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PubMed1993

Coating particles with a block co-polymer (poloxamine-908) suppresses opsonization but permits the activity of dysopsonins in the serum.

The surfaces of polystyrene microspheres (60 nm in diameter) and colloidal gold particles (17 nm in diameter) were coated with a polyoxyethylene (POE)/polyoxypropylene (POP) block co-polymer; poloxamine-908. The polymer adsorb strongly to the microspheres via its relatively hydrophobic POP segments. This leaves the POE chains in a mobile state as they extend outward from the surface and thereby provide stability to the particle suspension by suppressing aggregation. The blood clearance and biodistribution of uncoated vs. poloxamine-908-coated 125I-labelled polystyrene microspheres were compared 1 h after intravenous administration into rats. Poloxamine coating dramatically reduced liver accumulation of microspheres and kept them within the systemic circulation. These observations were further confirmed by electron microscopy, demonstrating that Kupffer cells were loaded with uncoated latex but had ingested few if any of the poloxamine-908-coated particles. The interaction of uncoated and poloxamine-coated gold particles with freshly isolated rat liver sinusoidal cells was examined by electron microscopy. The accumulation in Kupffer cells of gold particles after opsonization with autologous plasma was in accordance with previous observations where the dominant opsonizing activity had been identified as fibronectin. In contrast, coating of gold particles with poloxamine-908 prior to plasma opsonization prevented the adsorption of fibronectin onto their surface. Simultaneously, Kupffer cells failed to recognize poloxamine-908-coated gold particles before and after opsonization. Unlike Kupffer cells, liver endothelial cells endocytosed poloxamine-908-coated gold particles prior to opsonization but failed to recognize them after the opsonization process. This was taken as an indication of the presence of dysopsonic activity in plasma. This dysopsonic activity was studied using polystyrene latex microspheres, where the uptake of such particles by phagocytes is known to be independent of opsonization. The coating of 125I-labelled polystyrene microspheres with poloxamine-908 dramatically reduced their interaction with liver sinusoidal cells. This interaction was further reduced in the presence of either autologous plasma or serum. A heat-stable (60 degrees C for 15 min) serum component of molecular mass > 100 kDa was found to mediate this suppressive effect. Thus, we demonstrate that organ-specific receptors, opsonin activities and plasma dysopsonins regulate the in vivo clearance of particulate materials from the circulation. Poloxamine-908 coating modulates particle clearance by effectively blocking opsonization but still allowing for dysopsonization.

Animals↗
PubMed1993

Donor cell chimerism permitted by immunosuppressive drugs: a new view of organ transplantation.

One line of thought in organ transplantation feels that immunosuppressive drugs can lead to tolerance induction by allowing a previously unrecognized common mechanism of cell migration and microchimerism to occur, persist, and in some cases, become drug independent. It has been recognized that there is a spectrum of susceptibility of different organs to cellular rejection and that the variable ability of these organs to induce donor-specific nonreactivity reflects their comparative content of migratory leukocytes. Here, Thomas Starzl and colleagues discuss how many of the enigmas of transplantation immunology can be explained by this chimerism.

Animals↗
PubMed1993

Should physicians be permitted to 'balance bill' patients?

This paper studies the efficiency effects of physician fees when the insurer (possibly the government) pays a fee for each procedure, and the doctor may supplement the fee by an extra charge to the patient, a practice known as 'balance billing.' Monopolistically competitive physicians can discriminate among patients on the basis of both price and quality. Equilibria with and without balance billing are compared. The paper analyzes and recommends a new fee policy, a form of payer 'fee discrimination.'

Cost Sharing↗
PubMed1995

Problems and suggested solutions in creating an archive of clinical trials data to permit later meta-analysis: an example of methotrexate trials in rheumatoid arthritis.

