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Biomedical subjects

B J Thomas

Publications and source records attributed to B J Thomas.

At least 55 records · Page 3Linked to original sources
PubMed1999

Pharmaceutical and biological properties of poly(amino acid)/DNA polyplexes.

Physicochemical properties of polyplexes formed between pRSVlacZ and poly(amino acid)s were investigated as a paradigm of more complex, synthetic virus-like, DNA delivery systems, that are of interest to many gene delivery laboratories. We observed the interaction between polymer and DNA using ethidium exclusion, and determined the size distributions and the zeta potentials of polyplexes. We correlated these properties with their fundamental interactions with cultured B16 murine melanoma cells, and the resulting efficiency of transfection. A variety of poly(amino acid)s each condensed DNA to produce particles with mean hydrodynamic diameters of approximately 100 nm (a typical span of a population was 80-120nm). Poly(amino acid) polyplexes were unstable in electrolyte solutions such as cell culture media. The apparent particle size increased in electrolyte, depending on the charge ratio, to diameters up to 700 nm. This was thought to be due to aggregation, since neutral particles were most sensitive. When the charge ratio (+/-) exceeded unity polyplexes had positive zeta potentials (which peaked at approximately +30 mV), bound non-specifically to cells, were internalised and in the presence of an endosomolytic agent were able to transfect cells. Though all cationic poly(amino acid)s investigated formed polyplexes with similar physical properties, their biological properties were significantly different. Polyplexes prepared with poly-L-ornithine were the most effective transfection agents, but poly(lys-co-ala, 1: 1) systems appeared to be inactive. This may reflect the differences in uncoupling of DNA and polymer, which is expected to be necessary for passage through the nuclear pore. Uncoupling of polycation and DNA was investigated by exposing the complexes to dextran sulphate. Release of DNA was detected by increased fluorescence at 600 nm in the presence of ethidium. Release of DNA was incomplete from polyplexes formed with high molecular weight polylysine. This may explain the lower levels of transfection observed with high molecular weight polylysine. The significance of these observations for design of advanced non-viral gene delivery systems is discussed.

Amino Acids↗
PubMed1998

Polycation-DNA complexes for gene delivery: a comparison of the biopharmaceutical properties of cationic polypeptides and cationic lipids.

DNA plasmids formed particulate complexes with a variety of cationic polyamino acids and cationic lipids, which were used to transfect mammalian cells in culture. Complexation was studied by assaying for exclusion of ethidium using a fluorometric assay, which indicated that complexation with cationic polyamino acids took place with utilisation of the majority of charged functional groups. The particle sizes and zeta potentials of a range of complexes were determined. Generally polyamino acids formed uniform particles 80-120 nm in diameter in water, but their particle size increased on dilution of the particles in electrolytes or cell culture media. The efficiency of transfection was compared using complexes of pRSVlacZ, a reporter construct which expressed beta-galactosidase under the control of the Rous sarcoma virus promoter. Positively charged DNA/polyamino acid complexes were taken up by cells but required an endosomolytic agent, such as chloroquine, to facilitate transfection. Polyornithine complexes resulted in the highest levels of expression, in comparison with other homopolyamino acids (polyornithine>poly-L-lysine=poly-D-lysine>polyarginine). Copolyamino acids of lysine and alanine condensed DNA but were less active in transfection experiments. Copoly(L-Lys, L-Ala 1:1) was inactive even in the presence of chloroquine. In contrast DNA/cationic lipid complexes transfected cells spontaneously, and chloroquine did not improve the extent of expression, rather it usually reduced efficiency. There was little correlation between comparative efficiencies of lipid complexes between cell lines suggesting that the nature of the cell membrane and differences in mechanisms of internalisation were determinants of efficiency. In an effort to explore better cell culture models for gene delivery, monolayers of Caco-2 cells were transfected in filter culture. As the cells differentiated and formed a polarized monolayer, expression of beta-galactosidase was reduced until at day 27 expression was not significantly different from basal activity. The Caco-2 filter culture model merits further attention as a model of gene delivery to epithelial surfaces, such as would be encountered in the lung after inhalation.

Animals↗
PubMed1998

Prediction of the chemical composition of lamb carcasses from multi-frequency impedance data.

