Biomedical subjects
I Roberts
Publications and source records attributed to I Roberts.
Production of monoclonal human antibody to HLA-DR5 (DRw11) by mouse/human heterohybridomas.
We describe here the production of a human monoclonal antibody to the HLA-DR5 antigen. A human B-cell line secreting cytotoxic antibody that reacted preferentially with DR5-positive targets was fused to the mouse myeloma P3X63Ag8.653 and the resulting heterohybridomas cloned twice. The clones secreted human IgM (lambda light chain), which showed specificity for the DR5 antigen in cytotoxicity assays and reacted with DRw11-positive but not DRw12-positive targets. These results demonstrate the potential of this approach to the production of human monoclonal antibodies to transplantation antigens.
Legal aspects involved in the development of anti-progesterones for fertility control.
The development of medical means of inducing abortion requires a detailed knowledge of the abortion legislation. It is vital that these compounds are developed and used within the legal constraints of the country involved. In the United Kingdom, despite the Act of Union (1707), England and Scotland have not united under one legal system. In England the common law on abortion was superseded by the Offences Against the Persons Act (1861) which declared abortion illegal. This Act does not apply to Scotland where, up until 1967, common law applied. The Abortion Act of 1967 is an exemption act allowing abortion, under certain restrictions, to be legally performed. Whilst post-coital contraception is not covered by any of the abortion legislation, it is clear that contragestion and medical abortion is restricted by the current abortion legislation, hence medication will need to be taken in hospital.
Development of a new shuttle plasmid system for Escherichia coli and Clostridium perfringens.
We constructed a 7.9-kilobase-pair recombinant shuttle plasmid, designated pHR106, by combining desired segments of three plasmids: an Escherichia coli plasmid (pSL100) which provides a multiple cloning site, a Clostridium perfringens plasmid (pJU122) which provides a clostridial origin of replication, and an E. coli plasmid (pJIR62) which provides an E. coli origin of replication, an ampicillin resistance gene, and a chloramphenicol resistance gene of clostridial origin. The shuttle plasmid transformed E. coli HB101 with a frequency of 1 transformant per 10(4) viable cells and C. perfringens L-phase strain L-13 with a frequency of approximately 1 transformant per 10(6) viable cells. Because of the set of unique cloning sites and the chloramphenicol resistance marker, this shuttle plasmid should be particularly useful for studies of gene regulation and for enzyme production with C. perfringens.
DNA probes for K-antigen (capsule) typing of Escherichia coli.
DNA restriction fragments derived from the polysaccharide biosynthesis regions of cloned Escherichia coli K1, K5, and K12 capsular antigen genes hybridized only with DNA of strains determined by conventional methods to be of the same K serotype. A probe derived from the common transport region hybridized to all encapsulated E. coli strains.
Isolation of promoters from two anaerobic bacteria.
Promoters which function in Gram-positive organisms show, with few exceptions, remarkable conservation of sequences identical with those in Escherichia coli. An E. coli system was tested to select putative promoters of two anaerobes, the Gram-positive Clostridium absonum and the Gram-negative Bacteroides thetaiotaomicron. Random restriction fragments of chromosomal DNA from these organisms were fused to the galactokinase (galK) gene of E. coli within a plasmid vector. Approximately 10% of these fragments functioned as promoters in E. coli, and a broad range of activities was evident. A single 88 base pair (bp) C. absonum DNA fragment yielded, in the E. coli plasmid vector, approximately the same high activity as that provided by the E. coli galK promoter. Sequence analysis of this fragment showed typical -35 and -10 sequences, with about five -10-like sequences closely flanking each other, some overlapping, and this appears to result in multiple start sites for transcription. The transcriptions of E. coli plasmid fragments in vitro with both E. coli RNA polymerase and C. absonum RNA polymerase showed pairs of transcripts corresponding to two start sites. By colony hybridization with the 88 bp fragment, radioactively labelled, as a probe, a 4.2 kilobase segment of C. absonum chromosomal DNA containing the 88 bp fragment was isolated. About 375 bp of this fragment was sequenced. A putative Shine-Dalgarno sequence and ATG start site were detected, followed by an opening reading frame. Using a sequence about 100 bp downstream from the 88 bp sequence, a 17-base oligonucleotide was synthesized to serve as a primer. With C. absonum RNA as a template, a reverse transcriptase primer extension assay located a pair of transcription start sites just downstream from the 88 bp sequence, proving that the 88 bp sequence functions as a promoter in C. absonum.
Prostaglandins and antigestagens for the interruption of early pregnancy.