Because data archives contain patient-based rather than study-based data, they can address meta-analytic questions on uncommon outcomes and on predefined patient subsets, questions that are difficult to address using the traditional meta-analytic approach based on grouped data. We report the tasks involved in establishing the first data archive of rheumatoid arthritis trials. In general, problems stem from the heterogeneity of trials in the archive and we suggest some solutions. In the initial phases, difficulties include recruitment and incomplete participation of trial investigators, whereas later on, other issues arise, such as quality control, coping with different dataset designs, and incomplete documentation. Other issues include heterogeneous measures, missing variables, and comparing data across different visit intervals and trial lengths. Suggested solutions include requesting trial data in predefined archive-wide structures and asking for all possible documentation for each dataset. Data cleaning is necessary, as is rescaling of variables or developing unit-free outcomes, and estimating data for missing variables. Archive design should allow for referencing a patient's data among various datasets. Although one goal is to reduce the quantity of data in the archive while retaining information content, data from early stages of archive building must be accessible for developing new analysis datasets. Documentation of archive building and software choices are discussed. Our experience suggests data archiving for meta-analysis is time consuming and expensive, yet it provides a useful method for analyzing data from multiple trials.

Archives↗
PubMed1990

The in vivo erythrocyte micronucleus test: measurement at steady state increases assay efficiency and permits integration with toxicity studies.

The mouse erythrocyte micronucleus assay has been traditionally carried out using one or two exposures to the test agent, followed by sampling at two or three postexposure times to obtain a sample near the time of the transient peak of micronucleated polychromatic erythrocytes (PCEs). We have demonstrated that frequencies of micronucleated RNA-positive (PCEs) and RNA-negative erythrocytes in blood and bone marrow come to steady state during "continuous" exposure via diet or drinking water, or during repeated daily exposures to test agents by ip injection, gavage, or inhalation. Under these exposure conditions, frequencies of micronucleated cells in peripheral blood approached steady state within 2-3 days in RNA-positive erythrocytes and in 5-6 weeks in RNA-negative erythrocytes. With exposure durations of 6 days (monocrotaline or Crotalaria seeds in diet), 10 days (triethylenemelamine, mitomycin C, 7,12-dimethylbenzanthracene, or colchicine, ip daily), 90 days (triethylenemelamine or urethan in drinking water or 1,3-butadiene via inhalation), or 2 years (benezene by daily gavage), frequencies of micronucleated cells attained and remained at steady state for prolonged periods. At steady state, frequencies of micronucleated RNA-positive cells in bone marrow samples were similar to those in RNA-positive and RNA-negative cells in peripheral blood (e.g., triethylenemelamine in drinking water at 4 micrograms/ml resulted in frequencies of micronucleated RNA-negative erythrocytes in peripheral blood of 27/1000 after 45 days of exposure and 24/1000 after 90 days, with a frequency of 28/1000 in bone marrow RNA-positive erythrocytes after 90 days). The data suggest that the efficiency of the assay would be markedly improved by using a repeated dose schedule with a single sample taken at steady state, rather than scoring multiple samples at various times after a single dose. This approach allows the frequency of micronucleated cells to be measured in a sample of bone marrow or blood obtained at almost any stage of routine toxicity testing.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1995

Idiopathic haemochromatosis: magnetic resonance signal intensity ratios permit non-invasive diagnosis of low levels of iron overload.

OBJECTIVE: The detection of low levels of iron overload by magnetic resonance imaging. METHODS: Eight consecutive patients suspected of having idiopathic haemochromatosis. Comparison of signal intensity ratios and absolute iron content of liver. RESULTS: There was a good correlation between the signal intensity ratios and iron content in the range 2-30 micrograms Fe/mg dry weight. CONCLUSIONS: The ability of a non-invasive technique to detect low levels of iron overload could be useful in the assessment of therapy and in the screening of relatives of probands with idiopathic haemochromatosis.

Adult↗
PubMed1990

A high concentration of dopamine preferentially permitted release of newly synthesized prolactin.