Multi-frequency bioimpedance analysis (MFBIA) was used to determine the impedance, reactance and resistance of 103 lamb carcasses (17.1-34.2 kg) immediately after slaughter and evisceration. Carcasses were halved, frozen and one half subsequently homogenized and analysed for water, crude protein and fat content. Three measures of carcass length were obtained. Diagonal length between the electrodes (right side biceps femoris to left side of neck) explained a greater proportion of the variance in water mass than did estimates of spinal length and was selected for use in the index L2/Z to predict the mass of chemical components in the carcass. Use of impedance (Z) measured at the characteristic frequency (Zc) instead of 50 kHz (Z50) did not improve the power of the model to predict the mass of water, protein or fat in the carcass. While L2/Z50 explained a significant proportion of variation in the masses of body water (r(2) 0.64), protein (r(2) 0.34) and fat (r(2) 0.35), its inclusion in multi-variate indices offered small or no increases in predictive capacity when hot carcass weight (HCW) and a measure of rib fat-depth (GR) were present in the model. Optimized equations were able to account for 65-90% of the variance observed in the weight of chemical components in the carcass. It is concluded that single frequency impedance data do not provide better prediction of carcass composition than can be obtained from measures of HCW and GR. Indices of intracellular water mass derived from impedance at zero frequency and the characteristic frequency explained a similar proportion of the variance in carcass protein mass as did the index L2/Z50.

Animals↗
PubMed1998

Chlamydia pneumoniae in atheroma: consideration of criteria for causality.

AIMS: (1) To seek evidence of the existence of Chlamydia pneumoniae in a spectrum of atheromatous lesions in different types of arteries from individuals of different ages, using a polymerase chain reaction (PCR) assay supported by electron microscopy and immunocytochemistry; (2) to use electron microscopy to examine interactions between C pneumoniae and the cells present in the arterial tissue; (3) to assess the extent to which the data fulfil the criteria for causality. METHODS: At necropsy examination, 35 arterial specimens were taken from 25 subjects. The grade of atheroma was determined macroscopically and microscopically and the tissues coded and examined by the three techniques. RESULTS: Of the 35 specimens, 24 had macroscopic or microscopic atheromatous lesions of varying degree. Twenty two of the 35 specimens were examined by electron microscopy, C pneumoniae-like bodies being found in 11 (50%); seven specimens were examined by the immunocytochemical method, positive staining being detected in three; and all specimens were examined by the PCR technique, 15 (43%) being PCR positive. Overall, of the 24 specimens with lesions, 17 (71%) were positive by at least one of the three tests, whereas of the 11 specimens without lesions, only one was positive. The positive specimens comprised 10 of 19 aortas, three of six iliac arteries, and one coronary and one pulmonary artery. C pneumoniae was detected in four of six specimens in which there were early changes and in a 20 year old subject. Concerning the 25 subjects, of 17 who had atheromatous arteries, 14 (82%) were C pneumoniae positive and of the eight who had normal arteries, none was positive. CONCLUSIONS: There is a strong correlation between C pneumoniae and arterial atheromatous lesions. The organism may contribute to the disease process by damaging smooth muscle cells.

Adult↗
PubMed1998

Sensitivity of the ligase chain reaction assay for detecting Chlamydia trachomatis in vaginal swabs from women who are infected at other sites.

OBJECTIVE: To assess the sensitivity of the ligase chain reaction (LCR) assay for Chlamydia trachomatis in vaginal swabs from women who were positive in cervical samples and/or urines. SUBJECTS: 413 women attending the genitourinary medicine clinic, St. Mary's Hospital, Paddington. METHODS: The LCR assay was used to test vaginal swabs from 46 women who were C trachomatis positive at one or both of the other sites by direct fluorescent antibody (DFA) staining, by an enzyme immunoassay (EIA), or by the LCR assay. RESULTS: The LCR assay of vaginal swabs had the following sensitivity values using confirmed positive results: 93% (41/44) compared with DFA staining of cervical deposits, 93% (41/44) compared with the LCR assay of cervical samples, 93% (28/30) compared with an EIA for cervical samples, 91% (39/43) compared with DFA staining of urine deposits, and 93% (39/42) compared with the LCR assay of urine. Four women had vaginal swab samples negative by the LCR assay; one was positive only in the urine and two had cervical samples containing a small number of chlamydial elementary bodies. CONCLUSION: Testing vaginal swabs by the LCR assay is a sensitive method of detecting chlamydial infection; the results suggest that this procedure could be used as an alternative to examining urines in a screening programme for chlamydial infection in the community.

