• Transferrin receptor-mediated gene transfer to the corneal endothelium.

      Tan, Peng H; King, William J; Chen, Daxin; Awad, Hana M; Mackett, Mike; Lechler, Robert I; Larkin, D Frank; George, Andrew J; Department of Immunology, Imperial College, Hammersmith Hospital, London, UK. (2001-02-27)
      BACKGROUND: The application of gene therapy to prevent allograft rejection requires the development of noninflammatory vectors. We have therefore investigated the use of a nonviral system, transferrin-mediated lipofection, to transfer genes into the cornea with the aim of preventing corneal graft rejection. METHODS: Rabbit and human corneas were cultured ex vivo and transfected with either lipofection alone or in conjunction with transferrin. The efficiency of transfection, localization, and kinetics of marker gene expression were determined. Strategies to increase gene expression, using chloroquine and EDTA, were investigated. In addition to a marker gene, a gene construct encoding viral interleukin 10 (vIL-10) was transfected and its functional effects were examined in vitro. RESULTS: Transferrin, liposome, and DNA were demonstrated to interact with each other, forming a complex. This complex was found to deliver genes selectively to the endothelium of corneas resulting in gene expression. Treatment of corneas with chloroquine and EDTA increased the transfection efficiency eight-fold and threefold, respectively. We also demonstrated that constructs encoding vIL-10 could be delivered to the endothelium. Secreted vIL-10 was shown to be functionally active by inhibition of a mixed lymphocyte reaction. CONCLUSIONS: Our data indicate that transferrin-mediated lipofection is a comparatively efficient nonviral method for delivering genes to the corneal endothelium. Its potential for use in preventing graft rejection is shown by the ability of this system to induce vIL-10 expression at secreted levels high enough to be functional.
    • Transferrin-mediated uptake of plutonium by spermatogenic tubules.

      Hoyes, Katherine P; Bingham, D; Hendry, Jolyon H; Harrison, J D; Sharma, Harbans L; Morris, Ian D; School of Biological Sciences, University of Manchester, UK. (1996-10)
      Using isolated rat seminiferous tubules as an in vitro model, we have found that 238Pu can cross the blood-tubule barrier and accumulate within tubules in a time dependent manner. Furthermore, similar to 59Fe, tubule 238Pu uptake was inhibited by the addition of excess transferrin, suggesting that plutonium may utilize the physiological iron-transferrin pathway to cross the blood-tubule barrier. However unlike 59Fe, 238Pu was only transiently associated with the tubules, suggesting differences in the intracellular processing of these radionuclides. The assumptions made in the estimation of doses to the human testis from incorporated plutonium are considered.
    • Transferrin-mediated uptake of radionuclides by the testis.

      Hoyes, Katherine P; Morris, Ian D; Hendry, Jolyon H; Sharma, Harbans L; Department of Experimental Radiation Oncology, Paterson Institute for Cancer Research, Manchester, United Kingdom. (1996-02)
      In an attempt to explain the deleterious effects of gonadal radionuclide localization, we examined the role of transferrin in testicular radionuclide uptake. METHODS: In vivo testicular uptake and retention of the transferrin binding radionuclides 114mIn-citrate and 59Fe-citrate were compared with that of the nontransferrin binding isotopes 137Cs-citrate and Na125I for 63 days postinjection. Isotope uptake mechanisms were investigated in vitro using isolated seminiferous tubules and Sertoli cell monolayers grown in bicameral culture chambers. RESULTS: Indium-114m, 59Fe and 137Cs were localized in the testis by 24 hr postinjection, but accumulation of 125I was minimal. Although testicular 114mIn remained constant, 59Fe declined slowly over the following 63 days and 137Cs fell very rapidly. When 114mIn- or 59Fe-loaded testes were fractionated, and markedly more 114mIn was associated with the seminiferous tubules than 59Fe, suggesting that 114mIn may be retained. In vitro uptake of 59Fe, 67Ga and 114mIn by isolated seminiferous tubules was inhibited by transferrin, but uptake of 137Cs and 125I was unaffected. Iron-59, 67Ga and 114mIn were retained by isolated tubules in contrast to 137Cs and 125I. Whereas 137Cs, 59Fe and 114mIn all crossed Sertoli cell monolayers, the rate of transcellular transport of 137Cs was faster than that of 59Fe or 114mIn, suggesting differences in the intracellular processing of transferrin binding and nontransferrin binding radionuclides. CONCLUSION: These data suggest that some radionuclides may access the seminiferous epithelium through receptor-mediated endocytosis of transferrin. Such radionuclide localization could lead to continuous irradiation of the testes, resulting in mutagenic damage to spermatogenic cells.
    • Transforming growth factor beta 1 promotes the differentiation of endothelial cells into smooth muscle-like cells in vitro.

