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Biomedical subjects

C Arnold

Publications and source records attributed to C Arnold.

At least 37 records · Page 2Linked to original sources
PubMed2001

Phylogenetic analysis of multiple heterosexual transmission events involving subtype b of HIV type 1.

Between 1996 and 1999 thirteen cases of HIV infection were detected in Doncaster, a small town in the north of England (population approximately 250,000). A complex network of shared sexual histories involving local nightclubs linked these cases, with the only known risk factor being heterosexual intercourse. A series of frozen blood samples was collected in 1998-1999 and amplified by PCR to generate full-length gp120 clones. Sequencing demonstrated that all the transmission events in this heterosexual group involved the B subtype of HIV-1. When relationships between the samples were assessed it became clear that these 13 cases represented at least three separate strains of HIV-1, indicating that HIV is well established in this community. Eleven of the 13 cases were related, forming two distinct groups. Further investigation revealed that one group contained five patients whose general health was good and who were not receiving HAART. In contrast, the second group of six patients, including the putative index case, were symptomatic, receiving HAART, and may have been infected with a CXCR-4-utilizing virus. Several of the cases that were linked by genetic criteria were not linked by contact tracing, implying that further undiagnosed cases may exist in this community. To our knowledge, this is the largest outbreak of HIV studied within the heterosexual community in the United Kingdom to date, suggesting that this route of infection is becoming more common within the United Kingdom.

Disease Outbreaks↗
PubMed2001

Fluorescent amplified fragment length polymorphism genotyping of Salmonella Enteritidis: a method suitable for rapid outbreak recognition.

OBJECTIVE: To perform fluorescent amplified fragment length polymorphism (FAFLP) analysis on phage type (PT) reference strains of Salmonella enterica subsp. enterica serotype Enteritidis (S. Enteritidis), and S. Enteritidis PT 6 and 6a recent clinical isolates to determine its usefulness for primary characterization of clinical S. Enteritidis isolates, and then to determine whether FAFLP is suitable for rapid characterization of strains in an outbreak situation. METHODS: Twenty-five PT reference strains of S. Enteritidis and 20 S. Enteritidis PT 6 and 6a clinical isolates were subjected to FAFLP analysis using the selective primer combinations Eco + 0-Mse + T and Eco + 0-Mse + TA. RESULTS: FAFLP successfully separated each one of the 25 S. Enteritidis PT strains into distinct profiles, while macrorestriction and PFGE using XbaI identified 20 pulsed-field profiles. FAFLP also resolved cases and outbreaks due to S. Enteritidis PTs 6 and 6a. CONCLUSIONS: The resolving power of FAFLP was higher than that of PFGE. FAFLP is a highly discriminatory genotyping method and, in conjunction with phage typing for primary subdivision of S. Enteritidis, provides a rapid and powerful tool for strain differentiation, both for outbreak investigation and for epidemiologic surveillance.

Bacterial Typing Techniques↗
PubMed2001

Trypsinogen gene mutations in patients with chronic or recurrent acute pancreatitis.

Three-point mutations (R117H, N211, A16V) within the cationic trypsinogen gene have been identified in patients with hereditary pancreatitis (HP). A genetic background has also been discussed for idiopathic juvenile chronic pancreatitis (IJCP), which closely mimicks the clinical pattern of HP, and alcoholic chronic pancreatitis because only a small number of heavy drinkers develop pancreatitis. This prompted us to screen 104 patients in our well-defined pancreatitis cohort for the currently known cationic trypsinogen gene mutations. The R117H mutation was detected in seven patients (six patients of two clinically classified HP families, one patient with clinically classified IJCP) and the A16V mutation in one IJCP patient. No cationic trypsinogen gene mutations were found in the remaining 96 patients with chronic and recurrent acute pancreatitis of various etiologies. Our results demonstrate the need for genetic testing to exclude HP, particularly in the presence of an atypical or unknown family history. In addition, cationic trypsinogen gene mutations are no predisposing factor in patients with chronic and recurrent acute pancreatitis of different etiologies.

Acute Disease↗
PubMed2001

A comparison of multilocus sequence typing and fluorescent fragment-length polymorphism analysis genotyping of clone complex and other strains of Neisseria meningitidis.