During pregnancy the uterus is maintained in a quiescent state by the secretion of progesterone. Antigestagens antagonize the biological action of progesterone by binding to the nuclear receptor in the target organs. Administration of the antigestagen mifepristone to women induces bleeding during the luteal phase and in early pregnancy by releasing endogenous prostaglandins from the endometrium or decidua. In addition, the sensitivity of the myometrium to exogenous prostaglandins is markedly increased. Although mifepristone will induce bleeding in the majority of women in early pregnancy, the incidence of incomplete abortion or ongoing pregnancies increases with increasing gestational age and is too high to be clinically useful as an agent for therapeutic abortion. However, a single dose of mifepristone (400-600 mg) followed by a vaginal pessary of a prostaglandin analogue (0.5-1.0 mg), gemeprost, induced complete abortion in 95 of 100 women of gestational age less than 42 days (less than or equal to 56 days amenorrhoea). The incidence of diarrhoea (15%) and abdominal pain requiring opiate analgesia (10%) was much lower than when abortion was induced with prostaglandin alone. Vaginal bleeding continued for 13.8 +/- 0.8 days after administration of the prostaglandin. A combination of an antigestagen with a small dose of a prostaglandin analogue is an effective alternative to vacuum aspiration for the therapeutic termination of early pregnancy.
Serotyping and genotyping of encapsulated Escherichia coli K1 sepsis isolates with a monoclonal IgG anti K1 antibody and K1 gene probes.
Among infectious diseases caused by E. coli the capsular type K1 plays a predominant role. E. coli K1 isolates account for 80% of cases of E. coli neonatal meningitis and 30% of E. coli sepsis strains. Serotyping of K1 strains has conventionally relied upon the use of K1-specific bacteriophages or serum agar methods with polyvalent anti K1 serum. In the study present here, 187 E. coli sepsis isolates have been analysed for production of the K1 antigen using K1 phages, K1 serum agar plates and Latex agglutination and ELISA using an IgG2a anti K1 monoclonal antibody. In total, 33 sepsis isolates (about 18%) were identified as K1 positive, with three of these strains proving negative in all tests except those exploiting the monoclonal antibody. That these three strains elaborate the K1 antigen was confirmed by Southern blot experiments using cloned K1 antigen production genes as probes. The failure of the three strains in all the tests except those that use monoclonal antibody could be explained by apparent disruption of K1 gene sequences that encode functions essential for the export of capsular material to the cell surface. The superiority of tests based on monoclonal antibodies above the conventional methods for detection of K1 antigen is evident.
Contribution of capsular polysaccharide and surface properties to virulence of Escherichia coli K1.
We examined the surface properties, susceptibility to the bactericidal activity of serum, and susceptibility to phagocytosis of Escherichia coli K1, a laboratory strain of E. coli (LE392), and strain LE392 carrying a plasmid (pKT274) incorporating a 17-kilobase insert of DNA that encodes the ability to produce surface K1 antigen. As determined by electron microscopy, LE392 was nonencapsulated but both E. coli K1 and LE392(pKT274) possessed a thin capsule. By using charged aqueous two-phase polymer systems, both E. coli K1 and LE392(pKT274) were shown to be significantly more negatively charged than LE392. E. coli K1 was resistant to the bactericidal activity of serum, but both LE392 and LE392(pKT274) were completely inhibited by neonatal serum at a concentration of 20% (vol/vol). As measured by counting endocytosed and nonendocytosed bacteria and by chemiluminescent response, E. coli K1 was highly resistant to phagocytic uptake by polymorphonuclear leukocytes, whereas LE392 was rapidly taken up by such cells; LE392(pKT274) was resistant to endocytosis, although less so than E. coli K1. Most intraphagocytic E. coli LK1 remained structurally intact for up to 60 min, whereas both LE392 and LE392(pKT274) were rapidly degraded.
A high performance liquid chromatography method for the analysis of total and free indomethacin in serum.
A simple rapid and sensitive HPLC technique for the analysis of total and free plasma indomethacin concentrations in neonates is described. The advantages of the technique are small sample volumes (50 microL) enabling the use of capillary samples, direct extraction, low limits of detection (20 ng/mL) and good precision (CV less than 7% between-batch). Indomethacin and the internal standard (flufenamic acid) eluted within 12 min and the peaks were unaffected by indomethacin metabolites or by a number of commonly used drugs. The technique is suitable for the investigation into the pharmacokinetics of total and free indomethacin, in neonates with patent ductus arteriosus, and could be used to define a therapeutic window for free indomethacin levels to enable therapeutic drug monitoring to occur in this condition.
Modified plasmid isolation method for Clostridium perfringens and Clostridium absonum.
A rapid plasmid isolation procedure for Clostridium perfringens and C. absonum is described. The ratio of culture volume to lysis buffer volume was found to be crucial for efficient plasmid isolation. The method can be scaled up, without difficulty, for large-scale plasmid preparation.
Molecular cloning and analysis of genes for production of K5, K7, K12, and K92 capsular polysaccharides in Escherichia coli.
