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Biomedical subjects

A M Stevens

Publications and source records attributed to A M Stevens.

At least 19 recordsLinked to original sources
PubMed2005

Human leukocyte antigen class II alleles in Caucasian women with primary biliary cirrhosis.

In the current study, we investigated human leukocyte antigen (HLA) class II alleles in Caucasian women with primary biliary cirrhosis (PBC), a disease that preferentially affects women. Alleles of DRB1, DQA1, and DQB1 were determined by DNA-based HLA typing for women with PBC (n = 72) and healthy women (n = 381). All study subjects were Caucasian. HLA DRB1*08 was significantly increased in women with PBC compared to healthy women. The increase was primarily due to the DRB1*0801 allele, also the most common DRB1*08 allele among controls. DQB1*04 and DQA1*0401 were significantly increased. DRB1*1501, DQA1*0102, and DQB1*0602 were associated with decreased risk. Analyses conducted comparing parous women with PBC to parous healthy women (n = 68 and n = 282, respectively) yielded similar significant results. Although the DRB1*08-DQA1*0401-DQB1*04 haplotype was significantly associated with PBC, consistent with other studies, this haplotype nevertheless represented only 19% (14/72) of all PBC patients and can account for only a minority of the risk of PBC.

Adult↗
PubMed2004

Male microchimerism in women with systemic sclerosis and healthy women who have never given birth to a son.

BACKGROUND: Male DNA or cells are often used to measure microchimerism in a woman. In studies of autoimmune diseases male microchimerism is most often attributed to the previous birth of a son. OBJECTIVE: To determine the frequency of male microchimerism in healthy women or women with systemic sclerosis who had never given birth to a son. METHODS: Real time quantitative polymerase chain reaction targeting the Y chromosome specific sequence DYS14 was employed to test DNA extracted from peripheral blood mononuclear cells of 26 women with systemic sclerosis and 23 healthy women who had never given birth to a son. RESULTS: are expressed as the genome equivalent number of male cells per million host cells (gEq/mil).Results: Male DNA was found in 15% of women with systemic sclerosis (range 0 to 23.7 gEq/mil) and in 13% of healthy women (range 0 to 5.1 gEq/mil). Although two women with male DNA had an induced abortion, most had no history of spontaneous or induced abortion (either systemic sclerosis or healthy). CONCLUSIONS: Microchimerism with male DNA can be found in the circulation of women who have never given birth to a son. Thus sources other than a male birth must be considered when male DNA is used to measure microchimerism. Although other studies are needed, there was no apparent difference in women with systemic sclerosis and healthy women. Possible sources of male DNA include unrecognised male pregnancy or unrecognised male twin, an older male sibling with transfer through the maternal circulation, or sexual intercourse alone.

Abortion, Induced↗
PubMed2004

Maternal and sibling microchimerism in twins and triplets discordant for neonatal lupus syndrome-congenital heart block.

OBJECTIVE: Neonatal lupus syndrome-congenital heart block (NLS-CHB) is an acquired autoimmune disease in which maternal autoantibodies are necessary but not sufficient for disease. Maternal myocardial cells have been found in the hearts of patients with NLS-CHB, suggesting that maternal microchimerism may also play a role. In this study we asked whether levels of microchimerism in the blood are associated with NLS-CHB in discordant twins and triplets. METHODS: Human leucocyte antigen (HLA)-specific and Y-chromosome-specific real-time quantitative polymerase chain reaction (PCR) was used to quantitatively assay maternal and sibling microchimerism in peripheral blood. Because of HLA allele sharing in families, it was not always possible to distinguish between multiple sources of microchimerism. RESULTS: In one family, maternal and/or sibling microchimerism was detected in two triplets who had CHB, but not in the triplet with transient hepatitis. Levels ranged from 4 to 948 genome-equivalents of foreign deoxyribonucleic acid per million host genome-equivalents (gEq/million). Over the first year levels of sibling microchimerism decreased in the triplet with complete CHB and increased in the triplet who progressed from first- to second-degree CHB. In a second family, maternal and/or sibling microchimerism was detected in the healthy twin (1223 gEq/million) but not in the twin with CHB. CONCLUSIONS: Maternal and/or sibling microchimerism was detectable in the blood of infant twins and triplets discordant for NLS. Microchimerism in the blood was not specific for NLS-CHB, although in one family levels correlated with disease. Thus, microchimerism in the blood and/or tissues may be involved in the pathogenesis or progression of NLS-CHB, but additional factors must also contribute. Further investigation is warranted.

Chimerism↗
PubMed2003

Microbiological examination of ready-to-eat stuffing from retail premises in the north-east of England. The 'Get Stuffed' survey.