Five National Collection of Type Culture (NCTC) strains and 14 isolates of Neisseria meningitidis, representing 13 outbreak isolates from within the UK, were examined by multilocus sequence typing (MLST) for seven house-keeping genes. The results were compared with those of fluorescent amplified fragment-length polymorphism (FAFLP) analysis. Phylogenetic inferences were made from 3284-nucleotide lengths of sequence for the 19 isolates, by distance and parsimony methods. Two clusters of isolates were delineated. The larger, comprising eight isolates--S1, S3, Ironville, P9, ET-37 (M99-241951), P7, P10 and P60--shared 100-99.2% similarity and varied in only 40 nucleotides (approximately 1.22% variation) from the consensus sequence alignment. This cluster could be equated to the ET-37 complex because it had allelic signatures identical to MLST sequence types 11 and 50. These eight isolates were also assigned to one group by FAFLP. The reference ET-5 complex isolate 'ET-5 (NG144/82)' and an isolate (X9) from an outbreak in the north of England were also grouped together by MLST. They shared 99.2% similarity and differed within the aroE and fumC genes by 4 and 17 nucleotides, respectively. Their MLST sequence types were 32 and 661 and, therefore, these two isolates could be equated to the ET-5 complex. They also grouped together by FAFLP. A comparison of the resources required to apply MLST to the 19 isolates examined with those needed to characterise them by FAFLP indicated that FAFLP (a fragment-based genotyping method) is more cost-effective than the partial sequencing approach, MLST.

Bacterial Proteins↗
PubMed2001

Genotyping Neisseria gonorrhoeae using fluorescent amplified fragment length polymorphism analysis.

An evaluation of fluorescent amplified fragment length polymorphism (FAFLP) for typing Neisseria gonorrhoeae using 52 isolates revealed that its discriminatory power approached that of Opa-typing in identifying epidemiologically linked isolates. Automated, accurate sizing of FAFLP amplified fragments permits objective data analysis and storage, making it an attractive method for large surveillance projects.

Bacterial Typing Techniques↗
PubMed2000

Contortrostatin, a homodimeric disintegrin, binds to integrin alphavbeta5.

Contortrostatin is a homodimeric disintegrin from snake venom. We have shown that contortrostatin binds to integrins alphaIIbbeta3, alpha5beta1, and alphavbeta3. We now use several criteria to demonstrate the binding of contortrostatin to alphavbeta5. First, incubation of T24 cells, which express alphavbeta3 and alphavbeta5, with antibody against alphavbeta3 failed to completely inhibit adhesion of cells to vitronectin. However, pretreatment of the cells with contortrostatin or the combination of antibodies against alphavbeta3 and alphavbeta5 completely blocked adhesion to vitronectin. By contrast, either anti-alphavbeta5 alone or contortrostatin blocked adhesion of an alphavbeta3-negative T24 subline. Second, contortrostatin as well as anti-alphavbeta5 inhibits invasion of OVCAR-5, which express only alphavbeta5. Third, contortrostatin binds to purified alphavbeta5 in a saturable manner. Finally, radioligand binding assays yielded a K(d) value of 24 nM for [(125)I]contortrostatin binding to alphavbeta5. This investigation identifies alphavbeta5 as a binding site for contortrostatin. Blockage of alphavbeta5 by contortrostatin inhibits alphavbeta5-mediated adhesion and invasion.

Agkistrodon↗
PubMed2000

Fluorescent amplified-fragment length polymorphism genotyping of Neisseria meningitidis identifies clones associated with invasive disease.

Fluorescent amplified-fragment length polymorphism (FAFLP), a genotyping technique with phylogenetic significance, was applied to 123 isolates of Neisseria meningitidis. Nine of these were from an outbreak in a British university; 9 were from a recent outbreak in Pontypridd, Glamorgan; 15 were from sporadic cases of meningococcal disease; 26 were from the National Collection of Type Cultures; 58 were carrier isolates from Ironville, Derbyshire; 1 was a disease isolate from Ironville; and five were representatives of invasive clones of N. meningitidis. FAFLP analysis results were compared with previously published multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) results. FAFLP was able to identify hypervirulent, hyperendemic lineages (invasive clones) of N. meningitidis as well as did MLST. PFGE did not discriminate between two strains from the outbreak that were classified as similar but distinct by FAFLP. The results suggest that high resolution of N. meningitidis for outbreak and other epidemiological analyses is more cost efficient by FAFLP than by sequencing procedures.

Bacterial Typing Techniques↗
PubMed2000

Genotyping of verocytotoxin-producing Escherichia coli O157: comparison of isolates of a prevalent phage type by fluorescent amplified-fragment length polymorphism and pulsed-field gel electrophoresis analyses.

