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Biomedical subjects

L Robson

Publications and source records attributed to L Robson.

At least 91 records · Page 5Linked to original sources
PubMed1995

Purification of bacterially expressed single chain Fv antibodies for clinical applications using metal chelate chromatography.

A new procedure is described for the purification of an anti-carcinoembryonic antigen (CEA) single chain Fv (scFv), referred to as MFE-23, from bacterial supernatant. A simple insertion of a hexa-histidine tail fused at the C-terminus (MFE-23 His) provides an affinity tag which selectively binds to transition metal ions immobilised on an iminodiacetic acid (IDA) derivitised solid phase matrix. This method proved to be superior to standard CEA antigen affinity chromatography in the following ways. (1) A higher yield was produced (10 mg/l as opposed to 2.2 mg/l of bacterial supernatant). The latter figure was largely affected by the limited availability (size of the column) of immobilised CEA antigen. (2) Scale-up was relatively simple and less costly. (3) The risk of tumour derived antigen leaching from the column is eliminated. Results showed that immobilised Cu2+ ions were more effective than Ni2+ and Zn2+ ions in retaining the His tagged product giving a 90% pure product on elution. Clinical grade material was generated using size exclusion chromatography to remove aggregated material, and Detoxi gel to remove bacterial endotoxins. Validation assays to measure DNA, copper and endotoxins were performed to assess the levels of contaminants. MFE-23 His retained 84% antigen binding after 6 months storage at 4 degrees C and > 75% after radiolabelling. Further experiments confirmed that the His tail did not affect biodistribution and tumour localisation in nude mice bearing human colorectal tumour xenografts.

Amino Acid Sequence↗
PubMed1995

Investigations with fluorescence in situ hybridization (FISH) demonstrate loss of the telomeres on the reciprocal chromosome in three unbalanced translocations involving chromosome 15 in the Prader-Willi and Angelman syndromes.

Two patients with classical features of Angel-man syndrome (AS) and one with Prader-Willi syndrome (PWS) had unbalanced reciprocal translocations involving the chromosome 15 proximal long arm and the telomeric region of chromosomes 7, 8 and 10. Fluorescence in situ hybridization (FISH) was used for the detection of chromosome 15(q11-13) deletions (with probes from the PWS/AS region) and to define the involvement of the telomere in the derivative chromosomes (with library probes and telomere-specific probes). The 15(q11-13) region was not deleted in one patient but was deleted in the other two. The telomere on the derivative chromosomes 7, 8 and 10 was deleted in all three cases. Thus, these are true reciprocal translocations in which there has been loss of the small satellited reciprocal chromosome (15) fragment.

Adult↗
PubMed1995

Comparison of high resolution cytogenetics, fluorescence in situ hybridisation, and DNA studies to validate the diagnosis of Prader-Willi and Angelman's syndromes.

Eighty seven referrals with Prader-Willi syndrome and 49 with Angelman's syndrome were studied. High resolution cytogenetics was performed on all probands. Molecular studies, performed on the proband and both parents in each case, utilised multiple probes from within and distal to the 15(q11-13) region in order to establish the presence of DNA deletion or uniparental disomy. In addition, FISH, with probes at D15S11 and GABR beta 3 from the Prader-Willi syndrome/Angelman's syndrome region, was performed on a subset of 25 of these patients. In the referral group with Prader-Willi syndrome, 62 patients had a normal karyotype and 25 were deleted on high resolution cytogenetics. Twenty nine were found to be deleted with DNA techniques. In the Angelman's syndrome group, 37 had a normal karyotype and 12 were deleted on high resolution cytogenetics while 26 were deleted on molecular studies. The diagnosis was reassessed in 35 referrals with Prader-Willi syndrome and 11 with Angelman's syndrome following a non-deleted, non-disomic result. Of individuals who were neither deleted nor disomic on DNA studies, a false positive rate of 11.4% (4/35) for Prader-Willi syndrome and 16.7% (2/12) for Angelman's syndrome was found for a cytogenetically detected deletion. The false negative rate for deletion detected on high resolution cytogenetics was 19.5% (12/62) for Prader-Willi syndrome and 35% (13/37) for Angelman's syndrome. Thus high resolution cytogenetics was shown to be unreliable for deletion detection and should not be used alone to diagnose either syndrome. There were no discrepancies with FISH in 25 cases when FISH was compared with the DNA results, indicating that FISH can be used reliably for deletion detection in both syndromes.

