[Epigenetics: significance for tumor genesis and clinical features].
Explore the source record for details and available documents.
Cell Transformation, Neoplastic↗
Biomedical subjects
C Arnold
Publications and source records attributed to C Arnold.
Tissue levels, histologic changes and plasma pharmacokinetics of meta-Tetra (hydroxyphenyl) chlorin (mTHPC) in the cat.
The biodistribution and pharmacokinetics of meta-tetra(hydroxyphenyl)chlorin (mTHPC)(1) have been documented in humans, rats, dogs and rabbits. It has been demonstrated to be an effective photodynamic therapy agent for treatment of squamous cell carcinoma. Squamous cell carcinoma is a common feline neoplasm, causing significant morbidity and mortality in the feline population. The association between ultraviolet radiation exposure and occurrence of this neoplasm in the cat provides a useful model for the study of human cutaneous squamous cell carcinoma. In this study, we document the biodistribution, pharmacokinetics and toxicity of mTHPC in a group of normal cats. Four groups of cats were given the drug intravenously at dosages of 0, 0.15, 0.30 and 0.60 mg/kg. mTHPC levels were measured in plasma and tissues at 0, 24, 48, 72, 96 and 336 h after drug administration. Additionally, plasma samples were collected at 1 and 6 h post-injection and analysed. Biodistribution and pharmacokinetics of mTHPC in cats mirrors that in other animal species. There were no clinical or pathological changes associated with administration of the drug. The biodistribution and pharmacokinetics of mTHPC in cats mirrors that in other species studied. There were no clinical or pathological changes attributable to administration of the drug at the doses administered. mTHPC may be a useful photodynamic therapy drug in cats.
Animals↗
Isolation and characterization of human monoclonal autoantibodies to glutamic acid decarboxylase.
Production of human monoclonal autoantibodies to glutamic acid decarboxylase M(r) 65,000 (GAD65), characterization of their isotype, binding affinity, V region sequences and competition with autoantibodies in patients' sera is described. Lymphocytes from a patient with Addison's disease who had GAD65 autoantibodies without diabetes were immortalised and fused to a mouse/human hybridoma. In addition, mouse monoclonal antibodies to GAD65 were produced using standard techniques. F(ab')2S from our monoclonals and the GAD6 mouse monoclonal were used in competition with intact monoclonals and sera from diabetic patients for binding to 125I-labelled GAD65 (amino acids 46-586). Reactivities of the human monoclonals with GAD 65,000/67,000 M(r) chimeras were also studied. Variable region genes of human monoclonals were sequenced and analysed. The human monoclonals (n = 3) had affinity constants for GAD65 of 2.2 x 10(9), 5.8 x 10(9), 1.3 x 10(10) mol/l(-1); affinities of the mouse monoclonals (n = 5) ranged from 1.1 x 10(8) to 5.4 x 10(10) mol/l(-1). The binding of each of the human monoclonals was inhibited by GAD6 F(ab')2 and the binding of GAD6 antibody was inhibited by the human monoclonal F(ab')2S suggesting that the epitopes for these antibodies were overlapping. Studies with GAD65/GAD67 chimeras indicated that the human monoclonals reacted with C-terminal epitopes. The human monoclonals, GAD6 and 3/5 mouse monoclonals inhibited serum autoantibody binding to 125I-labelled GAD65. Overall, the human monoclonals were of high affinity, reacted with C-terminal epitopes and showed evidence of antigen driven maturation; they represented only a proportion of the repertoire of autoantibodies to GAD65 in the donor's serum and in the sera of patients with type-1 diabetes.
Addison Disease↗
Recombinant equine interleukin-1beta induces putative mediators of articular cartilage degradation in equine chondrocytes.