We applied the high-resolution genotyping technique fluorescent amplified-fragment length polymorphism (FAFLP) analysis to 71 isolates of a single phage type (PT8) of pulsed-field gel electrophoresis (PFGE)-characterized verocytotoxin-producing Escherichia coli O157. Twenty-seven similar, but not identical, groupings were defined by both FAFLP analysis and the PFGE profiles. Given the FAFLP analysis conditions described here, these two methods exhibited equivalent discriminatory powers.

Bacteriophage Typing↗
PubMed2000

Genome-sequence-based fluorescent amplified-fragment length polymorphism analysis of Mycobacterium tuberculosis.

The whole-genome fingerprinting technique, fluorescent amplified-fragment length polymorphism (FAFLP) analysis, was applied to Mycobacterium tuberculosis. Sixty-five clinical isolates were analyzed to determine the value of FAFLP as a stand-alone genotyping technique and to compare it with the well-established IS6110 typing system. The genome sequence of M. tuberculosis strain H37Rv (S. T. Cole et al., Nature 393:537-544, 1998) was used to model computer-generated informative primer combination(s), and the precision and reproducibility of FAFLP were evaluated by comparing the results of in vitro and computer-generated experiments. Multiplex FAFLP was used to increase resolving power in a predictable and systematic fashion. FAFLP analysis was broadly congruent with IS6110 typing for those strains with multiple IS6110 copies. It was also able to resolve an epidemiologically unlinked group of strains with only one copy of IS6110; up to 10% of clinical isolates may fall into this category. For certain epidemiological investigations, it was concluded that a combination of FAFLP and IS6110 typing would give higher resolution than would either alone. FAFLP data were digital, precise, reproducible, and suitable for rapid electronic dissemination, manipulation, interlaboratory comparison, and storage in national or international epidemiological databases. Because FAFLP samples and analyzes base substitution across the genome as a whole, FAFLP could generate new information about the microevolution of the M. tuberculosis complex.

Bacterial Typing Techniques↗
PubMed2000

The impact of loading on deformation about posteromedial meniscal tears.

To simulate the effects of partial weight bearing on meniscal repair, full-thickness tears were produced in the posteromedial aspect of seven ACL-intact cadaveric knees. Following suture repair, metal markers were imbedded to index the position of the tear. A radiolucent chamber was used to position and load each knee for computed tomography scanning at flexion angles of 0 degrees, 30 degrees, and 60 degrees with and without a load force of 100 lb. Sutures were removed and the tests repeated. Changes in marker distances were obtained for each test condition. Loading produced average dimensional changes of < or =0.5 +/-0.6 mm in either sutured or unsutured menisci. Based on the Friedman test, neither flexion angle, loading, nor suture exerted a significant impact (P> or =.52). These data support clinical accelerated rehabilitation programs previously published. Therefore, partial weight bearing during convalescence is tenable.

Adult↗
PubMed1999

Alterations in liver ATP homeostasis in human nonalcoholic steatohepatitis: a pilot study.

CONTEXT: The mechanisms that drive progression from fatty liver to steatohepatitis and cirrhosis are unknown. In animal models, obese mice with fatty livers are vulnerable to liver adenosine triphosphate (ATP) depletion and necrosis, suggesting that altered hepatic energy homeostasis may be involved. OBJECTIVE: To determine if patients with fatty liver disease exhibit impaired recovery from hepatic ATP depletion. DESIGN: Laboratory analysis of liver ATP stores monitored by nuclear magnetic resonance spectroscopy before and after transient hepatic ATP depletion was induced by fructose injection. The study was conducted between July 15 and August 30, 1998. SETTING: University hospital. PATIENTS: Eight consecutive adults with biopsy-proven nonalcoholic steatohepatitis and 7 healthy age- and sex-matched controls. MAIN OUTCOME MEASURE: Level of ATP 1 hour after fructose infusion in patients vs controls. RESULTS: In patients, serum aminotransferase levels were increased (P = .02 vs controls); albumin and bilirubin values were normal and clinical evidence of portal hypertension was absent in both groups. However, 2 patients had moderate fibrosis and 1 had cirrhosis on liver biopsy. Mean serum glucose, cholesterol, and triglyceride levels were similar between groups but patients weighed significantly more than controls (P = .02). Liver ATP levels were similar in the 2 groups before fructose infusion and decreased similarly in both after fructose infusion (P = .01 vs initial ATP levels). However, controls replenished their hepatic ATP stores during the 1-hour follow-up period (P<.02 vs minimum ATP) but patients did not. Hence, patients' hepatic ATP levels were lower than those of controls at the end of the study (P = .04). Body mass index (BMI) correlated inversely with ATP recovery, even in controls (R = -0.768; P = .07). Although BMI was greater in patients than controls (P = .02) and correlated strongly with fatty liver and serum aminotransferase elevations, neither of the latter 2 parameters nor the histologic severity of fibrosis strongly predicted hepatic ATP recovery. CONCLUSIONS: These data suggest that recovery from hepatic ATP depletion becomes progressively less efficient as body mass increases in healthy controls and is severely impaired in patients with obesity-related nonalcoholic steatohepatitis.