AIMS: To establish the microbiological quality of ready-to-eat stuffing from retail premises in the north-east of England. To establish threshold levels of bacteria in the product for acceptance as a ready-to-eat food. To determine the relationship between the microbiology of the product and production processes. METHODS AND RESULTS: A microbiological study of ready-to-eat stuffing using validated methods was performed on 147 samples from 139 retail premises. The determinants investigated were as follows: aerobic colony count, Escherichia coli, Enterobacteriaceae, Staphylococcus aureus, Bacillus spp., Salmonella spp. and Campylobacter spp. Results indicate that using current guidelines 76.3% were satisfactory, 15.6% were acceptable and 8.2% were of unsatisfactory quality. CONCLUSIONS: Unsatisfactory results were due to high aerobic colony counts, E. coli, Enterobacteriaceae and S. aureus. There were significant associations between bacteriological quality and temperature of storage, food hygiene training, product discard policy and confidence in management scores. SIGNIFICANCE AND IMPACT OF THE STUDY: The microbiology of ready-to-eat stuffing suggests that this is a relatively safe product. It is suggested that the product be placed in food category 3 in the current guidelines for ready-to-eat foods.

Bacteria↗
PubMed2003

Polymyositis as a manifestation of chronic graft-versus-host disease.

OBJECTIVE: Chronic graft-versus-host disease (GVHD) after haematopoietic stem cell transplantation (HSCT) has similarities to some idiopathic autoimmune diseases, including polymyositis. To investigate the relationship between chronic GVHD and idiopathic myositis we conducted a detailed analysis of all cases of myositis occurring in a large series of HSCT patients. METHODS: We conducted a retrospective chart review of all cases of myositis that developed in 7161 patients who underwent HSCT at the Fred Hutchinson Cancer Research Center between 1969 and 1999. RESULTS: Among 1859 individuals who developed chronic GVHD, 12 developed myositis. No patients developed myositis without chronic GVHD. Myositis was first identified between 7 and 55 months after transplantation. In histopathology, electromyography, laboratory values and response to immunosuppressive therapy, the cases resembled idiopathic polymyositis. Autoantibodies were found in eight cases. CONCLUSIONS: Myositis in the chronic GVHD population occurred with an incidence higher than expected by chance, suggesting that muscle may be a target tissue for chronic GVHD. Recent studies have implicated allogeneic cells persisting after maternal-fetal cell transfer in selected autoimmune diseases, including myositis. This report lends support to the possibility that both idiopathic myositis and chronic GVHD-related myositis could involve allo-autoimmune responses.

Adult↗
PubMed2002

Cat scratch disease (CSD) in patients with stellate neuroretinitis: 3 cases.

This case series describes three patients with a similar clinical picture: unilateral abrupt visual loss, optic nerve edema, and a macular star exudate. In all cases we found significant antibody titers to Bartonella henselae, the causative agent of cat scratch disease. Cat scratch disease seems to be the most common cause of stellate neuroretinitis, formerly known as Leber's idiopathic stellate retinopathy. A review of the pertinent literature shows that serologic evidence of B. henselae is sufficient to confirm the diagnosis given the low incidence of significant titers in the general population. Cat scratch disease is usually a self limiting disorder in immunocompetent patients, but treatment with doxycycline is recommended.

Adult↗
PubMed2001

From the simple detection of microchimerism in patients with autoimmune diseases to its implication in pathogenesis.

Long-term persistence of fetal cells in parous women (fetal microchimerism, FM) as well as maternal cells in their offspring (maternal microchimerism, MM) have been reported. Systemic sclerosis (SSc), primary biliary cirrhosis (PBC), and Sjögren's syndrome (SS) share similar epidemiology with a predilection for females following childbearing years, with clinical similarities to chronic graft-versus-host disease, a known condition of chimerism. This led to the hypothesis that FM could be involved in the pathogenesis of autoimmune diseases. Initial investigations were conducted in SSc, where the hypothesis was supported by the more frequent occurrence and, quantitatively, a greater degree of FM in women with SSc compared to matched healthy women. Long-term persistence, however, of fetal cells in healthy women indicates that FM per se is not sufficient for causing SSc, but may be important in the context of other risk factors, such as genetic susceptibility and HLA relationship among host and nonhost cells. Contradictory results have recently been published for both PBC and SS and cause difficulty in drawing any conclusions about the role of FM in their pathogenesis. On the other hand, MM has been investigated as a risk factor in patients with systemic lupus (SLE) and juvenile dermatomyositis (JDM). A potential role of MM has been suggested in the pathogenesis of SLE. Recent publications also support the hypothesis that MM might lead to increased risks for JDM. In conclusion, contradictory results have been observed. This reflects a need for standardization of protocols and the selection of control populations. Detection of microchimerism has to be quantitatively studied in the context of genetic factors in order to study its relationship to the pathogenesis of autoimmune diseases.