Adolescent↗
PubMed1995

Carboplatin in combination with paclitaxel in advanced ovarian cancer: dose determination and pharmacokinetic and pharmacodynamic interactions.

Data from various phase I/II studies of carboplatin in combination with paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) have suggested that the degree of thrombocytopenia seen is less than that expected when carboplatin is given alone. However, some studies also have suggested that the area under the plasma concentration-time curve (AUC) of carboplatin is lower than that expected, raising the possibility of a pharmacokinetic interaction. Patients with advanced epithelial ovarian cancer were treated with first-line carboplatin (AUC = 7, using the 51Cr EDTA [edetic acid] clearance method) and escalating doses of paclitaxel. Thrombocytopenia was mild and was significantly less when the paclitaxel dose was 175 mg/m2 versus 150 mg/m2. Paclitaxel kinetics were nonlinear, as previously reported. The achieved carboplatin AUC was 7 +/- 1 mg/mL.min, indicating that the pharmacokinetics of carboplatin are not affected by paclitaxel. Glomerular filtration rates measured in 184 patients using the 51Cr EDTA clearance method were compared with rates estimated from the plasma creatinine level using the Cockcroft-Gault or Jeliffe formulas and showed a significant bias of these two formulas. Clearances above 50 mL/min were underestimated by an amount that became greater as the clearance increased and was approximately 25% to 35% for patients with clearances in the normal range. Since creatinine-based methods have been used in many previous studies, care is needed in interpreting the predicted AUC values from these studies. Carboplatin and paclitaxel may be given safely in combination at full doses, and the thrombocytopenia seen is significantly less than that observed with single-agent carboplatin. No evidence exists of a pharmacokinetic interaction, and the observation in some studies that the carboplatin AUC was lower than expected was probably due to the methodology used to estimate the glomerular filtration rate.

Adult↗
PubMed1994

Role of cell volume and protein kinase C in regulation of a Cl- conductance in single proximal tubule cells of Rana temporaria.

1. The whole-cell patch clamp technique was used to investigate Cl- currents in single proximal tubule cells isolated from kidneys of Rana temporaria. 2. Immediately following establishment of the whole-cell clamp, the Cl- conductance (gCl) of the cells was low. However, with 2 mM ATP in the pipette there was a time-dependent activation of gCl. Such activation was inhibited when the bath contained a hypertonic Ringer solution. 3. The Cl- conductance was not directly dependent on cell volume; gCl increased with hypotonic shock and decreased with hypertonic shock, but only in the presence of ATP. 4. Activation of gCl by ATP was dependent on extracellular Ca2+; however, the conductance was not directly Ca2+ sensitive. Activation was inhibited by Gd3+, which also had a direct inhibitory action on gCl. 5. Inhibition of protein kinase C (PKC), by 10 microM PKC pseudo-substrate (PKC-ps), completely abolished the ATP-dependent activation of gCl, while stimulation of PKC, by the PKC activator 4 beta-phorbol 12-myristate, 13-acetate (PMA), increased the degree of activation typically observed with ATP. 6. We propose that gCl is activated by PKC-mediated phosphorylation and plays a role in volume regulation of the cells.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

A variety of genetic mechanisms are associated with the Prader-Willi syndrome.