Adenosine Triphosphate↗
PubMed1999

Monoclonal antibodies recognize at least five epitopes on the SIV Nef protein and identify an in vitro-induced mutation.

Eleven monoclonal antibodies (MAbs) to SIV Nef were produced and characterized. Five antibody-binding sites on SIV Nef were identified on the basis of the reactivity of the antibodies with recombinant proteins. Two of the five epitopes were defined using overlapping peptides. A further three epitopes could not be defined with peptides but all antibodies reacted in Western blot, suggesting that the epitopes were at least partially conformation dependent. Antibodies in two of the five epitope groups were further differentiated by competition analysis. The panel of MAbs described is able to distinguish between a number of recombinant Nef proteins currently under investigation in vivo in macaques. Two of the MAbs described are able to distinguish between the Nef protein from pathogenic (J5) and attenuated (C8) strains of SIV, thus providing useful tools for studying the relevance of the Nef protein in the pathogenesis of SIV infection. In FACScan analysis two of the MAbs, KK70 and KK75, were used to identify an in vitro-induced mutation in J5 Nef grown in C8166 cells. Sequence analysis of the phenotypic variants identified a mutation of the tryptophan (TGG) at amino acid 214 to a stop codon (TGA), thus truncating the Nef protein. The functional significance of this observation remains unclear but highlights the need to interpret data with caution if virus has been cultured in vitro even for a short period of time.

Animals↗
PubMed1999

Non-peptide corticotropin-releasing hormone antagonists: syntheses and structure-activity relationships of 2-anilinopyrimidines and -triazines.

Screening of our chemical library using a rat corticotropin-releasing hormone (CRH) receptor assay led to the discovery that 2-anilinopyrimidine 15-1 weakly displaced [125I]-0-Tyr-oCRH from rat frontal cortex homogenates when compared to the known peptide antagonist alpha-helical CRH(9-41) (Ki = 5700 nM vs 1 nM). Furthermore, 15-1 weakly inhibited CRH-stimulated adenylate cyclase activity in the same tissue, but it was less potent than alpha-helical CRH(9-41) (IC50 = 20 000 nM vs 250 nM). Systematic structure-activity relationship studies, using the cloned human CRH1 receptor assay, defined the pharmacophore for optimal binding to hCRH1 receptors. Several high-affinity 2-anilinopyrimidines and -triazines were discovered, some of which had superior pharmacokinetic profiles in the rat. This paper describes the structure-activity studies which improved hCRH1 receptor binding affinity and pharmacokinetic parameters in the rat. Compound 28-17 (mean hCRH1 Ki = 32 nM) had a significantly improved pharmacokinetic profile in the rat (19% oral bioavailability at 30 mg/kg) as well as in the dog (20% oral bioavailability at 5 mg/kg) relative to the early lead structures.

Animals↗
PubMed1999

Contortrostatin, a dimeric disintegrin from Agkistrodon contortrix contortrix, inhibits angiogenesis.

Contortrostatin, a 13.5 kDa disulfide-linked homodimeric polypeptide possessing an Arg-Gly-Asp sequence, was isolated from venom of the southern copperhead snake. Daily injection of contortrostatin into the primary tumor of human breast cancer MDA-MB-435 carried in nude mice significantly inhibited tumor growth and neovascularization of the tumor tissue. On the chick embryo chorioallantoic membrane, contortrostatin inhibited angiogenesis induced by MDA-MB-435 cells, basic fibroblast growth factor, and vascular endothelial growth factor. In addition, contortrostatin effectively blocked adhesion of human umbilical vein endothelial cells (HUVEC) to immobilized vitronectin and significantly inhibited invasion of HUVEC through a Matrigel barrier. Competitive binding assays and adhesion assays with different integrin antibodies suggested that integrin alpha(v)beta3 is a binding site for contortrostatin on vascular endothelial cells. Detachment of HUVEC from vitronectin by contortrostatin induced apoptosis. HUVEC adhered and spread well on immobilized contortrostatin without undergoing apoptosis, suggesting that it is the inhibition of adhesion and spreading of HUVEC on extracellular matrix proteins, rather than binding of contortrostatin to integrins per se, that triggers apoptosis. We conclude that contortrostatin binds to alpha(v)beta3, and interferes with the anchorage-dependent survival mechanism of the vascular endothelial cells, and the mobility of the cells. The consequent suppression of angiogenesis is an important component of the antineoplastic activity of contortrostatin.

