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C D Gregory

Publications and source records attributed to C D Gregory.

At least 19 recordsLinked to original sources
PubMed2000

Distinct role of follicular dendritic cells and T cells in the proliferation, differentiation, and apoptosis of a centroblast cell line, L3055.

Germinal center (GC) B cells undergo complex interactions with follicular dendritic cells (FDC) and T cells in the course of differentiation into memory B and plasma cells. To delineate the individual roles of FDC and T cells at each stage of GC B cell differentiation at the clonal level and to analyze the signals involved, we adopted a unique experimental model using an FDC line, HK, and a lymphoma cell line, L3055, that resembles centroblasts. A detailed phenotypic analysis revealed L3055 cells to be a clonal population originating from the GC. Like freshly isolated centroblasts, L3055 cells underwent spontaneous apoptosis when cultured in the absence of fresh FDC or HK cells. L3055 cells proliferated continuously in the presence of HK cells, while they differentiated into a population with the phenotype of centrocytes after stimulation with CD40 ligand (CD40L) and IL-4. The CD40L-stimulated L3055 cells underwent CD95-mediated apoptosis, which was reminiscent of the feature of CD40L-stimulated tonsillar GC B cells. In contrast to HK cells that did not protect L3055 cells from anti-Ig killing, CD40L plus IL-2, IL-4, and IL-10 prevented anti-Ig-induced apoptosis. These experimental results demonstrate a distinct function of FDC and activated T cells, in that FDC provide signals for rapid proliferation of centroblasts, whereas T cells confer signals for differentiation of centroblasts into centrocytes and resistance to B cell receptor-mediated apoptosis. T cells collaborate with FDC in the protection and expansion of the Ag-specific GC B cells.

Adolescent↗
PubMed1999

Macrophage recognition of ICAM-3 on apoptotic leukocytes.

Cells undergoing apoptosis are cleared rapidly by phagocytes, thus preventing tissue damage caused by loss of plasma membrane integrity. In this study, we show that the surface of leukocytes is altered during apoptosis such that the first Ig-like domain of ICAM-3 (CD50) can participate in the recognition and phagocytosis of the apoptotic cells by macrophages. Macrophage recognition of apoptotic cell-associated ICAM-3 was demonstrated both on leukocytes and, following transfection of exogenous ICAM-3, on nonleukocytes. The change in ICAM-3 was a consistent consequence of apoptosis triggered by various stimuli, suggesting that it occurs as part of a final common pathway of apoptosis. Alteration of ICAM-3 on apoptotic cells permitting recognition by macrophages resulted in a switch in ICAM-3-binding preference from the prototypic ICAM-3 counterreceptor, LFA-1, to an alternative macrophage receptor. Using mAbs to block macrophage/apoptotic cell interactions, we were unable to obtain evidence that either the alternative ICAM-3 counterreceptor alpha d beta 2 or the apoptotic cell receptor alpha v beta 3 was involved in the recognition of ICAM-3. By contrast, mAb blockade of macrophage CD14 inhibited ICAM-3-dependent recognition of apoptotic cells. These results show that ICAM-3 can function as a phagocytic marker of apoptotic leukocytes on which it acquires altered macrophage receptor-binding activity.

Antibodies, Monoclonal↗
PubMed1999

Lactate quantitation in a gerbil brain stroke model by GSLIM of multiple-quantum-filtered signals. Generalized spectral localization by imaging.

Quantitative magnetic resonance imaging of lactate using a zero-quantum/double-quantum filter and generalized spectral localization by imaging (GSLIM) was applied to a model of unilateral stroke in gerbil brain. GSLIM lactate images at 4T clearly reveal elevated concentrations of lactate in the ischemic compared with the normal hemisphere 100-175 minutes after unilateral carotid ligation. These results indicate that the technique is capable of studies of brain infarcts, and that application to human ischemic pathology in brain and other tissues may be possible.

Animals↗
PubMed1999

Minimal cross-linking and epitope requirements for CD40-dependent suppression of apoptosis contrast with those for promotion of the cell cycle and homotypic adhesions in human B cells.