Autoimmune Diseases↗
PubMed2001

Amino acid residues in LuxR critical for its mechanism of transcriptional activation during quorum sensing in Vibrio fischeri.

PCR-based site-directed mutagenesis has been used to generate 38 alanine-substitution mutations in the C-terminal 41 amino acid residues of LuxR. This region plays a critical role in the mechanism of LuxR-dependent transcriptional activation of the Vibrio fischeri lux operon during quorum sensing. The ability of the variant forms of LuxR to activate transcription of the lux operon was examined by using in vivo assays in recombinant Escherichia coli. Eight recombinant strains produced luciferase at levels less than 50% of that of a strain expressing wild-type LuxR. Western immunoblotting analysis verified that the altered forms of LuxR were expressed at levels equivalent to those of the wild type. An in vivo DNA binding-repression assay in recombinant E. coli was subsequently used to measure the ability of the variant forms of LuxR to bind to the lux box, the binding site of LuxR at the lux operon promoter. All eight LuxR variants found to affect cellular luciferase levels were unable to bind to the lux box. An additional 11 constructs that had no effect on cellular luciferase levels were also found to exhibit a defect in DNA binding. None of the alanine substitutions in LuxR affected activation of transcription of the lux operon without also affecting DNA binding. These results support the conclusion that the C-terminal 41 amino acids of LuxR are important for DNA recognition and binding of the lux box rather than positive control of the process of transcription initiation.

Alanine↗
PubMed2000

Structural insights into the stereochemistry of the cyclooxygenase reaction.

Cyclooxygenases are bifunctional enzymes that catalyse the first committed step in the synthesis of prostaglandins, thromboxanes and other eicosanoids. The two known cyclooxygenases isoforms share a high degree of amino-acid sequence similarity, structural topology and an identical catalytic mechanism. Cyclooxygenase enzymes catalyse two sequential reactions in spatially distinct, but mechanistically coupled active sites. The initial cyclooxygenase reaction converts arachidonic acid (which is achiral) to prostaglandin G2 (which has five chiral centres). The subsequent peroxidase reaction reduces prostaglandin G2 to prostaglandin H2. Here we report the co-crystal structures of murine apo-cyclooxygenase-2 in complex with arachidonic acid and prostaglandin. These structures suggest the molecular basis for the stereospecificity of prostaglandin G2 synthesis.

Animals↗
PubMed1999

Involvement of the RNA polymerase alpha-subunit C-terminal domain in LuxR-dependent activation of the Vibrio fischeri luminescence genes.

LuxR is a sigma(70) RNA polymerase (RNAP)-dependent transcriptional activator that controls expression of the Vibrio fischeri lux operon in response to an acylhomoserine lactone-cell density signal. We have investigated whether the alpha-subunit C-terminal domain (alphaCTD) of RNAP is required for LuxR activity. A purified signal-independent, LuxR C-terminal domain-containing polypeptide (LuxRDeltaN) was used to study the activation of transcription from the luxI promoter in vitro. Initiation of lux operon transcription was observed in the presence of LuxRDeltaN and wild-type RNAP but not in the presence of LuxRDeltaN and RNAPs with truncated alphaCTDs. We also studied the in vivo role of the RNAP alphaCTD in activation of lux transcription in Escherichia coli. This enabled a comparison of results obtained with full-length LuxR to those obtained with LuxRDeltaN. These in vivo studies indicated that both LuxR and LuxRDeltaN require the RNAP alphaCTD for activity. The results of DNase I protection studies showed that LuxRDeltaN-RNAP complexes can bind and protect the luxI promoter, but with less efficacy when the alphaCTD is truncated in comparison to the wild type. Thus, both in vitro and in vivo experiments demonstrated that LuxR-dependent transcriptional activation of the lux operon involves the RNAP alphaCTD and suggest that alphaCTD-LuxR interactions may play a role in recruitment of RNAP to the luxI promoter.

Bacterial Proteins↗
PubMed1997

Cross-species induction of luminescence in the quorum-sensing bacterium Vibrio harveyi.

Different species of bacteria were tested for production of extracellular autoinducer-like activities that could stimulate the expression of the luminescence genes in Vibrio harveyi. Several species of bacteria, including the pathogens Vibrio cholerae and Vibrio parahaemolyticus, were found to produce such activities. Possible physiological roles for the two V. harveyi detection-response systems and their joint regulation are discussed.

