Cell communication and the "bystander effect".
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Alpha Particles↗
Biomedical subjects
C Seymour
Publications and source records attributed to C Seymour.
Peripheral blood CD34+ cell count reliably predicts autograft yield.
A reliable measure to predict peripheral blood progenitor cell (PBPC) autograft CD34+ cell content is required to optimize the timing of PBPC collection. We prospectively examined the peripheral blood (PB) CD34+ cell count in 59 consecutive patients with various malignancies and analyzed the correlation between the PB CD34+ cell count and various parameters in the PBPC autograft. Two hundred and thirty-five collections were performed with a median of 4.0 collections per patient (range, 2-10). The median PB CD34+ cell count at the time of collection was 39 x 10(6)/1 (range, 0.0-285.6). The PBPC autograft parameters measured were the CD34+ cell, colony-forming unit granulocyte-macrophage (CFU-GM) and mononuclear cell (MNC) content. There was a strong linear correlation between PB CD34+ cells/l and autograft CD34+ cells/kg (r = 0.8477). The correlation with CFU-GM/kg (r = 0.5512) was weaker. There was no correlation between autograft CD34+ cells/kg and PB WBC (r= 0.0684), PB MNC (r = 0.1518) or PB platelet count (r = 0.2010). At our institution we aim to obtain a minimum of 0.5 x 10(6) CD34+ cells/kg with each day of collection. We demonstrate that such a collection can be reliably obtained if the PB CD34+ cell count exceeds 5.0 x 10(6)/l.
Blood Cell Count↗
Radiation-induced carcinogenesis: studies using human epithelial cell lines.
It has proved difficult to develop suitable models to study radiation-induced carcinogenesis by using human epithelial cells. However, immortalised human epithelial cell lines have proved useful. Unirradiated cells from the human keratinocyte cell line (HPV-G) and the human embryonic lung cell line (L132) were found to be tumourigenic in T-cell-deficient mice; thus, they are not suitable for transformation studies. Human urothelial cell lines (SV-HUC-1, NT11, BC16) and the human thyroid epithelial cell line (HTori-3) were nontumourigenic. The urothelial cell lines were refractory to radiation-induced carcinogenesis, and only one small tumour was observed in 57 mice that received irradiated cells. Whereas tumours were not produced following irradiation of these urothelial cells, changes in anchorage-independent growth were observed after a single dose of 8 Gy gamma-irradiation but not after 2 or 4 Gy. Irradiation of the human thyroid epithelial cell line (HTori-3) in vitro resulted in tumour formation. Passaging of the cells in vitro before injection did not seem to be critical. Some of the cell lines derived from the primary thyroid tumours exhibited p53 mutations in exons 5, 6, 7, and 8, as detected by single-stranded conformational polymorphism (SSCP) analysis. Thus, the human thyroid epithelial cell line (HTori-3) looks promising as a model for investigating the molecular events in radiation-induced carcinogenesis.
Animals↗
Survival of human epithelial cells irradiated with cobalt 60 as microcolonies or single cells.
Microcolonies of one to >50 cells were irradiated. They were assayed for survival using the Puck and Marcus clonogenic technique and the distant progeny were tested for expression of lethal mutations. The results show that epithelial cell colonies appear to respond as a unit rather than as individual cells to a radiation dose and the uncorrected initial surviving fraction is relatively constant irrespective of the number of cells present at the time the microcolony was irradiated. Irradiation of colonies or monolayers, which were then dispersed, confirmed this and showed slight sparing of the cells irradiated in contact compared with single cells but no sparing effect when the gap junctions were closed. Measurement of apoptosis 2 h post-irradiation showed higher levels in clones derived from cells irradiated in contact but delayed apoptosis in the progeny and lethal mutations appear to be associated with irradiation of single cells. Lethal mutations occurred in the progeny of cells irradiated as single cells for at least 30 cell generations but if cell microcolonies were irradiated the progeny survival showed a complex relationship with progenitor dose. When gap junction intercellular communication (GJIC) was blocked during and immediately post-irradiation using nitrosamines or TPA, cultures regained the initial survival and lethal mutation frequency seen with single cells. It is concluded that the presence of more than one cell in a microcolony at the time of irradiation does result in an altered and possibly a co-ordinated pattern of survival and lethal mutation expression but that inhibition of GJIC can reverse the effects of contact. The results may have implications for investigations of normal tissue response.
Apoptosis↗
Induction of multiple PCR-SSCPE mobility shifts in p53 exons in cultures of normal human urothelium exposed to low-dose gamma-radiation.