Eight different CD40 mAb shared with soluble trimeric CD40 ligand (sCD40LT) the capacity to rescue germinal center (GC) B cells from spontaneous apoptosis and to suppress antigen receptor-driven apoptosis in group I Burkitt's lymphoma cells. Three mAb (G28-5, M2 and M3) mimicked sCD40LT in its ability to promote strong homotypic adhesion in resting B cells, whereas others (EA5, BL-OGY/C4 and 5C3) failed to stimulate strong clustering. Binding studies revealed that only those mAb that promoted strong B cell clustering bound at, or near to, the CD40L binding site. While all eight mAb and sCD40LT were capable of synergizing with IL-4 or phorbol ester for promoting DNA synthesis in resting B cells, co-stimulus-independent activation of the cells into cycle through CD40 related directly to the extent of receptor cross-linking. Thus, mAb which bound outside the CD40L binding site synergized with sCD40LT for promoting DNA synthesis; maximal levels of stimulation were achieved by presenting any of the mAb on CD32 transfectants in the absence of sCD40LT or by cross-linking bound sCD40LT with a second antibody. Monomeric sCD40L, which was able to promote rescue of GC B cells from apoptosis, was unable to drive resting B cells into cycle. These studies demonstrate that CD40-dependent rescue of human B cells from apoptosis requires minimal cross-linking and is essentially epitope independent, whereas the requirements for promoting cell cycle progression and homotypic adhesion are more stringent. Possible mechanisms underlying these differences and their physiological significance are discussed.

Apoptosis↗
PubMed1998

Prolonged phenotypic, functional, and molecular change in group I Burkitt lymphoma cells on short-term exposure to CD40 ligand.

Group I Burkitt lymphoma (BL) cell lines (L3055, Elijah, Louckes, BL2, and BL29) retaining the original biopsy phenotype were found to undergo prolonged phenotypic, functional, and molecular change on short-term exposure to soluble recombinant CD40L trimer. Sensitivity to, extent of, and duration of change appeared to reflect passage number in that the earliest passaged lines, L3055 and BL29, were generally the most susceptible. Culture of group I BL lines with CD40L resulted in significant growth arrest (without apoptosis) that, for L3055 cells, was sustained for 7 to 9 days after 72 hours of exposure. This was accompanied by the formation of large homotypic aggregates together with gross changes in individual cell morphology and a concomitant upregulation of CD54 (ICAM-1). Three of the five group I BL lines exhibited rapid downregulation of the hallmark CD77 surface antigen, which, for L3055 cells, was maintained for at least 12 days after 72 hours of incubation with CD40L. With the exception of BL2, all group I BL lines were induced to express CD40 homodimers on CD40-stimulation, whereas only monomers were detected in unstimulated cells. Experiments using CD40-transfected Rat-1 fibroblasts showed that the ability to signal for dimer formation required Thr234 of CD40. For L3055 and BL29 cells, an initial 72 hours of exposure to CD40L resulted in the maintenance of homodimers for up to 14 and 10 days, respectively. There was a close correlation between the induction and duration of CD40 homodimers and the appearance of Bcl-2. For L3055 cells, which are sensitive to apoptosis-induction on BCR-engagement, exposure to CD40L for 72 hours was found to provide considerable protection from anti-IgM, which was still significant to 20 days. The implications of such sustained effects on relatively short-term exposure of tumor B cells to CD40L are discussed.

Antibodies, Anti-Idiotypic↗
PubMed1998

Human cells arrest in S phase in response to adenovirus 12 E1A.

It has previously been shown that following viral infection, Ad5 E1A induces cell cycle progression of quiescent rodent cells, leading to DNA synthesis and mitosis. Here we have examined the effect of Ad12 E1A on the cell cycle characteristics of human cells. Human tumor (A549, KB, and HeLa) cells were infected with Ad12 d/620, a mutant virus which has a lesion in the E1B gene and essentially expresses only E1A. These infected cells progressed from being largely in G1 into S phase, where they arrested. Even up to 96 h postinfection (p.i.) the cells remained blocked in S phase. DNA synthesis did, however, proceed in Ad12 d/620-infected cells, giving rise to multiple copies of cellular DNA. Similar results were obtained when primary human skin fibroblasts were infected, although the polyploidy was less marked. The expression of cyclins A, B1, and E in the tumor cells increased appreciably in response to E1A. In contrast, there was a dramatic reduction in the levels of cyclin D1 and D3. Increases in cyclin D1 expression could be detected at very late times p.i. In those cell lines expressing low levels of cdc2 and cdk2 an appreciable increase in expression was seen soon after Ad12 E1A could be detected. The elevated levels of cyclins A, B1, and E were associated with increased protein kinase activity directed against histone H1. An increase in cyclin D1-associated kinase activity against Rb1 was also observed at late times. This deregulation of the cell cycle was not solely dependent on E1A inactivation of Rb, since similar effects were seen in Ad12 d/620-infected retinoblastoma (Y-79) cells, implicating p107 and p130 in E1A-mediated changes in cell cycle progression. We propose that the E1A-induced levels of cyclins A, B1, and E by Ad12 E1A in human cells may lead to an uncoupling of S phase from cell cycle progression.