Journal Article↗
PubMed1999

The appearance of escape variants in vivo does not account for the failure of recombinant envelope vaccines to protect against simian immunodeficiency virus.

The presence or evolution of immune escape variants has been proposed to account for the failure of recombinant envelope vaccines to protect macaques against challenge with simian immunodeficiency virus (SIVmac). To address this issue, two groups of three cynomolgus macaques were immunized with recombinant SIV Env vaccines using two different vaccine schedules. One group of macaques received four injections of recombinant SIV gp120 in SAF-1 containing threonyl muramyl dipeptide as adjuvant. A second group were primed twice with recombinant vaccinia virus expressing SIV gp160 and then boosted twice with recombinant SIV gp120. Both vaccine schedules elicited neutralizing antibodies to Env. However, on the day of challenge, titres of anti-Env antibodies measured by ELISA were higher in macaques primed with recombinant vaccinia virus. Following intravenous challenge with 10 monkey infectious doses of the SIVmac J5M challenge stock, five of the six immunized macaques and all four naive controls became infected. The virus burdens in PBMC of macaques that were primed with recombinant vaccinia virus were lower than those of naive controls, as determined by virus titration and quantitative DNA PCR. Sequence analysis was performed on SIV env amplified from the blood of immunized and naive infected macaques. No variation of SIV env sequence was observed, even in macaques with a reduced virus load, suggesting that the appearance of immune escape variants does not account for the incomplete protection observed. In addition, this study indicates that the measurement of serum neutralizing antibodies may not provide a useful correlate for protection elicited by recombinant envelope vaccines.

Amino Acid Sequence↗
PubMed1999

Predictive fluorescent amplified-fragment length polymorphism analysis of Escherichia coli: high-resolution typing method with phylogenetic significance.

The fluorescent amplified-fragment length polymorphism (FAFLP) assay potentially amplifies a unique set of genome fragments from each bacterial clone. It uses stringently hybridizing primers which carry a fluorescent label. Precise fragment sizing is achieved by the inclusion of an internal size standard in every lane. Therefore, a unique genotype identifier(s) can be found in the form of fragments of precise size or sizes, and these can be generated reproducibly. In order to evaluate the potential of FAFLP as an epidemiological typing method with a valid phylogenetic basis, we applied it to 87 strains of Escherichia coli. These comprised the EcoR collection, which has previously been classified by multilocus enzyme electrophoresis (MLEE) and which represents the genetic diversity of the species E. coli, plus 15 strains of the clinically important serogroup O157. FAFLP with an unlabelled nonselective EcoRI primer (Eco+0) and a labelled selective MseI primer (Mse+TA) gave strain-specific profiles. Fragments of identical sizes (in base pairs) were assumed to be identical, and the genetic distances between the strains were calculated. A phylogenetic tree derived from measure of distance correlated closely with the MLEE groupings of the EcoR collection and placed the verocytotoxin-producing O157 strains on an outlier branch. Our data indicate that FAFLP is suitable for epidemiological investigation of E. coli infection, providing well-defined and reproducible identifiers of genotype for each strain. Since FAFLP objectively samples the whole genome, each strain or isolate can be assigned a place within the broad context of the whole species and can also be subjected to a high-resolution comparison with closely related strains to investigate epidemiological clonality.

Bacterial Typing Techniques↗
PubMed1998

[Molecular biology analysis: hereditary hemochromatosis, Wilson disease, alpha 1-antitrypsin deficiency and Dubin-Johnson syndrome].

The molecular pathology of hereditary hemochromatosis, Wilson's disease, alpha 1-antitrypsin-deficiency and Dubin-Johnson syndrome could be well characterised during the last years. Diagnosis of hereditary hemochromatosis is reliably confirmed by PCR-augmentation and restriction-analysis. Wilson's disease is a monogenetic disease, which is characterised by over 50 mutations. Molecular diagnosis is complicated by the lack of a single specific mutation. Diagnosis of Dubin-Johnson syndrome and alpha 1-antitrypsin-deficiency is possible by PCR-analysis and hybridisation with specific oligonucleotides.

DNA Mutational Analysis↗