Chlamydia Infections↗
PubMed1998

Bioelectrical impedance analysis for the estimation of body composition in rats.

Bioelectrical impedance analysis (BIA) was used to assess body composition in rats fed on either standard laboratory diet or on high-fat diet designed to induce obesity. Bioelectrical impedance analysis predictions of total body water and thus fat-free mass (FFM) for the group mean values were generally within 5% of the measured values by tritiated water (3H2O) dilution. The limits of agreement for the procedure were, however, large, approximately +/- 25%, limiting the applicability of the technique for measurement of body composition in individual animals.

Adipocytes↗
PubMed1998

Quantification of Chlamydia trachomatis in cervical and urine specimens from women attending a genitourinary medicine clinic: implications for screening strategies.

Population screening and intervention programmes can reduce the prevalence and incidence of infection with Chlamydia trachomatis, especially if sensitive molecular diagnostic tests are used. However, diagnostic tests that perform well on genitourinary medicine (GUM) clinic populations may be less useful for screening, particularly if the majority of infected subjects are asymptomatic and their samples contain fewer organisms. We have compared the extent of low organism load in cervical and urine samples from symptomatic and asymptomatic chlamydia-positive women, by using a direct fluorescent antibody staining method and counting the chlamydial elementary bodies (EBs). We have investigated the ability of an enzyme immunoassay (EIA; MicroTrak) and a DNA amplification (ligase chain reaction; LCR) assay to detect low numbers of organisms in cervical samples and the ability of the LCR assay to detect low numbers of organisms in urine. A low organism load (< 10 EBs) was seen by direct fluorescent antibody (DFA) staining in about 30% of cervical samples and in about 75% of urines from chlamydia-positive women; the proportions in symptomatic women were not significantly different from those in asymptomatic women. The EIA identified only 16% of cervical samples that contained < 10 EBs by DFA staining; the LCR identified 100% of cervical samples and 93% of urine samples that contained < 10 EBs by DFA staining. The findings suggest that the ability of chlamydial diagnostic tests to identify positive women should be similar among patients attending a GUM clinic and those taking part in a population screening programme, and that sensitive molecular assays such as the LCR should identify subjects with a low organism load in both groups.

Chlamydia Infections↗
PubMed1997

roughex down-regulates G2 cyclins in G1.

Cell cycle arrest in G1 at the onset of patterning in the Drosophila eye is mediated by roughex. In roughex mutants, cells accumulate Cyclin A protein in early G1 and progress into S phase precociously. When Roughex is overexpressed in S/G2 cells, Cyclin A is mislocalized to the nucleus and degraded, preventing mitosis. Whereas Roughex inhibits Cyclin A accumulation, Cyclin E down-regulates Roughex protein in vivo. Roughex binds to Cyclin E and is a substrate for a Cyclin E-Cdk complex in vitro. These data argue that Roughex inhibits Cyclin A accumulation in early G1 by targeting Cyclin A for destruction. In late G1, Roughex is destabilized in a Cyclin E-dependent process, releasing Cyclin A for its role in S/G2.

Animals↗
PubMed1997

Control of G1 in the developing Drosophila eye: rca1 regulates Cyclin A.

In the developing eye of Drosophila melanogaster, cells become synchronized in the G1 phase of the cell cycle just prior to the onset of cellular differentiation and morphogenesis. In roughex (rux) mutants, cells enter S phase precociously because of ectopic activation of a Cyclin A/Cdk complex in early G1. This leads to defects in cell fate and pattern formation, and results in abnormalities in the morphology of the adult eye. A screen for dominant suppressors of the rux eye phenotype led to the identification of mutations in cyclin A, string (cdc25), and new cell cycle genes. One of these genes, regulator of cyclin A (rca1), encodes a novel protein required for both mitotic and meiotic cell cycle progression. rca1 mutants arrest in G2 of embryonic cell cycle 16 with a phenotype very similar to cyclin A loss of function mutants. Expression of rca1 transgenes in G1 or in postmitotic neurons promotes Cyclin A protein accumulation and drives cells into S phase in a Cyclin A-dependent fashion.