      Arciniegas, E; Sutton, Andrew B; Allen, Terence D; Schor, Ana M; CRC Department of Medical Oncology, Christie Hospital, Manchester, UK. (1992-10)
      Alpha-smooth muscle actin is considered a reliable marker for distinguishing between arterial smooth muscle and endothelial cells. Several authors have reported heterogeneity in the expression of this actin isoform in atherosclerotic lesions. Such heterogeneity appears to result from the presence of different smooth muscle cell phenotypes (contractile and synthetic) in these lesions. In the present study, we show that bovine aortic endothelial cells, which are characterised by the presence of Factor VIII-related antigen (FVIII) and by the absence of alpha-smooth muscle actin (alpha-SM actin) may be induced to express the latter when exposed to TGF-beta 1. FVIII was detected by immunofluorescence, alpha-SM actin was detected by immunofluorescence and immunoblotting. The number of cells expressing alpha-SM actin increased with time of incubation with TGF-beta 1, and this increase occurred concomitantly with a decrease in the expression of FVIII. Double immunofluorescence demonstrated the presence of cells that expressed both FVIII and alpha-SM actin after 5 days of incubation with TGF-beta 1. With longer incubation times (10-20 days) the loss of FVIII expression was complete and over 90% of the cells expressed alpha-SM actin. Ultrastructurally, cells in control cultures showed the typical features of endothelial cells. In the TGF-beta 1-treated cultures, cells which appeared indistinguishable from contractile and synthetic smooth muscle cells were observed. Withdrawal of TGF-beta 1 after 10 days incubation resulted in the re-appearance of polygonal cells which were FVIII-positive and alpha-SM actin-negative.(ABSTRACT TRUNCATED AT 250 WORDS)
    • Transforming growth factor-B3 protects murine small intestinal crypt stem cells and animal survival after irradiation, possibly by reducing stem-cell cycling.

      Booth, Dawn; Haley, John D; Bruskin, Arthur M; Potten, Christopher S; CRC Epithelial Biology Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. (2000-04-01)
      Damage to the normal replacing tissues of the body, specifically the gastro-intestinal tract, limits the treatment and hence, cure rate of cancer patients. Here, we investigate the possibility that the sensitivity of the gastro-intestinal tract can be manipulated by transforming growth factor beta3 (TGF-beta3), making it more resistant to radiation in a murine model. The effects of TGF-beta3 were assessed using the crypt microcolony assay, a test of crypt stem-cell functional competence, in animal survival studies examining diarrhoea severity, labelling index and crypt size. Prior treatment with TGF-beta3 can result in a 3- to 4-fold increase (protection factor, PF) in surviving crypts, whilst longer exposure can raise the PF to almost 12. Protection of intestinal clonogenic stem cells results in marked protection of survival with a corresponding reduction in the duration and level of diarrhoea and ultimate restoration of normal histology in surviving mice. Inhibition of proliferation can be demonstrated when sufficient TGF-beta3 exposure is studied. Crypt size is also reduced. In conclusion, TGF-beta3 protects small intestinal clonogenic stem cells from radiation damage, reducing diarrhoea and animal mortality. The mode of action is believed to be specific inhibition of stem-cell proliferation.
    • Transforming growth factor-beta 1 induces apoptosis independently of p53 and selectively reduces expression of Bcl-2 in multipotent hematopoietic cells.

