolysaccharide also were studied. Cells were incubated for intervals of 3-108 h, washed and frozen, harvested, and assayed for endothelial cell procoagulant activity. Units of endothelial cell procoagulant activity were derived from a standard thromboplastin curve. Our results show that amino acid and hypertonic dextrose total parenteral nutrition solutions are able to strongly induce endothelial cell procoagulant activity expression in vitro. In contrast, lipid emulsion significantly inhibited the induction of endothelial cell procoagulant activity by 20% dextrose, N-amino acid, and lipopolysaccharide. These results provide further evidence for the role of the cellular pathways of coagulation in total parenteral nutrition-induced thrombosis. Furthermore, the inhibitory properties of lipid emulsion may be of practical advantage in reducing total parenteral nut induced thrombosis.

Animals↗
PubMed1989

Monocyte/macrophage procoagulant activity as a measure of immune responsiveness in Lewis and brown Norway inbred rats. Discordance with lymphocyte proliferative assays.

In vitro lymphocyte proliferative assays were performed using Lewis (Lew) and Brown Norway (BN) rats, and compared to induction of monocyte/macrophage procoagulant activity (PCA) in a mixed lymphocyte culture and by endotoxin (LPS) (E. Coli 0111:B4). Splenic mononuclear cells from Lew rats had significantly greater mitogen-induced proliferation to concanavalin A (P = .002) and phytohemagglutinin (P = 0.007). The Lew cells also showed greater allogeneically induced proliferation by BN cells in a one-way MLC in comparison to the reciprocal BN proliferative response (P less than 0.04). PCA induction in peripheral blood mononuclear cells (PBM) by allogeneic stimulation in MLC or total content PCA by LPS did not vary significantly between the 2 strains (P greater than 0.5). Induction of PCA by LPS was rapid, with a moderate rise over basal activity at 3 hr and maximal activity at 6 hr. Two-way allogeneic induction of PCA in PBM from BN and Lew rats resulted in PCA elevation by 3 hr, which became maximal at 18 hr. One-way MLC with Lew or BN cells as responders resulted in moderate increases in PCA by 3-6 hr, with equivalent maximal activities recorded at 18 hr. Viable PCA accounted for 26-32% of total content PCA in both Lew and BN rats. Maximal allogeneic PCA induction by MLC was 14-18% of PCA induced by LPS and required a longer incubation for its expression. Our results indicate that in vitro PCA expression by Lew and BN PBM following allogeneic or endotoxin stimulation shows little interstrain variability in comparison to lymphocyte proliferative responses. Thus PCA appears to more closely reflect the observed in vivo responses of these strains to allogeneic challenge.

Animals↗
PubMed1988

The fluorescence of serum proteins in diabetic patients with and without retinopathy.

Non-enzymatic glycosylation of long-lived proteins results in a characteristic fluorescence, known as protein 'browning'. The degree of fluorescence of skin collagen correlates with retinopathy in diabetes. We have therefore measured the fluorescence of serum albumin and IgG by a sensitive HPLC technique in 69 diabetic patients, 38 with retinopathy and 31 without complications, and in 26 age-matched controls. The fluorescence of the IgG fraction, calculated as the ratio of fluorescence (excitation 360 nm, emission 454 nm) to optical density (280 nm), was elevated in diabetic patients with retinopathy (1.5 +/- 0.5) compared with those without retinopathy (1.1 +/- 0.2, p less than 0.005) and control subjects (1.1 +/- 0.2, p less than 0.005). The fluorescence ratio for the albumin fraction was increased in all diabetic patients (11.2 +/- 2.7) compared with control subjects (8.9 +/- 1.5, p less than 0.001). There was no significant difference in the fluorescence ratios of albumin in those with and without retinopathy. The fluorescence of serum albumin and IgG were not significantly correlated with serum protein glycosylation measured by the fructosamine method.

Biomarkers↗