Interleukin-1 is considered a central mediator of cartilage loss in osteoarthritis in several species, however an equine recombinant form of this cytokine is not readily available for in vitro use in equine osteoarthritis research. Equine recombinant interleukin-1beta was cloned and expressed and its effects on the expression and activity of selected chondrocytic proteins implicated in cartilage matrix degradation were characterized. Reverse transcriptase polymerase chain reaction methods were used to amplify the entire coding region of the equine IL-1beta mRNA, which was cloned into an expression vector, expressed in E. coli, and purified using a Ni2+ chromatographic method. The effects of the recombinant peptide on chondrocyte gene expression were determined by Northern blotting using RNA from equine chondrocyte cultures hybridized to probes for matrix metalloproteinases (MMP 1, MMP 3, MMP 13), tissue inhibitor of matrix metalloproteinases 1 (TIMP 1) and cyclooxygenase 2 (COX 2). Effects on selected mediators of cartilage degradation (nitrite concentrations and MMP activity) were determined using conditioned medium from reIL-1beta-treated equine cartilage explant cultures. A recombinant peptide of approximately 21 kd was obtained. Northern blotting analyses revealed a marked up-regulation of expression of all MMPs, TIMP 1, and COX 2 in mRNA from treated chondrocytes. Furthermore, cartilage explants exposed to reIL-1beta had augmented collagenase/gelatinase and stromelysin activities as well as increased concentration of nitrite in conditioned media. The development of a biologically active, species-specific IL-1beta provides a valuable tool in the study of osteoarthritis pathophysiology and its treatment in horses.
Animals↗
Specific proliferative T cell responses and antibodies elicited by vaccination with simian immunodeficiency virus Nef do not confer protection against virus challenge.
The efficacy of immunizing with a combination of simian immunodeficiency virus (SIV) Nef vaccines was evaluated. Four vaccinates received three intradermal immunizations with recombinant vaccinia virus that expressed SIV Nef, followed by three intramuscular immunizations with rDNA also expressing SIV Nef. Finally, the four vaccinates received two subcutaneous boosts with recombinant SIV Nef protein. This immunization protocol elicited anti-Nef antibodies in all of the vaccinates as well as specific proliferative responses. However, specific cytotoxic T cell responses were not detected before virus challenge. All vaccinates were challenged intravenously with 10 MID(50) of SIVmacJ5 along with four controls. All eight subjects became infected after SIV challenge and there were no group-specific differences in virus load as measured by virus titration and vRNA analysis. The results of this study support indirectly the report from Gallimore and colleagues (Nat Med 1995;1:1667) suggesting that CD8(+) T lymphocyte responses are required for Nef-based vaccines to restrict SIV infection. If Nef-based vaccines are to be beneficial in controlling infection with immunodeficiency viruses, then it will be necessary to develop more effective immunization protocols that elicit potent CD8(+) cell responses reproducibly.
Animals↗
Overexpression of laminin alpha1 chain in colonic cancer cells induces an increase in tumor growth.
Laminins represent a growing family of glycoproteins constituting the basement membrane. They are known to direct many biological processes. With respect to carcinogenesis, laminins play an important role in cell adhesion, mitogenesis, differentiation and even metastasis. To further study the biological significance of laminin-1 (composed of alpha1, beta1 and gamma1 chains) in intestinal cell differentiation or tumorigenesis, an alpha1-laminin expression vector was introduced into the HT29 colonic cancer cells, in which laminin alpha1 chain is not expressed. Upon transfection of the alpha1 chain, the alpha1beta1gamma1 trimer was found secreted in the media along with free alpha1 chain as assessed by immunoprecipitation. The presence of the laminin alpha1 chain did not significantly modify the levels of the other laminin chains nor the integrins expressed by the HT29 cells. In spite of similar growth properties with the control cells in vitro (plastic dish, soft agar), the laminin alpha1 transfectants showed a significantly increased tumor growth when injected in nude mice. Histologic and immunohistochemic examination of the laminin alpha1-expressing tumors points to an increased recruitment of the host stromal and vascular cells, without modification in the differentiation profile and invasion potential. In parallel, a clear accumulation of laminin-10 (alpha5beta1gamma1) at the carcinoma/stromal interface and a segregation of the integrin beta4 subunit at the basal pole of the cancer cells occurred, compared to control tumors. Overall, our observations emphasize the importance of laminin-1 as a chemoattractant of both stromal and vascular cells and in epithelial/stromal cell interactions for the organization of the basement membrane and segregation of integrins leading to an epithelial cell growth signal. Such a sequence of events is reminiscent of what occurs during development.
Animals↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