An extensive set of chromosome 15 DNA polymorphisms and densitometric analysis with four markers mapping to the Prader-Willi chromosome region (PWCR) of chromosome 15 have been used to characterize a cohort of 30 subjects with classical Prader-Willi syndrome (PWS). Molecular analysis enabled the classification of the PWS subjects into four groups: (A) 18 subjects (60%) had deletions of paternal 15q11-13 involving a common set of DNA markers. Two subjects had differently sized deletions, one larger and one smaller than the other cases. (B) Eight (27%) had maternal uniparental disomy for chromosome 15. (C) One (3%) had a marker chromosome carrying an extra copy of the PWCR. The marker chromosome was demonstrated to be of paternal origin and the two intact chromosomes were maternally derived. This case represents an apparent exception to the generally held view that PWS is associated with an absence of paternally inherited gene(s) located in the PWCR. (D) The remaining three cases (10%) had none of the above abnormalities. This last subgroup of patients has not previously been well characterized but could represent limited deletions not detectable with the markers used or abnormalities in the imprinting process. These cases represent potentially valuable resources to elucidate more precisely the fundamental disorders responsible for PWS.

Adolescent↗
PubMed1994

The use of fluorescence in-situ hybridisation to clarify abnormal Y chromosomes in two infertile men.

OBJECTIVE: To characterise the structurally abnormal Y chromosomes present in two infertile men by means of the molecular technique of fluorescence in-situ hybridisation (FISH). METHODS: Both men were phenotypically normal and had azoospermia. In Case 1 the karyotype with routine cytogenetics was 46,XX; in Case 2 it was complex, with 3 cell lines--45,X/46,X,i(Yp)/46,X,+?del(Y)(q11). We used probes from the Y chromosome and FISH to clarify these karyotypes. RESULTS: In Case 1 a translocation of Y chromosome to the X chromosome was identified with FISH--t(X;Y). In Case 2, an isochromosome of the short arm of the Y chromosome was confirmed in one cell line, and another cell line was shown to contain a small Y chromosome with heterochromatic deletion. These findings explained the underlying pathogenesis in both cases. CONCLUSIONS: Molecular investigation with FISH should become part of the routine investigation of infertile men with an abnormal Y chromosome.

Adult↗
PubMed1994

Novel karyotype in the Ullrich-Turner syndrome--45,X/46,X,r(X)/46,X, dic(X)--investigated with fluorescence in situ hybridization.

A 10-year-old girl with Ullrich-Turner syndrome was found to have the novel karyotype 45,X/46,X,r(X)(p11q11)/46,X,dic(X)(p11). Fluorescence in situ hybridization (FISH) with the alpha satellite X centromere probe established the origin of the small ring chromosome. Scanning a large number of cells by interphase FISH showed that the dicentric (X) was the least prevalent cell line. The common breakpoint of Xp11 suggests a sequence of errors as the mechanism whereby these 3 distinct cell lines have arisen.

Cells, Cultured↗
PubMed1994

Production and tumour-binding characterization of a chimeric anti-CEA Fab expressed in Escherichia coli.

A recombinant chimeric Fab (rcFab), with Fv derived from the monoclonal A5B7 antibody to carcinoembryonic antigen (CEA), and with human CHI and C kappa was cloned into pUC 19 and expressed in Escherichia coli. rcFab (10 to 12 mg per litre) was produced in bacterial culture fluid, and functional purified rcFab was isolated by affinity chromatography (using antibody to human C kappa) and size-exclusion gel filtration. The rcFab did not show reduced affinity for CEA, and reacted with human colorectal tumours showing a typical anti-CEA pattern by immunocytochemistry; it was also stable after iodination. Biodistribution studies in nude mice bearing human tumour xenografts showed no toxicity and good tumour localization. Therapeutic ratios at early time points were better than those obtained with whole murine antibody. The results demonstrate that bacterially produced anti-CEA Fab is of use for tumour targeting.

Adenocarcinoma↗
PubMed1994

Phage libraries for generation of clinically useful antibodies.