We used continuous labelling ([3H]leucine) of cultured adenohypophysial cells to investigate the relationship between the storage and release of newly synthesized and stored prolactin in response to dopamine (1 mumol/l) and thyrotropin-releasing hormone (TRH) (0.1 mumol/l) challenge. Newly synthesized prolactin was identified by the tritium radiation activity incorporated in prolactin. A maximal dose of dopamine (1 mumol/l) could not completely block prolactin release from a primary culture of lactotrophs. During 3 h of continuous labelling under maximal dopaminergic inhibition, newly synthesized prolactin was released which was of a significantly higher specific activity than control groups. In contrast, TRH stimulation produced results consistent with previous observations of the release of predominantly old, stored hormone. However, the absolute amount of the newly synthesized prolactin was increased by the TRH administration, and the increased release of the newly synthesized prolactin could be accounted for by increased levels of synthesis. Our results are consistent with the concept of the existence of a regulated route and a dopamine-insensitive constitutive route of prolactin release which predominantly encompasses newly synthesized hormone. However, the possibility that cellular heterogeneity or that non-dopaminergic prolactin-release inhibiting factor(s) (PIF) is responsible for this observed release cannot be ruled out.

Animals↗
PubMed1984

Butyrate prevents Harvey Sarcoma virus focus formation but permits oncogene expression.

Since butyrate inhibits DNA synthesis and cell growth, we adapted NIH 3T3 cells to grow in butyrate to study its effects on retroviral cell transformation. In cultures of butyrate-adapted NIH 3T3 cells infected with Harvey Sarcoma Virus, no foci were formed; however, there was evidence of retroviral replication, and the P21 oncogene product was demonstrated by immunofluorescence in its characteristic localization beneath the cytoplasmic membrane of single cells and pairs of cells. When the butyrate was removed, these cells proliferated to form foci. Butyrate therefore does not prevent oncogene expression but does reversibly inhibit focus formation.

Animals↗
PubMed1986

Voltammetry in vivo with a single working electrode may permit detection of striatal dopamine-serotonin interactions in anesthetized and freely moving rats.

We have recently improved the technique of differential pulse voltammetry to detect extracellular 3,4-dihydroxyphenylacetic and 5-hydroxyindoleacetic acid concentrations in vivo with a single monopyrolytic carbon fibre electrode (working electrode). Thus it is now possible to perform a simultaneous evaluation of the turnover of dopamine (DA) and serotonin (5-HT) in a specific brain area of anaesthetized or conscious freely moving rats. We have attempted to determine whether there is an interaction between the two neuronal systems in the striatum. Our results show that various pharmacological manipulations in anaesthetized or conscious freely moving rats alter the activity of both systems suggesting the presence of interactions between 5-HT and DA systems in brain.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1990

A four-day period of bimodality auditory and visual experience is sufficient to permit normal emergence of the map of auditory space in the guinea pig superior colliculus.

Previous work has shown that if guinea pigs are deprived of simultaneous visual and auditory experience during a 4-day period 26-30 days after birth (DAB), this prevents the normal emergence of an auditory space map in the superior colliculus. The present work reports that if this 4-day period is the only developmental period in which the animals have simultaneous bimodality experience, the auditory space map appears at the normal time. Apart from this time-window animals were deprived of either visual experience (by dark-rearing) or of normal auditory experience (by rearing in an environment of omnidirectional masking noise). We conclude that the period 26-30 DAB is a crucial developmental period during which coincident auditory and visual experience is utilized in the initial formation of the collicular map of auditory space.

Animals↗
PubMed1987

Acetylcholine permits long-term enhancement of neuronal responsiveness in cat primary somatosensory cortex.

Acetylcholine (ACh) was administered iontophoretically to single neurons in cat somatosensory cortex. Using extracellular recording techniques, neuronal responsiveness was determined at regular intervals from the number of action potentials produced either by iontophoretically applied glutamate or by tactile stimulation of the cutaneous receptive field. The responses were altered in only 21% (13/61) of the neurons following the application of ACh alone. In contrast, 75% (66/88) of the neurons displayed altered responses during administration of ACh simultaneously with either iontophoretically administered glutamate or with tactile stimulation of the receptive field. Forty-seven percent (29/62) of the responses potentiated in the presence of ACh remained enhanced for periods lasting from 8 min to over 1 h. The responsiveness of cortical neurons to afferent inputs changes during the reorganization of somatotopic maps that occurs after deafferentation, and perhaps during some forms of learning. As ACh has been implicated in some of these processes, it may be that the changes in responsiveness observed here following iontophoretically applied ACh are similar to those which facilitate the acquisition of neuronal responses to altered or novel afferent inputs.