Adenoviridae↗
PubMed1998

Homology between a human apoptosis specific protein and the product of APG5, a gene involved in autophagy in yeast.

Apoptosis specific proteins (ASP) are expressed in the cytoplasm of cultured mammalian cells of various lineages following induction of apoptosis. The cDNA encoding ASP has been cloned from a human expression library and has significant homology to the Saccharomyces cerevisiae APG5 gene which is essential for yeast autophagy. The ASP gene, known as hAPG5, can be transcribed to give mRNAs of 3.3 kbp, 2.5 kbp and 1.8 kbp which are present at comparable levels in viable and apoptotic cells, demonstrating that protein expression must be regulated at the translational level. These data indicate a possible relationship between apoptosis and autophagy and suggest evolutionary conservation in mammalian apoptosis of a degradative process present in yeast.

Animals↗
PubMed1998

Human CD14 mediates recognition and phagocytosis of apoptotic cells.

Cells undergoing programmed cell death (apoptosis) are cleared rapidly in vivo by phagocytes without inducing inflammation. Here we show that the glycosylphosphatidylinositol-linked plasma-membrane glycoprotein CD14 on the surface of human macrophages is important for the recognition and clearance of apoptotic cells. CD14 can also act as a receptor that binds bacterial lipopolysaccharide (LPS), triggering inflammatory responses. Overstimulation of CD14 by LPS can cause the often fatal toxic-shock syndrome. Here we show that apoptotic cells interact with CD14, triggering phagocytosis of the apoptotic cells. This interaction depends on a region of CD14 that is identical to, or at least closely associated with, a region known to bind LPS. However, apoptotic cells, unlike LPS, do not provoke the release of pro-inflammatory cytokines from macrophages. These results indicate that clearance of apoptotic cells is mediated by a receptor whose interactions with 'non-self' components (LPS) and 'self' components (apoptotic cells) produce distinct macrophage responses.

Animals↗
PubMed1998

Offset-dependent partial saturation in binomial solvent suppression sequences.

In vivo spectroscopic experiments are commonly done under partially T1 saturated conditions and saturation factors (observed signal intensity divided by fully relaxed intensity) used for quantitative analysis of absolute and relative metabolite concentrations. The conventional calculation of the saturation factor assumes uniform excitation across the spectrum. This assumption is violated when selective-excitation solvent peak suppression techniques, such as binomial sequences, are used. In this case, the degree of partial saturation, and hence the saturation factor, is dependent on the resonance offset. This has implications for the frequency offset of maximum excitation, solvent suppression effectiveness, absolute and relative quantitative measurements, and progressive saturation T1 measurements. In this paper, the jump and return (JR) and jump and return echo (JRE) sequences are examined in detail with regard to these implications. It is shown that offset-dependent partial saturation depends on the sequence used and can become significant. A saturation factor which is a function of offset can be used to correct for this effect and allow for proper quantitative interpretation of experimental results under partially saturated conditions.

Algorithms↗
PubMed1997

Mechanisms of antigen receptor-dependent apoptosis of human B lymphoma cells probed with a panel of 27 monoclonal antibodies.

The present study has used a panel of 23 monoclonal antibodies to IgM and 4 to IgD in order to probe parameters influencing sIg-dependent apoptosis in an IgM/IgD-expressing Burkitt lymphoma line. No direct correlation was observed between the capacity of the different anti-mu to drive cells into apoptosis and either their domain specificity or their affinity for sIgM. There was, however, a direct correlation between the functional outcome and the ability of the monoclonal antibodies to elicit a rise in intracellular Ca2+. For apoptosis to occur, the Ca2+ response had to attain a threshold value of approximately 100 nM. A direct role for Ca2+ in the delivery of the apoptotic signal was demonstrated using thapsigargin to raise intracellular Ca2+ levels. Antigen receptor ligation was linked to Ca2+ increases by tyrosine kinases as revealed by direct analysis of protein tyrosine phosphorylation and the effects of selective protein tyrosine kinase-inhibiting tyrphostins. These findings reveal a central role for the antigen receptor-generated Ca2+ signal in driving apoptosis in human B lymphoma cells and stresses the need to use a panel of reagents when probing function with presumed ligand-mimetic monoclonal antibodies.

Antibodies, Monoclonal↗
PubMed1997

Differential effects of BCL-2 on survival and proliferation of human B-lymphoma cells following gamma-irradiation.