Insertion of antibody genes into filamentous bacteriophage makes it possible to generate and screen libraries of 10(7) or more antibodies. Each phage expresses an antibody on its surface and contains the corresponding antibody gene. Genes that encode antibodies with desired characteristics are readily selected and their antibodies expressed as soluble proteins in Escherichia coli. We used this system to produce an antibody to carcinoembryonic antigen with higher affinity and better tumour specificity than antibodies currently in use.

Animals↗
PubMed1994

Volume regulatory responses in frog isolated proximal cells.

Cells respond to increase in volume by activating solute efflux pathways, resulting in water loss and restoration of the original cell volume. The solute efflux pathways underlying these volume regulatory decrease (VRD) responses have been relatively well studied. However, the transduction pathways whereby the change in cell volume is converted into an intracellular signal resulting in VRD are much less well understood. We have examined VRD in isolated proximal tubule cells from the frog, with particular attention to the roles of stretch-activated channels, Ca2+ and protein kinases. Cell length was taken as an index of cell volume, and was measured continuously using a photodiode array. VRD was observed in approximately 50% of cells, and was inhibited by Ba2+, Gd3+ and 4,4'-diisothiocyanatostilbene 2,2'-disulphonic acid (DIDS), and removal of extracellular Ca2+. VRD was accelerated by the active phorbol ester, phorbol 12-myristate 13-acetate (PMA), and the phosphatase inhibitor F-; on the other hand, VRD was prolonged by 4 alpha-phorbol 12,13-didecanoate (PDC), an inactive phorbol ester), and inhibited by PMA and Gd3+, PMA and 0 Ca2+, and staurosporine. Volume regulation was unaffected by di-butyryl cAMP and 3-isobutyl-1-methyl-xanthene (IBMX). These data suggest that Ca2+ and PKC, via protein phosphorylation, play a stimulatory role in VRD.

Animals↗
PubMed1994

Volume-activated, gadolinium-sensitive whole-cell currents in single proximal cells of frog kidney.

Stretch-activated channels (SACs) have been implicated in the control of epithelial cell volume. Such channels are generally sensitive to the trivalent lanthanide, gadolinium (Gd3+). In this study, using Gd3+ sensitivity and volume activation as indices, we have looked for ionic currents attributable to SACs using the whole-cell-patch clamp technique in freshly isolated proximal tubule cells of the frog. Hypotonic shock caused a reversible increase in whole-cell conductance, which was inhibited by Gd3+. In conjunction with this increase in conductance, cell length (measured using an optical technique) also increased. We observed two types of volume- and Gd(3+)-sensitive currents: voltage-dependent IVD and voltage-independent IVI. IVD was found in all cells, activated by depolarisation and hypotonic shock, and was inhibited reversibly by 10 microM Gd3+. The conductance did not discriminate between Na+ and K+ but was slightly anion-selective and was Ca(2+)-permeable. IVI was observed in only 50% of cells and was also inhibited by Gd3+. Although the inhibition was irreversible, it was dose-dependent, suggesting a specific effect of Gd3+ on IVI. Cells that showed IVI had a significantly higher conductance than those that did not (38.7 +/- 4.4, n = 20, and 20.5 +/- 0.7, n = 15, microS.cm-2 respectively). In contrast to IVD, IVI was mildly cation-selective, Ca(2+)-permeable, and also selective for Na+ over K+. As with IVD, volume-induced increases in IVI were inhibited by Gd3+. Both of these currents are activated during hypotonic shock and may be involved in volume-regulatory processes in frog proximal cells.

Animals↗
PubMed1993

Effect of warm and cold applications on the resolution of i.v. infiltrations.

Differences in pain intensity, surface area measurements of induration and erythema, and interstitial fluid volume when warm versus cold applications were randomly made to an intentional intravenous infiltrate of 5 mL of a designated solution were examined. Three solutions were used: 1/2 saline (154 mOsm), normal saline (308 mOsm), and 3% saline (1027 mOsm). Differences in volume were determined by magnetic resonance imaging (MRI) at three intervals postinfiltration. The sample was 18 healthy adults between 20 and 45 years. There was no difference in remaining infiltrate when 1/2 saline or normal saline were used, but a significant (p < .001) difference was found with 3% saline. For all solutions there was a significant (p < .001) difference in the volume of infiltrate remaining when warmth was applied and this effect held across MRI readings and solutions. Pain intensity did not differ by treatment but a significant (p < .005) difference was found by solution, with 3% saline producing the greatest difference. Erythema was absent with all solutions. Surface induration was affected by solution and decreased over time (p = .001). There was no effect of warmth or cold on surface area induration.