With a DNA fragment from within the region encoding the transport functions for K1 production as a hybridization probe in Southern blot experiments, homologous DNA sequences were detected in the DNA from Escherichia coli strains producing K5, K7, K92, and K100 capsular polysaccharides. No homology with the laboratory strain LE392 was detected. The same DNA probe was used to prescreen cosmid libraries in LE392 by colony hybridization, as a rapid method to isolate clones encoding the genes for K5, K7, K12, and K92 antigen production. Clones carrying sequences homologous to the probe that also produced capsular material were identified by using polyclonal and monoclonal antibodies raised against the K antigen in question and K antigen-specific phages. By restriction enzyme mapping of the appropriate cosmid clones it was possible to align the genes for the production of different K antigens in terms of common restriction endonuclease cleavage sites. A DNA fragment encoding the postulated transport functions for K7 antigen production could complement deletion mutations in the transport functions for K1 antigen production. Thus the transport to the cell surface of chemically distinct polysaccharides may be by a common process. Analysis in E. coli of the proteins produced by plasmids carrying the likely transport functions for K1, K5, and K7 antigen production revealed that each region coded for a similar polypeptide.
Thyroid function tests in patients with familial dysalbuminaemic hyperthyroxinaemia (FDH).
Two patients with familial dysalbuminaemic hyperthyroxinaemia (FDH) are described in whom the albumin variant resulted in raised total T4 levels, and artefactually raised free T4 using a 'single-step' technique employing an analogue of T4 as tracer. The first patient was clinically euthyroid and presented with relapse of schizophrenia and abnormal thyroid function tests (total T4 336 nmol/L, total T3 4.2 nmol/L, TSH 1.8 mU/L, free T4 73 pmol/L). These results led to diagnostic confusion and the patient was treated with a short course of anti-thyroid drugs. The second patient had signs and symptoms of thyrotoxicosis at her first visit but was clinically euthyroid 5 months later when she was 10 weeks pregnant. Thyroid function tests were total T4 259 nmol/L, total T3 3.6 nmol/L, TSH 3.8 mU/L, free T4 46 pmol/L. Further studies showed both patients to be biochemically euthyroid. A variant albumin was confirmed in both patients by a screening test for FDH and by reverse-flow electrophoresis. Family studies on 10 relatives of the first patient identified eight with FDH. A simple screening procedure for the indentification of FDH is described and the use of laboratory tests in suspected cases is discussed.
A simple method for the measurement of glycerol in serum.
An automated enzymic assay for the measurement of glycerol in serum is described. As it uses a centrifugal analyser, this method does not require prior extraction. It is precise, sensitive, and can be performed rapidly at low cost.
Toxicological studies in a Distalgesic addict.
The disposition and kinetics of paracetamol, dextropropoxyphene and their metabolites were investigated in an addict who claimed to be taking 80-100 Distalgesic tablets daily regularly. Plasma concentrations of paracetamol, dextropropoxyphene and their principal metabolites were measured after an oral dose of 15 Distalgesic tablets. The absorption of paracetamol and dextropropoxyphene was rapid with peak plasma concentrations at 15 and 30 min respectively. The elimination half-life for paracetamol was 2.3 h. Nordextropropoxyphene remained at steady state with very high plasma concentrations (5 mg/l). The urinary excretion of paracetamol and metabolites was not abnormal. The total recovery of paracetamol was only 31% of the dose. Apart from raised plasma gamma-glutamyltransferase activity there was no biochemical evidence for paracetamol-induced hepatocellular damage despite ingestion of 97.5 g of paracetamol over the 11 days of the withdrawal period.
Characterisation of antibodies to insulin to help diagnose factitious hypoglycaemia.
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Multiple hormone secretion by a human pancreatic glucagonoma in culture.
A patient presenting clinically with the glucagonoma syndrome had high plasma glucagon levels (1920 ng/l) and at laparotomy, a pancreatic islet cell tumour was removed. The tumour was dispersed and placed in culture where it remained viable for 63 days. The tumour cells secreted immunoreactive (IR) glucagon at levels up to 2400 ng/l as detected by a C-terminal glucagon specific antibody and 85 400 ngequiv./l as measured by an N-terminal glucagon specific antibody. The difference between these two levels was attributed to the presence of different molecular forms of glucagon measured with the N-terminal specific antibody. IR insulin (up to 302 mU/l) and IR somatostatin (up to 2500 ng/l) were also detected. There was no direct or inverse correlation between different hormone levels. Small but significant levels of N-terminal and C-terminal vasoactive intestinal peptide (VIP) were detected in some cultures but there was no evidence of gastrin or ACTH. Glucagon and somatostatin secretion persisted for the duration of the culture (63 days) but insulin concentrations declined. Incubation of cultures with somatostatin (1 ng/ml) caused a 75% decrease in glucagon levels, while insulin (1000 mU/l) produced a 70% inhibition of somatostatin.
Cloning and deletion analysis of a genomic segment of Bacillus subtilis coding for the sdhA, B, C (succinate dehydrogenase) and gerE (spore germination) loci.
From a Bacillus subtilis gene bank constructed in Escherichia coli and based on a low copy number cloning vector we have isolated a hybrid plasmid, pSH1047, containing an 8.0 kb segment of B. subtilis DNA coding for the sdhA, B and C genes, which code for the component polypeptides of succinate dehydrogenase, and the gerE gene, which may code for or regulate a protease involved in producing spores which germinate normally. We report the restriction map of this segment and the analysis of deletion derivatives which allow us to correlate the physical and genetic maps of these chromosomal segments.