      Francis, Julia M; Heyworth, Clare M; Spooncer, Elaine; Pierce, Andrew; Dexter, T Michael; Whetton, Anthony D; Leukaemia Research Fund Cellular Development Unit, Department of Biomolecular Sciences, UMIST, Sackville St., Manchester, M60 1QD, United Kingdom. (2000-12-15)
      Transforming growth factor-beta1 (TGF-beta1) can inhibit cell proliferation or induce apoptosis in multipotent hematopoietic cells. To study the mechanisms of TGF-beta1 action on primitive hematopoietic cells, we used the interleukin-3 (IL-3)-dependent, multipotent FDCP-Mix cell line. TGF-beta1-mediated growth inhibition was observed in high concentrations of IL-3, while at lower IL-3 concentrations TGF-beta1 induced apoptosis. The proapoptotic effects of TGF-beta1 occur via a p53-independent pathway, since p53(null) FDCP-Mix demonstrated the same responses to TGF-beta1. IL-3 has been suggested to enhance survival via an increase in (antiapoptotic) Bcl-x(L) expression. In FDCP-Mix cells, neither IL-3 nor TGF-beta1 induced any change in Bcl-x(L) protein levels or the proapoptotic proteins Bad or Bax. However, TGF-beta1 had a major effect on Bcl-2 levels, reducing them in the presence of high and low concentrations of IL-3. Overexpression of Bcl-2 in FDCP-Mix cells rescued them from TGF-beta1-induced apoptosis but was incapable of inhibiting TGF-beta1-mediated growth arrest. We conclude that TGF-beta1-induced cell death is independent of p53 and inhibited by Bcl-2, with no effect on Bcl-x(L). The significance of these results for stem cell survival in bone marrow are discussed.
    • Transgenerational effects of preconception paternal contamination with (55)Fe.

      Hoyes, Katherine P; Lord, Brian I; McCann, Christine; Hendry, Jolyon H; Morris, Ian D; Cancer Research Campaign Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, M20 4BX, United Kingdom. (2001-11)
      The conjecture that germline mutations induced by radiation exposure before conception may predispose subsequent offspring to cancer remains contentious. Previous experimental studies have shown that preconception paternal irradiation with (239)Pu induces perturbations in the hemopoietic systems of offspring and influences sensitivity to a secondary carcinogen. In the present study, male DBA2 mice were injected intravenously with the Auger electron emitter (55)Fe (4 kBq g(-1)) 18 or 84 days before mating with normal females. Comet analysis showed an increased incidence of DNA strand breaks in sperm from contaminated animals after 84 days, but not after 18 days, indicating spermatogonial rather than spermatid damage. Offspring were either assayed for changes in bone marrow stem cells and committed progenitors or challenged with the chemical carcinogen methyl nitrosourea (MNU, 50 mg/kg) at 10 weeks of age and monitored for the onset of malignancy. Offspring from irradiated fathers had normal peripheral blood profiles, although the stem cell population was amplified in offspring arising from those exposed to (55)Fe at 84 days before conception. Exposure to MNU significantly increased the incidence of lympho-hemopoietic malignancies in offspring from the 84-day group, but not in those from the 18-day group. These findings support the hypothesis that aberrations that are potentially leukemogenic may be transmitted to offspring after radiation damage to the paternal germline.
    • Transgenerational susceptibility to leukaemia induction resulting from preconception, paternal irradiation.

      Lord, Brian I; CRC Section of Haemopoietic Cell and Gene Therapeutics, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. blord@picr.man.ac.uk (1999-07)
      The clustered excess of childhood leukaemia and non-Hodgkin's lymphoma at Seascale, close to the nuclear reprocessing plant at Sellafield in the UK is well authenticated and has remained a 'current topic' for over a decade. Its root cause has not been established. Following a study suggesting that parental irradiation exposure prior to conception was a factor, a recent laboratory-based report reopened the debate by indicating the potential for preconception, paternal irradiation (PPI) to result in increased or accelerated induction of lympho-myeloid malignancy in offspring subjected to a recognized leukaemogen. This short commentary presents those new findings in the light of the many and diverse epidemiological investigations of first generation malignancies following parental exposure, the majority of which indicate no real evidence to support the concept that patterns of lympho-myeloid malignancy reflect levels of PPI. Other experimental work supporting PPI are considered against unsuccessful attempts to reproduce them. The alternative, and more popular, hypothesis of infection spread via population mixing, which is more ubiquitous than confinement to nuclear localities, is introduced. Mechanisms of potentiation by PPI are considered, though the danger of applying these current findings to explain the enigma of Seascale, or any other cluster, is recognized.
    • A transient assay for regulatory gene function in haemopoietic progenitor cells.