Bcl-2 can inhibit apoptosis induced by a variety of stimuli, including radiation and its presence in tumour cells would be expected to indicate poor prognosis. Bcl-2-expressing tumours, however, are often low-grade and highly responsive to therapy. To investigate this apparent paradox, we analysed in vitro the responses of Burkitt lymphoma (BL) cells to gamma-irradiation in the presence and absence of Bcl-2. High-level expression of Bcl-2 was shown to promote BL cell survival following irradiation. However, a significant proportion of Bcl-2-rescued cells subsequently underwent apoptosis after an extended period in culture. In addition, in different BL lines, Bcl-2 was found either to promote or to inhibit long-term proliferative activity following gamma-irradiation. This differential regulation of proliferation correlated both with differential effects of Bcl-2 on the cell cycle and with differences in p53 status. Thus, by one week after irradiation, BL cells expressing only wild-type p53 (wt/wt) had arrested in G1, whereas those with a mutant allele (wt/mu) were arrested in all phases of the cell cycle. The proportion of Bcl-2-rescued cells that subsequently underwent apoptosis was reduced by ligation of CD40 at the time of irradiation in wt/wt BL cells, but not in wt/mu cells. CD40-ligation reduced both G1-arrest and apoptosis in parallel. These results indicate that, whilst Bcl-2 can delay apoptosis in BL cells following gamma-irradiation, the protein can also cause growth-arrest and thereby promote apoptosis. Long-term survival following Bcl-2-mediated rescue of gamma-irradiated cells may depend on p53 status and require additional death-repressing or growth-promoting signals.

CD40 Antigens↗
PubMed1997

Biochemical heterogeneity in hysterectomized uterus measured by 31P NMR using SLIM localization.

Ultrasound and magnetic resonance imaging show contrast between the inner and outer myometrium, which is useful in the diagnosis of gynecological disorders. To determine whether the image contrast is associated with biochemical differences between these myometrial regions, phosphorus metabolite concentrations in the inner one third of the myometrium (the junctional zone; JZ) were compared with the outermost one third of the myometrium (OM) in hysterectomized uteri using 31P spectral localization by imaging (SLIM). The technique was validated by comparing the results of SLIM with the results of standard Fourier-encoded spectroscopic imaging (FSI) analysis using phantoms, and by nonlocalized spectroscopy on biopsies taken from the same hysterectomy specimens. As expected theoretically, SLIM yielded better localization than FSI, as judged by spectral intensity and leakage measurements on phantom compartments of known composition. SLIM localization revealed that the JZ has a higher intracellular phosphomonoester (PME) concentration than does the OM, which was confirmed by nonlocalized spectroscopy, and that there is very little NMR-visible phosphorus in the cervix.

Adult↗
PubMed1997

Quantitative lactate-specific MR imaging and 1H spectroscopy of skeletal muscle at macroscopic and microscopic resolutions using a zero-quantum/double-quantum coherence filter and SLIM/GSLIM localization.

Quantitative lactate imaging and spectroscopy were performed on phantoms and on electrically stimulated, excised frog skeletal muscle at macroscopic and microscopic resolutions. Lactate selectivity was achieved by use of a zero-quantum/double-quantum coherence (ZQC/DQC) lactate filter, which suppressed all signals besides lactate, including water and lipid, to below noise level. Three-dimensional lactate data sets were acquired in 1-3 h; one of these spatial dimensions was frequency-encoded and the other two were phase-encoded. High-resolution images were reconstructed using the spectral localization by imaging (SLIM) and generalized SLIM (GSLIM) techniques. Lactate quantitation was achieved by employing an external lactate concentration standard and was verified by comparison to quantitative STEAM-localized and nonlocalized spectra that used total creatine as an internal concentration reference. Additionally, quantitatively accurate behavior of the SLIM and GSLIM techniques as applied to data sets of low signal-to-noise ratio and to macroscopically heterogeneous objects was verified using simulations and real muscle lactate data sets with known heterogeneity.

Animals↗
PubMed1997

Projection-reconstruction spectroscopic imaging for B0 field plotting and shimming without pulsed gradients.

Shimming is important. Noniterative methods are desirable. Such methods exist for shimming a spectrometer with pulsed field gradients, generally based on field maps made by spin-warp Fourier imaging. For spectrometers with no pulsed gradients (or for cases where T2 is too short to permit echo imaging), an alternative method is presented: projection-reconstruction spectroscopic imaging, which can be accomplished using only the shim coils of a conventional spectrometer. Images so acquired can be used to map the field, even in the presence of multiple spectral components. Noniterative optimization of the axial shims of a GN-300 spectrometer is demonstrated using 1D + 1D spectroscopic images. Prospects for extending the technique to include the radial shims using 3D + 1D spectroscopic images are discussed.

Fourier Analysis↗