Amino Acid Sequence↗
PubMed1997

Low-dose warfarin prophylaxis to prevent symptomatic pulmonary embolism after total knee arthroplasty.

Pulmonary embolism poses a risk to patients undergoing total knee arthroplasty. The selection of an appropriate prophylaxis agent and its implementation have been influenced by decreased duration of hospital stay and the pressures of cost containment. The purpose of this study was to determine the inpatient and outpatient pulmonary embolism rates, the number of days required to attain the target level of anticoagulation, and complications associated with the use of a low-dose warfarin prophylaxis protocol after primary and revision total knee arthroplasty. Between 1984 and 1993, there were 815 primary and revision total knee arthroplasties that received low-dose warfarin prophylaxis at our institution. The average time to attainment of the target level of anticoagulation was 3 days. The average duration of warfarin prophylaxis was 12 days. Overall, there were a total of three symptomatic pulmonary embolisms (0.3%; 95% confidence interval, 0.08%-1.1%). There were eight (1%) symptomatic deep vein thromboses (all distal). There were two deaths (0.3%), but neither one was secondary to a pulmonary embolism. Seventeen knees (2.5%) developed a hematoma after surgery, and two of these patients required drainage of the knee. Low-dose warfarin prophylaxis is safe and effective in preventing symptomatic pulmonary embolism after total knee arthroplasty.

Adult↗
PubMed1997

Outcome after total hip arthroplasty. Comparison of a traditional disease-specific and a quality-of-life measurement of outcome.

The purpose of this study was to examine the relationship between the Harris Hip Score (HHS), a traditional method of patient assessment of a total hip arthroplasty (THA), and the Medical Outcomes Study 36-Item Short-Form Health Survey (SF-36), a commonly used health-related quality-of-life survey. One hundred forty patients returning for routine clinical follow-up evaluation of a primary THA were asked to fill out the SF-36 quality-of-life survey, as well as questions concerning their perceptions of their THA. The patient's surgeon assessed the THA with the traditional HHS. The correlations between the HHS and the SF-36 domains were highest in the physical component summary scores for male patients of all ages and female patients 65 years of age or older. The correlations were lower for the mental component summary scores of all patients, but particularly in female patients younger than 65. When the SF-36 scores were compared with age and sex-matched population norms, both age and sex were found to be important. Men younger than 65 had scores lower than norms in the physical function domains, but were comparable in the mental health domains. The older men had scores comparable to the norms in all domains. Female patients of all ages, however, had lower scores in the physical function domains. The greatest differences were noted in the female patients younger than 65. The HHS is commonly used to assess disease-specific pain and function in THA patients; however, the results of this study suggest that the SF-36 health survey can capture additional important quality-of-life domains that are influenced by a THA and that these domains are influenced by the age and sex of the patient. The combination of a disease-specific scoring system and a quality-of-life survey would allow a more global assessment of a THA in all patients. Studies evaluating the results of THAs should either assess the results of male and female patients separately when sample size is sufficiently large or use sex as a possible covariate in a multivariate analysis.

Adolescent↗
PubMed1997

Impact of declining reimbursement and rising hospital costs on the feasibility of total hip arthroplasty.

Total hip arthroplasty, although a very successful clinical treatment, remains an expensive procedure in an era of constrained health care resources. Hospitalization cost, charge, and reimbursement data were compared between all patients who underwent elective, primary, unilateral total hip arthroplasty in 1988 and 1993 at the UCLA Medical Center. Although length of hospitalization decreased by 36%, increases both in unit supply costs and in the intensity of hospital services prevented a statistically significant reduction in total hospitalization cost. Reimbursement declined by 27% after calculating inflation with the Consumer Price Index for Medical Care. Further, the margin by which reimbursement exceeded cost decreased from 66% in 1988 to 8% in 1993. These trends constitute a serious threat to the financial feasibility of total hip arthroplasty.

Aged↗
PubMed1997

Evaluation of bioelectrical impedance for prospective nutritional assessment in cystic fibrosis.