      McIvor, Zoe J; Heyworth, Clare M; Johnson, Barbra A; Pearson, Stella; Fiegler, Heike; Hampson, Lynne; Dexter, T Michael; Cross, Michael A; Laboratory of Molecular Medicine, IZKF University of Leipzig, Leipzig, Germany. zoem@medizin.uni-leipzig.de (2000-09)
      This work aimed to provide a means of assaying directly the effects of transient expression of introduced genes on the survival, proliferation, lineage commitment and differentiation of haemopoietic progenitor cells. For this purpose, we have developed a system that allows isolation of productively transfected, mulitipotent haemopoietic cells within a few hours of the introduction of test genes. We have shown that FDCP-mix cells productively transfected with expression plasmids encoding green fluorescent protein (GFP) differentiate normally and retain colony-forming potential. We constructed an expression vector consisting of a bicistronic cassette in which a GFP marker gene and a test gene are driven from the same promoter. The vector design has been optimized for co-expression and the test gene was shown to be biologically active. The expression profile from a transiently transfected template under different growth conditions reveals that active expression continues for at least 2 d after transfection. The transient transfection of FDCP-mix cells with the vectors described provides a powerful tool for analysis of the immediate early effects of test gene overexpression during haemopoietic differentiation.
    • Transient quinonimines and 1,4-benzothiazines of pheomelanogenesis: new pulse radiolytic and spectrophotometric evidence.

      Napolitano, Alessandra; Di Donato, Paola; Prota, Giuseppe; Land, Edward J; The Department of Organic and Biological Chemistry, University of Naples Federico II, Italy. (1999-09)
      Biosynthetic and model in vitro studies have shown that pheomelanins, the distinctive pigments of red human hair, arise by oxidative cyclization of cysteinyldopas mainly 5-S-cysteinyldopa (1) via a critical o-quinonimine intermediate, which rearranges to unstable 1,4-benzothiazines. To get new evidence for these labile species, fast time resolution pulse radiolytic oxidation by dibromide radical anion of a suitable precursor, the dihydro-1,4-benzothiazine-3-carboxylic acid 7 was performed in comparison with that of 1. In the case of 7, dibromide radical anion oxidation leads over a few microseconds (k = 2.1 x 10(9) M(-1) s(-1)) to a phenoxyl radical (lambda(max) 330 nm, epsilon = 6300 M(-1) cm(-1)) which within tens of milliseconds gives rise with second-order kinetics (2k = 2.7 x 10(7) M(-1) s(-1)) to a species exhibiting an absorption maximum at 540 nm (epsilon = 2200 M(-1) cm(-1)). This was formulated as the o-quinonimine 3 arising from disproportionation of the initial radical. The quinonimine chromophore is converted over hundreds of milliseconds (k = 6.0 s(-1)) to a broad maximum at around 330 nm interpreted as due to a 1,4-benzothiazine or a mixture of 1,4-benzothiazines, which as expected are unstable and subsequently decay over a few seconds (k = 0.5 s(-1)). Interestingly, the quinonimine is observed as a labile intermediate also in the alternative reaction route examined, involving cyclization of the o-quinone (lambda(max) 390 nm, epsilon = 6900 M(-1) cm(-1)) arising by disproportionation (2k = 1.7 x 10(8) M(-1) s(-1)) of an o-semiquinone (lambda(max) 320 nm, epsilon = 4700 M(-1) cm(-1)) directly generated by dibromide radical anion oxidation of 1. Structural formulation of the 540 nm species as an o-quinonimine was further supported by rapid scanning diode array spectrophotometric monitoring of the ferricyanide oxidation of a series of model dihydrobenzothiazines.
    • Transient structure associated with the spindle pole body directs meiotic microtubule reorganization in S. pombe.

      Funaya, C; Samarasinghe, S; Pruggnaller, S; Ohta, M; Connolly, Yvonne; Müller, J; Murakami, H; Grallert, Agnes; Yamamoto, M; Smith, Duncan L; et al. (2012-04-10)
      Vigorous chromosome movements driven by cytoskeletal assemblies are a widely conserved feature of sexual differentiation to facilitate meiotic recombination. In fission yeast, this process involves the dramatic conversion of arrays of cytoplasmic microtubules (MTs), generated from multiple MT organizing centers (MTOCs), into a single radial MT (rMT) array associated with the spindle pole body (SPB), the major MTOC during meiotic prophase. The rMT is then dissolved upon the onset of meiosis I when a bipolar spindle emerges to conduct chromosome segregation. Structural features and molecular mechanisms that govern these dynamic MT rearrangements are poorly understood.
    • The translaminal fibrils of the human amnion basement membrane.