We have previously shown that primary explant cultures of human urothelium exposed to low doses of gamma-radiation subsequently accumulate a high level of stable p53 but it was not clear from those studies whether this protein stabilization occurred through an event in another gene involved in p53 protein control or possibly an epigenetic event. In these experiments, primary urothelial cultures from five different patients were exposed to either 0.5 or 5 Gy gamma-radiation from a 60 Cobalt source and allowed to grow for 7-10 division cycles to allow development of any radiation-induced, non-lethal changes in the cells. C-myc, Bcl-2 and stable p53 proteins were found to be elevated in cultures following both radiation doses. PCR-SSCPE analysis of the p53 gene was performed on cultures in order to determine whether genetic mutations could be the underlying basis for persistent increased stable p53 expression. Following 0.5 Gy exposure, the cultures also developed multiple distinct 'foci' of rapidly dividing cells which strongly overexpressed p53. These grew on a background of morphologically normal cells. When such foci were selectively analysed for their p53 mutation status by PCR-SSCPE, there was evidence that they contained cells which had developed changes to the p53 gene post-irradiation. These changes appeared to occur more frequently in focal cells than in cells of normal morphological appearance in the same culture. These results may have mechanistic importance given the controversy regarding low-dose radiation effects and p53-related genomic instability.
Culture Techniques↗
Lethal mutations and genomic instability.
The delayed expression of cell death in progeny of irradiated survivors was christened 'lethal mutations' by Tikvah Alper in 1984. The effect occurs when clones, or populations of cells grown up from irradiated progenitor cells, are replated and reassessed for cloning efficiency or population doubling time. The effect has been shown to be associated with the low dose shoulder region of the survival curve and is due to events occurring in the first two hours post irradiation, i.e. the 'fast repair' period. In this review the lethal mutation data accumulated over the past ten years is discussed in relation to our modern understanding of cellular and molecular events in radiation carcinogenesis and genomic instability research. It is suggested that lethal mutations are associated with a general epigenetic or field effect occurring in all irradiated cells, which makes them more prone to mutations, some of which are lethal. The implications of this for our current approach to risk estimates and therapeutic dose calculation, need to be addressed.
Animals↗
Medium from irradiated human epithelial cells but not human fibroblasts reduces the clonogenic survival of unirradiated cells.
The results reported in this paper show a highly significant fall in cloning efficiency in unirradiated normal and malignant epithelial cell lines receiving medium from irradiated cultures. Medium irradiated in the absence of cells had no effect nor did irradiated medium from a fibroblast line, but irradiated medium from epithelial cells had an extremely toxic effect on unirradiated fibroblasts. Cells from four different cell lines were seeded, using standard techniques, 6 h prior to irradiation with cobalt 60 (Co60). At various times ranging from 1-60 h after irradiation, medium was removed, passed through a 0.22 mu filter and then used to replace the medium from parallel cultures seeded at cloning densities which had not been irradiated. The effect produced by the epithelial cell cultures was dependent on the cell number present at the time of irradiation, suggesting that a cell-derived factor is involved. The effect could be observed using medium taken from irradiated cultures as soon as 30 min/post irradiation. Examination of unirradiated cultures 48 h after receiving irradiated medium revealed the presence of high numbers of apoptotic bodies and other morphological evidence suggesting apoptosis may be a prominent mechanism of cell death responsible for the reduced cloning efficiency.
Cell Line↗
p53 protein expression and increased SSCP mobility shifts in the p53 gene in normal urothelium cultured from smokers.
This study provides evidence of a significantly (P = 0.018) increased level of expression of the stable conformation of p53 in normal urothelial cells, cultured in vitro from bladder biopsies obtained from normal smokers without malignant disease of any site. With two significant exceptions, non-smokers showed low or no expression of this protein. Past smokers appeared to segregate into high or low p53 expressers, but the expression was not correlated with years since quitting smoking or with pack years smoked. The mean data in this group were not quite significantly different (P = 0.08) from the non-smoker group, due to the wide inter-patient variation. For most of the smoker group, pack years correlated with p53 expression with a mean unit of 1.7 +/- 0.37% p53 per pack year but there was a small group of very heavy smokers who showed lower than expected expression (approximately 0.3-0.8% p53 per pack year). These were statistical outliers (Grubbs test). No explanation could be found for this. Over-expression of p53 protein, often correlates with mutations in the gene, but may also indicate that breakdown of wild-type p53 has slowed. SSCP analysis of the biopsy material was not possible on all patients due to ethical constraints on the amounts of tissue which could be taken but in the cases where it was possible the association between loss of p53 protein function and mobility shifts in p53 exons 5-8 was confirmed with smokers having 3.5 times the number of mobility shifts detected in non-smoker DNA. Thus the results may point to a role for the early abrogation of p53 protein function in bladder carcinogenesis induced by cigarette smoking.