We have compared the use of bioelectrical impedance analysis (BIA) with anthropometry for the prediction of changes in total body potassium (TBK) in a group (n = 31) of children with cystic fibrosis. Linear regression analysis showed that TBK was highly correlated (r > 0.93) with height2/impedance, weight, height, and fat-free mass (FFM) estimated from skin-fold measurements. Changes in TBK were also correlated, but less well, with changes in height2/impedance, weight, height, and FFM (r = 0.69, 0.59, 0.44, and 0.40, respectively). The children were divided into two groups: those who had normal accretion of TBK (> 5%/y) and those who had suboptimal accretion of TBK (< 5%/y). Analysis of variance showed that the significant difference in the change in TBK between the groups was detectable by concomitant changes in impedance and weight but not by changes in height, FFM, or weight and height Z scores. The results of this study suggest that serial BIA measures may be useful as a predictor of progressive undernutrition and poor growth in children with cystic fibrosis.

Adolescent↗
PubMed1997

Extraction of electrical characteristics from pixels of multifrequency EIT images.

Computer modelling has shown that electrical characteristics of individual pixels may be extracted from within multiple-frequency electrical impedance tomography (MFEIT) images formed using a reference data set obtained from a purely resistive, homogeneous medium. In some applications it is desirable to extract the electrical characteristics of individual pixels from images where a purely resistive, homogeneous reference data set is not available. One such application of the technique of MFEIT is to allow the acquisition of in vivo images using reference data sets obtained from a non-homogeneous medium with a reactive component. However, the reactive component of the reference data set introduces difficulties with the extraction of the true electrical characteristics from the image pixels. This study was a preliminary investigation of a technique to extract electrical parameters from multifrequency images when the reference data set has a reactive component. Unlike the situation in which a homogeneous, resistive data set is available, it is not possible to obtain the impedance and phase information directly from the image pixel values of the MFEIT images data set, as the phase of the reactive reference is not known. The method reported here to extract the electrical characteristics (the Cole-Cole plot) initially assumes that this phase angle is zero. With this assumption, an impedance spectrum can be directly extracted from the image set. To obtain the true Cole-Cole plot a correction must be applied to account for the inherent rotation of the extracted impedance spectrum about the origin, which is a result of the assumption. This work shows that the angle of rotation associated with the reactive component of the reference data set may be determined using a priori knowledge of the distribution of frequencies of the Cole-Cole plot. Using this angle of rotation, the true Cole-Cole plot can be obtained from the impedance spectrum extracted from the MFEIT image data set. The method was investigated using simulated data, both with and without noise, and also for image data obtained in vitro. The in vitro studies involved 32 logarithmically spaced frequencies from 4 kHz up to 1 MHz and demonstrated that differences between the true characteristics and those of the impedance spectrum were reduced significantly after application of the correction technique. The differences between the extracted parameters and the true values prior to correction were in the range from 16% to 70%. Following application of the correction technique the differences were reduced to less than 5%. The parameters obtained from the Cole-Cole plot may be useful as a characterization of the nature and health of the imaged tissues.

Biometry↗
PubMed1997

Association of antibodies to Chlamydia trachomatis heat-shock protein 60 kD with chronic nongonococcal urethritis.

Ninety male patients with acute nongonococcal urethritis (NGU) who presented for follow-up 10-92 days after initiation of treatment were evaluated. A polymerase chain reaction assay and direct fluorescence antibody test were used to detect Chlamydia trachomatis at presentation and during follow-up. Chlamydial heat-shock protein 60 kD (hsp60) serology with an enzyme-linked immunosorbent assay and C. trachomatis serology with a microimmunofluoresence test were undertaken. In 62 (69%) of the men, evidence of chronic urethritis was noted during follow-up. C. trachomatis was detected in only two patients during follow-up. Chlamydial hsp60 antibody was associated with the development of chronic urethritis between 10 and 92 days after treatment began (P < .04), that is, at 10-29 days (P < .02) and at 30-92 days (P < .008). These results are consistent with the theory that immune response to hsp60 is important in the development of this chronic disease. The results also suggest that chronic NGU is not a consequence of continued production of hsp60 by C. trachomatis.

Adult↗