      Campbell, S; Allen, Terence D; Moser, B B; Aplin, J D; Department of Medical Microbiology, University of Manchester, UK. (1989-10)
      The organisation of extracellular matrix beneath the human amniotic epithelium was investigated in order that the co-ordinate synthesis of basal lamina and stroma by these cells could be better understood. Transmission electron microscopy of intact tissue confirmed that stromal matrix fibrils are located between the cell surface and the basal lamina, and also penetrate the lamina. The distribution of the supralaminal fibrils and their association with the lamina was further investigated by scanning electron microscopy (SEM) after removal of the overlying epithelium. Five complementary procedures were used to remove the cells from the underlying lamina. Trypsin-EDTA treatment caused the epithelial cells to retract or detach from the lamina. SDS or ammonium hydroxide was used to extract the epithelium, which was then removed by physical shearing. Transmission electron microscopy (TEM) confirmed that the lamina densa and supralaminal fibres were present after extraction by these agents. Incubation in CHAPS, a zwiterionic detergent, did not remove the epithelium but permitted exposure of the basal lamina by mechanical scoring. Extraction with boric acid followed by osmium tetroxide produced epithelial disruption and revealed the lamina and stroma in different areas. Although the extraction pattern was different in each case, all of the five methods confirmed that individual fibrils and fibril bundles are present on the apical surface of, and enter, the lamina densa. Examination of the stromal surface of the basal lamina after fracture revealed fibrils passing from the stroma into the lamina densa. We therefore suggest that, in this tissue, newly synthesised stromal matrix components appear in an assembled fibrillar form between the basal cell surface and the basal lamina before becoming associated with the sublaminal stroma.
    • Translation of the human papillomavirus type 16 E7 oncoprotein from bicistronic mRNA is independent of splicing events within the E6 open reading frame.

      Stacey, Simon N; Jordan, Deborah; Snijders, P J; Mackett, Mike; Walboomers, J M; Arrand, John R; Department of Molecular Biology, Paterson Institute for Cancer Research, Christie Hospital, Manchester, United Kingdom. (1995-11)
      In this study we investigated the translational capacities of bicistronic and spliced mRNAs originating from the E6 and E7 regions of the high-risk genital human papillomavirus type 16 (HPV-16) and the low-risk HPV-11. For HPV-16 it was found, unexpectedly, that E7 protein could be translated from full-length bicistronic E6-E7 mRNAs. E6*I and E6*II splicing events were not required for E7 synthesis, nor did splicing increase the efficiency of E7 translation significantly. In cells, E7 synthesis from all known naturally occurring mRNA structures was very inefficient compared with that from synthetic monocistronic controls, suggesting that HPV-16 employs translational mechanisms to restrict E7 protein levels. For HPV-11, only RNAs initiated at the P264 promoter, located within the E6 open reading frame, were capable of providing an efficient template for E7 synthesis. P264-initiated mRNAs were as efficient in vivo as monocistronic controls, suggesting that the low-risk HPV-11 does not limit E7 synthesis by translational mechanisms. A detailed analysis of HPV-16 templates by using site-directed mutagenesis showed that the majority of ribosomes which ultimately translate E7 have not reinitiated after translating some or all of the upstream open reading frames. The data support a model in which the failure of 40S ribosomal initiation complexes to recognize the E6 AUG renders them capable of proceeding efficiently to translate E7.
    • Translocation t(11;14) in multiple myeloma: Analysis of translocation breakpoints on der(11) and der(14) chromosomes suggests complex molecular mechanisms of recombination.