Acetylcholine↗
PubMed1984

Pseudogene IFN-alpha L: removal of the stop codon in the signal sequence permits expression of active human interferon.

Biologically active interferon (10(6)-10(7) units/liter) was produced in Escherichia coli from modified human alpha interferon (IFN-alpha) pseudogene L. IFN-alpha pseudogene L has a stop codon in the signal peptide coding region. The region that contains the stop codon was replaced with the corresponding region of another human IFN-alpha gene, WA, that does not have a stop codon and was previously engineered for expression by fusion to the M13mp11 lac promoter. The interferon L fusion product was induced with IPTG after infecting E. coli JM103 with the M13 bacteriophage that contained the modified human IFN-alpha pseudogene L. Hence, the IFN-alpha L mature interferon coding sequence, which is not identical to any other alpha-interferon gene, has been conserved for active interferon coding information.

Amino Acid Sequence↗
PubMed1985

Plasmids supplying the Q-qut-controlled gam function permit growth of lambda red- gam- (Fec-) bacteriophages on recA- hosts.

A plasmid, pgam, has been constructed which expresses the phage lambda gene, gam, under the control of the lambda late promoter, p'R, contained in a form of a p'R-qut-t'R1 module. Lambda red- gam-, which normally do not grow on recA- hosts, are able to grow on recA- hosts containing pgam, because their Q function can turn on the gam gene expression. This facilitates cloning with lambda red- gam- vectors in recA- hosts.

Bacteriophage lambda↗
PubMed1988

Escherichia coli sbcC mutants permit stable propagation of DNA replicons containing a long palindrome.

Recombinant DNA libraries generated in vitro should in theory contain all of the sequences of the genomes from which they are derived. However, the literature is dotted with reports of sequences that cannot be recovered, are under-represented, or are highly unstable. In particular, long palindromic nucleotide sequences of perfect or near-perfect symmetry are either lethal to the vector or suffer deletions or other rearrangements that remove symmetry [Collins, Cold Spring Harbor Symp. Quant. Biol. 45 (1981) 409-416; Collins et al., Gene 19 (1982) 139-146; Hagan and Warren, Gene 24 (1983) 317-326]. We report here that mutation of a single gene, namely sbcC, can overcome this inviability and allow for the stable propagation of a 571-bp nearly perfect palindrome in Escherichia coli. This has implications for the choice of strains used for the recovery and analysis of cloned nucleotide sequences.

Bacteriophage lambda↗
PubMed1990

Of mice and yeast: versatile vectors which permit gene expression in both budding yeast and higher eukaryotic cells.

Gene expression in heterologous species is a powerful tool for the cloning and characterization of genes. Here, we present a family of expression shuttle vectors which work in the budding yeast, Saccharomyces cerevisiae, and also in mammalian cells. The quantity of product expressed by the gene under study can be modulated in yeast by virtue of the control over plasmid copy number and culture conditions.

Animals↗
PubMed1991

Cellular transcripts encoded at a locus which permits retrovirus expression in mouse embryonic cells.

Three independent recombinant retroviruses have been activated on insertion into the F2 locus of mouse F9 embryonal carcinoma cells. Each provirus has integrated downstream from the cellular F2 promoter, which is active in transient transfection assays using a chloramphenicol acetyltransferase reporter enzyme. The F2 promoter drives expression of a series of related transcripts in F9 and 3T3 cells, and a single 450-nt transcript in mouse tissues. F2 homologous sequences have been detected in the genomes of all mammalian species tested, and the 450-nucleotide (nt) F2 transcript is expressed in rat and human cells. Three pairs of differently sized F2 cDNA clones have been isolated and analyzed. The largest clones possess two 199-nt 98.5% identical repeats, one of which is present in the smaller clones, as well as the major 450-nt transcript. Activated proviral integration sites map to introns of the largest F2 cDNA clone. While none of the F2 cDNA contains a long open reading frame or homology to databank sequences, evidence suggests that the F2 locus encodes a constitutive function required at high levels, or represents an expressed but nonfunctional, single-copy element, conserved among mammals.

Animals↗