Adult↗
Role of elevated alpha-fetoprotein in prenatal diagnosis of junctional epidermolysis bullosa and pyloric atresia.
A case of junctional epidermolysis bullosa, Herlitz variant, and pyloric atresia in a 33 weeks' gestation male infant is reported. The second trimester amniotic fluid exhibited elevated concentrations of alpha-fetoprotein and presence of acetylcholinesterase; however, the fetus appeared anatomically normal by multiple high-resolution ultrasound examinations. This case, as well as others previously reported, shows that serious fetal skin disease should be considered as part of the differential diagnosis whenever persistent elevation of alpha-fetoprotein and presence of acetylcholinesterase are found in the amniotic fluid of a fetus that appears anatomically normal by ultrasound. Prenatal diagnosis may be established by fetal skin biopsy and extensive prenatal counseling should be offered to families on the basis of the prognosis and severity of this disease.
Acetylcholinesterase↗
Phylogeny of Helicobacter isolates from bird and swine feces and description of Helicobacter pametensis sp. nov.
Previously, nine fecal isolates from wild birds and a domestic swine were identified as helicobacters by phenotypic characterization and reaction with a helicobacter genus-specific DNA probe. These isolates fell into three biotypes by analysis of phenotypic traits. To further characterize these isolates, full 16S rRNA sequences were determined for strains representing each biotype, and sequence comparison indicated that the strains represented three novel, phylogenetically defined Helicobacter species. Three 16S rRNA-based DNA probes were designed and used to identify the remaining strains. Probe reactivity divided the strains into the same three groups identified phenotypically. Six of the isolates represented a new species of the genus Helicobacter for which we propose the name Helicobacter pametensis sp. nov. The following phenotypic features distinguished H. pametensis from other Helicobacter and Campylobacter species: positive tests for oxidase, catalase, alkaline phosphatase, nitrate reduction, growth at 42 degrees C, and growth in the presence of 1% glycine; negative tests for urease, gamma glutamyl transpeptidase, indoxyl acetate hydrolysis, and hippurate hydrolysis; and susceptibility to nalidixic acid and cephalothin. H. pametensis cells were motile and possessed one subterminal sheathed flagellum at each end. The two additional Helicobacter species were similar to H. pametensis except that they were urease positive, hydrolyzed indoxyl acetate, and were resistant to cephalothin. Because these two additional species are phenotypically similar and are represented by only two isolates for one species and one isolate for the other, they are not formally named but are referred to as Helicobacter sp. "Bird-B" and Helicobacter sp. "Bird-C."(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Isolation of Helicobacter strains from wild bird and swine feces.
We report the first isolations of Helicobacter strains from wild birds and swine. Genus-specific oligonucleotide probes identified nine Cape Cod isolates from gull, tern, house sparrow, and pig feces as Helicobacter spp. and not Campylobacter spp. Antibiotic sensitivity and urease tests distinguished three phenotypes. Strains examined rapidly lost culturability under simulated natural conditions.
Animals↗
Comparative virulence of blood and stool isolates of Shigella sonnei.
Shigellemia is rare in developed countries and might result from the emergence of unusually virulent strains. We compared systemic invasiveness markers of isolates from the blood of 3 temporally clustered patients with Shigella sonnei bacteremia in Boston with those of 11 unrelated contemporaneous strains from stools of people in New England. We found no difference between the two groups in O-chain length by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, mouse 50% lethal dose, in vivo response to iron, and susceptibility to serum, which varied from moderately susceptible to ultrasusceptible. Mean intraperitoneal 50% lethal doses of smooth form I colonies for mice were equally low (10(5.8) CFU) in both groups, and the 50% lethal doses were lowered equally further in the two groups by predosing with iron to levels useful in mouse model sepsis studies. S. sonnei bacteremia may reflect compromised host defenses, not bacterial virulence.
Adult↗
All colonies of CHO-K1 cells surviving gamma-irradiation contain non-viable cells.