      Fenton, James A L; Pratt, Guy; Rothwell, Dominic G; Rawstron, Andy C; Morgan, Gareth J; Academic Unit of Oncology and Haematology, University of Leeds, Leeds, United Kingdom. jamesf@lrf.leeds.ac.uk (2004-02)
      We describe the characterization of the genomic DNA breakpoints of two multiple myeloma (MM) patients with t(11;14). IGH translocation events are present in many MM tumors, and it is proposed that they occur early in the pathogenesis, based on the assumption that the translocations are simple reciprocal events mediated by errors in class-switch recombination (CSR). We provide evidence from two patients that the translocation event can be more complex, with DNA from chromosome band 11q13 joined to apparently already recombined hybrid (Smu/Sgamma) switch region sequences. In one case, there was also evidence that a further rearrangement had occurred at the t(11;14) recombination site, resulting in an inversion of 40 bp of the 5'Smu flanking sequence. This suggests that primary IGH arrangements in MM may be more complex than previous myeloma models have suggested, but that they essentially occur through illegitimate CSR events.
    • Transmembrane potentials in cells: a diS-C3(3) assay for relative potentials as an indicator of real changes.

      Plásek, J; Dale, Robert E; Sigler, K; Laskay, Gabor; Institute of Physics of Charles University, Prague, Czech Republic. (1994-12-30)
      The mechanism by which the fluorescent cationic dye diS-C3(3) reports on cellular transmembrane potential has been investigated in murine haemopoietic cells. Due to the large molar absorbance of diS-C3(3) and its high quantum yield of fluorescence in cells, this dye can be used at very low labelling concentrations (5 x 10(-8) to 2 x 10(-7) M). In contrast to the quenching of fluorescence observed for the most commonly used voltage-sensitive dyes of the carbocyanine class, the fluorescence intensity of diS-C3(3) increases when the dye accumulates in the cells. The method of synchronous emission spectroscopy was used to resolve the intracellular and extracellular components of the diS-C3(3) fluorescence of suspensions of labelled cells. In comparing changes in these signals consequent on changes in transmembrane potential induced by varying the extracellular concentration of potassium ions in the presence of valinomycin, the logarithm of the ratio of intensities of these two components, as predicted theoretically, was found to be a good linear measure of transmembrane potential under these conditions. The dye was also demonstrated to be suitable for flow-cytofluorimetric analysis, the logarithm of the mean population signal similarly being found to provide a good linear measure of the transmembrane potential. The conditions under which such linearity may be expected with respect to possible effects due to changes in the capacity for binding of the dye to proteins and various cytosolic structures are delineated and their validity with respect to the possibly contentious role of mitochondria in such measurements examined in particular. The use of the method in indicating changes in the transmembrane potential and/or changes in the transport numbers of the major ions determining transmembrane potential between different physiological states, the possible extension to determinations of absolute differences in potential between different cell states without calibration or comparison with potassium-ion potentials, and the conditions for validity and limitations of these partially complementary measurements, are discussed.
    • Transmission FT-IR chemical imaging on glass substrates: applications in infrared spectral histopathology.

      Bassan, P; Mellor, J; Shapiro, J; Williams, K; Lisanti, Michael P; Gardner, P; Manchester Institute of Biotechnology, University of Manchester , 131 Princess Street, Manchester (2014-02-04)
      Fourier transform-infrared (FT-IR) chemical imaging in transmission mode has traditionally been performed on expensive mid-IR transparent windows such as barium/calcium fluoride, which are more fragile than glass, making preparation in the histopathology laboratories more cumbersome. A solution is presented here by using cheap glass substrates for the FT-IR chemical imaging, which has a high-wavenumber transmission window allowing measurement of the C-H, N-H, and O-H stretches occurring at ca. 2500-3800 cm(-1). The "fingerprint" region of the IR spectrum occurring below 1800 cm(-1) is not obtainable; however, we demonstrate that a wealth of information is contained in the high wavenumber range using 71 patients on a breast tissue microarray (TMA) as a model for investigation. Importantly, we demonstrate that the tissue can be classified into four basic tissue cell types and that using just the epithelial cells, reasonable discrimination of normal and malignant tissue can be found.
    • Transplantation of cultured small bowel enterocytes.

      Campbell, F C; Tait, I S; Flint, Neil; Evans, Gareth S; Department of Surgery, University of Dundee. (1993-09)
    • Transplantation potential of hematopoietic cells released into the circulation during routine chemotherapy for non-Hodgkin's lymphoma.