Luminescent Measurements↗
PubMed1997

Mutational analysis of the Vibrio fischeri LuxI polypeptide: critical regions of an autoinducer synthase.

Synthesis of the Vibrio fischeri autoinducer, a signal involved in the cell density-dependent activation of bioluminescence, is directed by the luxI gene product. The LuxI protein catalyzes the synthesis of N-acyl-homoserine lactones from S-adenosylmethionine and acylated-acyl carrier protein. We have gained an appreciation of the LuxI regions and amino acid residues involved in autoinducer synthesis by isolating and analyzing mutations generated by random and site-specific mutagenesis of luxI. By random mutagenesis we isolated 13 different single amino acid substitutions in the LuxI polypeptide. Eleven of these substitutions resulted in no detectable autoinducer synthase activity, while the remaining two amino acid substitutions resulted in reduced but detectable activity. The substitutions that resulted in no detectable autoinducer synthase activity mapped to two small regions of LuxI. In Escherichia coli, wild-type luxI showed dominance over all of the mutations. Because autoinducer synthesis has been proposed to involve formation of a covalent bond between an acyl group and an active-site cysteine, we constructed site-directed mutations that altered each of the three cysteine residues in LuxI. All of the cysteine mutants retained substantial activity as an autoinducer synthase in E. coli. Based on the analysis of random mutations we propose a model in which there are two critical regions of LuxI, at least one of which is an intimate part of an active site, and based on the analysis of site-directed mutations we conclude that an active-site cysteine is not essential for autoinducer synthase activity.

Bacterial Proteins↗
PubMed1997

Quorum sensing in Vibrio fischeri: essential elements for activation of the luminescence genes.

LuxR is required for cell density-dependent activation of the Vibrio fischeri luminescence (lux) genes. It has not been possible to study full-length LuxR in vitro, but a polypeptide containing the C-terminal transcriptional-activator domain of LuxR (LuxRdeltaN) has been purified, and its binding to lux regulatory DNA has been investigated. By itself, LuxRdeltaN interacts with a region of lux regulatory DNA that is upstream of the lux box, which is a 20-bp element that is required for LuxR activation of the luminescence operon. Individually, neither the purified LuxRdeltaN nor RNA polymerase binds to the lux box region, but together the two proteins bind in synergy to the lux box-luxI promoter region. We show that binding of LuxRdeltaN to the upstream region is not a prerequisite for its synergistic binding with RNA polymerase to the lux box and the luxI promoter region. We also show that LuxRdeltaN and RNA polymerase are both required and sufficient for transcriptional activation of the lux operon. This argues against the hypothesis that LuxR functions to alleviate repression of the lux operon by another cellular factor. Rather, our data support the view that LuxR functions as an accessory factor that enables RNA polymerase to bind to and initiate transcription from the promoter of the lux operon.

Bacterial Proteins↗
PubMed1997

Adenocarcinoma of the cervix: a regional retrospective study.

A retrospective study using data from the Yorkshire Cancer Registry was undertaken to investigate the incidence, characteristics and prognosis of cases of cervical adenocarcinoma in the Yorkshire Region. Three hundred and twenty-six women resident in the Yorkshire Region formed the basis of the study. The overall five year survival for adenocarcinoma of the cervix is 53%. This was influenced by stage, lymph node involvement and differentiation. 37 out of 122 surgically treated patients received inappropriate treatment. There is a need for improved data collection in the treatment of these patients. This lends weight to the argument that cancer of the cervix should be managed in cancer units.

Adenocarcinoma↗
PubMed1996

Three-dimensional structure of human cytomegalovirus protease.

Herpesviruses encode a serine protease that specifically cleaves assembly protein. This protease is critical for replication, and represents a new target for antiviral drug design. Here we report the three-dimensional structure of the protease from human cytomegalovirus (hCMV) at 2.27 angstroms resolution. The structure reveals a unique fold and new catalytic strategy for cleavage. The monomer fold of the enzyme, a seven-stranded beta-barrel encircled by a chain of helices that form the carboxy terminus of the molecule, is unrelated to those observed in classic serine proteases such as chymotrypsin and subtilisin. The serine nucleophile at position 132 is activated by two juxtaposed histidine residues at positions 63 and 157. Dimerization, which seems to be necessary for activity, is observed in the crystals. Correlations of the structure with the sequences of herpesvirus proteases suggest that dimerization may confer specificity and recognition in substrate binding.

Binding Sites↗