This paper addresses the problem of the production of defective cells within clones arising from irradiated progenitor cells and is specifically aimed at answering the question of whether lethal mutations result from a generalised effect which lowers the ability of all the progeny to divide successfully or whether it represents a late expressed but unique lethal defect induced by radiation which occurs in some cells only and which causes those cells only to cease dividing. The results obtained from autoradiographic analysis of cells within individual surviving colonies (i.e. containing more than 150 cells) suggests that some cells in all clones are not synthesizing DNA over a 9-h period and that the proportion of non-synthesising cells rises with increasing dose of radiation from less than 3% in the controls to 80-85% after a progenitor dose of 12.5 Gy. Because of the possibility that cells had longer division times post irradiation, these results were repeated using Ki67 antibody labelling, a technique which identifies cells which are in cycle. The results were similar. This suggests the non-labelled cells were not reproducing. Both techniques were also used to look at the % labelling of morphologically abnormal cells in the colonies. The results suggested that up to 35% of these abnormal cells were actively cycling and about 20% were synthesising DNA. Abnormal cells did not appear in subcultures of survivor progeny suggesting that they may have failed to replate successfully and may contribute to the lethally mutated population. The idea that radiation induces a general instability in the cell population was supported by experiments where growth and the plating efficiency of irradiated progeny was measured daily. This revealed that the growth curves deviated from the control by a constant factor suggesting a division probability of about 70% of the control level after a progenitor dose of 10 Gy. The results are discussed in the context of their significance for survival curve analysis and for radiotherapy and radiation protection results.
Animals↗
Demonstration by western blotting of antiheart antibodies before and after cardiac transplantation.
Western blotting has been used to detect antiheart antibodies in two groups of patients: two who required retransplantation for hyperacute rejection, and 22 consecutive patients, whose serum was tested at monthly intervals for three months following transplantation. Pretransplant and posttransplant serum samples were tested for IgM and IgG reactivity against the patients own heart and donor heart. In all patients the pretransplant lymphocytic crossmatch had been negative. In the two patients requiring retransplantation, both had multiple bands of strong IgM and IgG against their own heart prior to transplantation as well as antibodies against the donor heart. The study of 22 consecutive patients revealed that (1) the presence of strong antibody prior to transplantation is associated with unusually severe or frequent rejection episodes, (2) 20/22 patients made antiheart antibody following transplantation, but in 12 patients it was IgM only, and (3) most of the antiheart antibodies made posttransplant were not specific for the donor heart. Comparison of Western blotting with immunofluorescent detection of antibodies on frozen sections revealed that the Western blotting procedure is more sensitive and results are easier to interpret.
Adolescent↗
Use of western blotting to detect anti-heart antibodies before and after cardiac transplantation.
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Adolescent↗
Biochemical and immunological characterization of the major structural proteins of feline immunodeficiency virus.
Feline immunodeficiency virus (FIV) structural proteins were identified using sera obtained from experimentally inoculated cats. Proteins analysed by both radioimmunoprecipitation and Western blotting were specific for FIV infection and failed to cross-react with either antisera to feline leukaemia virus of feline syncytium-forming virus. Western blot analysis of purified virus revealed immunoreactive proteins with apparent Mr of 65K, 50K, 40K, 32K, 24K, 15K and 10K. The major core structural proteins of the virus were isolated by reverse phase HPLC and the aminoterminal sequences of p10 and p24 were determined. Monoclonal antibodies specific for p24 suggested the presence of a precursor protein that could be detected in 35[S]methionine/cysteine-labelled, virus-infected cell extracts. This putative precursor protein possessed an apparent Mr of 50K (Pr50gag). Further analysis revealed the presence of two additional proteins of 130K and 40K. Experiments utilizing tunicamycin, endoglycosidase H and glycopeptidase F revealed that p130 and p40 exhibited properties characteristic of glycoproteins. Our studies also indicated that FIV is immunologically related to other lentiviruses.
Amino Acid Sequence↗
The effects of L-glutamate on cultured insect neurones.
The effects of L-glutamate on insect cultured neurones were studied under current and voltage-clamp conditions using conventional and whole-cell patch-clamp techniques. Brief pressure or iontophoretic application of L-glutamate produced either a depolarisation or hyperpolarisation. The current underlying the depolarisation was inwardly directed and reversed at around 0 mV while the hyperpolarisation was caused by an outward current that reversed between -60 and -80 mV. Single channel currents underlying the depolarisation were readily recorded from cell attached patches and showed multiple conductance states. Channel activity corresponding to the hyperpolarising response has not yet been observed.
Animals↗
The influence of lethal mutations on the quantification of radiation transformation frequencies.
Transformation frequencies for gamma irradiated C3H 10T1/2 cells have been analysed, taking account of the occurrence of lethal mutations in these cells. Lethal mutations already noted by these authors in primary thyroid and established CHO K1 cells occur at high levels in C3H 10T1/2 cells and lead, therefore, to considerable underestimates of transformation frequency, particularly at high doses where this is expressed on a per surviving cell basis. The results may help to provide an explanation for the dose response plateau which is seen when these cells are irradiated and transformed foci per surviving cell are scored.
Animals↗