Adult↗
PubMed1993

Age-related behavioural, neurochemical and radioligand binding changes in the central 5-HT system of Sprague-Dawley rats.

Mature (3-4 months) and aged (18-19 months) Sprague-Dawley (SD) rats were treated with 5-HT receptor agonists and drug-induced behaviours monitored. The 5-HT2/1C agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), induced wet dog shakes and back muscle contractions which were significantly increased in aged, compared to mature, rats, suggesting an age-related enhancement of 5-HT2 receptor function. In contrast, the selective 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) induced forepaw treading, flat body posture, hypothermia and hyperactivity which were not significantly different in aged compared to mature rats. Levels of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) in the hippocampus and frontal cortex were measured using high performance liquid chromatography with electrochemical detection. There were no age-related changes in hippocampal 5-HT or 5-HIAA. However both 5-HT and 5-HIAA were increased in the frontal cortex of aged SD rats. 8-OH-DPAT reduced 5-HIAA in both regions examined in mature rats, an effect which was attenuated in the aged rats, suggesting an age-related reduction in presynaptic 5-HT1A receptor function. DOI did not induce any changes in 5-HT or 5-HIAA in either of the regions examined. Radioligand binding studies with [3H] ketanserin showed there to be no significant age-related changes in cortical 5-HT2 receptor density or affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1993

Fluorescence in-situ hybridisation and molecular studies used in the characterisation of a Robertsonian translocation (13q15q) in Prader-Willi syndrome.

A patient with classical Prader-Willi syndrome was found to have a Robertsonian translocation 45,XY,t(13q15q)mat. On CBG banding, the translocation chromosome had a large centromere with one primary constriction. Using fluorescence in situ hybridisation, positive signals were obtained with chromosome 13 and chromosome 15 centromere probes, proving that the translocation was dicentric. NOR banding was negative in this chromosome, suggesting that the breakpoints were at 13p11 and 15p11. DNA studies showed that, while there was no deletion involving 15(q11-13), maternal uniparental disomy for chromosome 15 was present. We compare our findings with the five other cases of familial Robertsonian translocation PWS that have been reported.

Adult↗
PubMed1992

Increased expression of c-myc proto-oncogene in biopsies of ulcerative colitis and Crohn's colitis.

The steady state levels of c-myc mRNA have been measured in RNA samples extracted from colonoscopic biopsies of inflammatory bowel disease patients obtained at routine endoscopy sessions. Biopsies were immediately frozen in liquid nitrogen limiting the ischaemic time to less than 15 seconds, and can be stored for up to 96 hours before separation of RNA. Yields of RNA using biopsies were 0.137 (0.041)% wet wt (mean (SD), n = 68), these are significantly better than those obtained from surgical material (0.064 (0.063)% wet wt (mean (SD), n = 21) where the tissue ischaemic time was 45 minutes to one hour 40 minutes. Functional activity of RNA extracted was demonstrated by the ability to direct in vitro protein translation in the rabbit reticulocyte system. We have used this technique to show that there is an increased ratio of steady state c-myc proto-oncogene expression in inflamed tissue from 18 patients with left sided ulcerative colitis and five patients with segmental Crohn's colitis, compared with an uninvolved region of the colon in each case. No difference in c-myc expression was seen in biopsies at least 30 cm apart in 11 control patients with no macroscopic or histological abnormalities. Increased expression of c-myc in inflammatory bowel disease is consistent with the activation of this proto-oncogene during altered cell cycle control resulting from the inflammatory process.

Adult↗