      Pettengell, Ruth; Testa, Nydia G; Swindell, Ric; Crowther, Derek; Dexter, T Michael; Department of Experimental Haematology, Paterson Institute for Cancer Research and Christie Hospital, Manchester, UK. (1993-10-01)
      Primitive hematopoietic cells released into the peripheral blood (PB) were studied in 50 patients with high-grade non-Hodgkin's lymphoma enrolled in a phase III trial of intensive weekly chemotherapy (VAPEC-B) alone or with granulocyte colony-stimulating factor (G-CSF). Mononuclear cells numbers were monitored and their in vitro growth potential assessed in clonogenic progenitor cell assays and in long-term culture. Total colony-forming cells (granulocyte-macrophage [GM], burst-forming unit, erythroid [BFU-E], Mix-CFC) were increased 40-fold (median) over baseline with chemotherapy alone and 106-fold with chemotherapy and G-CSF after the final dose. CD34+ cells were increased to a median of 4%, equivalent to that in normal bone marrow (BM) controls. Circulating colony-forming cell levels were maximal when the recovering total white blood cell (WBC) count reached 5 to 10 x 10(9)/L. The timing of the maximum was reproducible in individual patients. Therefore the WBC count can be used as a guide to the timing of leukapheresis. PB cells from normal controls' and patients' prechemotherapy were unable to sustain hemopoiesis in two-stage long-term cultures. In contrast, PB cells collected from patients primed with chemotherapy alone or chemotherapy with G-CSF at the time of predicted maximal colony-forming cell release were able to generate and sustain hematopoiesis in long-term cultures at a level comparable or superior to normal BM. These findings indicate that the use of G-CSF after routine outpatient chemotherapy stimulates maximal release of primitive hemopoietic cells into the circulation, including colony-forming cells and long-term culture-initiating cells. Their numbers are comparable with those in normal BM and are such that a single leukapheresis will usually yield enough cells for hemopoietic reconstitution after myeloablative chemotherapy.
    • Transplantation potential of peripheral blood stem cells induced by granulocyte colony-stimulating factor.

      Molineux, Graham; Pojda, Z; Hampson, Ian N; Lord, Brian I; Dexter, T Michael; Department of Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute, Manchester, UK. (1990-11-15)
      The major effect of granulocyte colony-stimulating factor (G-CSF) is to induce neutrophilia in previously untreated animals or after chemotherapy or marrow transplantation in humans, primates and rodents. In addition, it has been reported that migration of committed progenitor cells to the blood occurs during G-CSF therapy. In this article, by using sex mismatched transplants and a molecular probe for Y-chromosome specific DNA sequences, we show that among the peripheral blood cells during G-CSF therapy are substantial numbers of primitive stem cells capable of (1) reconstituting the hematopoietic system in the long term, and (2) making a contribution to the lymphoid populations of the thymus, in radiation ablated recipients. These data suggest that blood from patients treated with G-CSF may provide a convenient source of the most primitive stem cells for autologous or allogeneic bone marrow transplantation.
    • Transverse location of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene in model lipid bilayer membrane systems by resonance excitation energy transfer.

      Davenport, L; Dale, Robert E; Bisby, R; Cundall, R (1985-07-16)
      A fluorescent phospholipid derivative, the fluoresceinthiocarbamyl adduct of a natural phosphatidylethanolamine, has been synthesized and incorporated into sonicated single-bilayer vesicles of egg lecithin and dipalmitoyllecithin. The surface location of this probe has been confirmed by using extrinsic fluorescence quenching studies together with steady-state emission anisotropy measurements. Electronic excitation energy transfer between 1,6-diphenyl-1,3,5-hexatriene incorporated within the hydrophobic core of the bilayer and the novel derivative has been investigated to estimate the depth within the bilayer at which the former is located. Efficiencies have been measured for two different phospholipids, egg lecithin and dipalmitoyllecithin, in the latter case both above and below the phospholipid phase transition, with and without added cholesterol. The observed dependence of the transfer efficiency on the acceptor concentration was compared with that calculated according to Förster theory applied to random two-dimensional distributions of donor and acceptor molecules in parallel planes for various interplanar separations, taking into account orientational effects. The Förster R0 of about 45 A for this donor-acceptor pair is particularly well suited to such studies since it is of the order of the width of the bilayer. The experiments showed that energy-transfer spectroscopy can provide useful quantitative information as to the transverse location of diphenylhexatriene in homogeneous phospholipid bilayers and may also reflect lateral partitioning of donor or of both donor and acceptor into different phases in systems exhibiting phase separations.