Human hepatitis delta virus: an agent with similarities to certain satellite RNAs of plants.
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Animals↗
Biomedical subjects
J M Taylor
Publications and source records attributed to J M Taylor.
The effect of heterogeneity in tumor cell kinetics on radiation dose-response. An exploratory investigation of a plateau effect.
PURPOSE: To investigate the effect of heterogeneity in tumor cell kinetics on radiation dose-response curves for a population of patients. MATERIALS AND METHODS: A series of exploratory calculations have been performed using an improved geometric-stochastic model of tumor cure. RESULTS: Radiation therapy dose-response curves may plateau, or nearly so, at tumor control levels well below 100%, if a proportion of tumors would grow sufficiently fast to counterbalance the effect of fractionated radiotherapy. If the model assumptions of doubling time heterogeneity are correct, the difference between a short and protracted radiation regimen would be not only in the position and steepness of the radiation dose-response curve, but also in the level of the predicted plateau. CONCLUSIONS: For a given rate of dose accumulation, the one-sided flattening in dose-response curves at high doses is predicted from the modeling, and determined by the proportion of most radioresistant and rapidly growing tumors. This shows that empirical models of tumor control probability which assume a symmetric sigmoid relationship from 0 to 100% have apparent limitations, seemingly not well acknowledged in the literature.
Algorithms↗
Stability of plasma levels of cytokines and soluble activation markers in patients with human immunodeficiency virus infection.
Cytokine and immune activation marker levels in plasma are valuable measurements of immune status and treatment effects in human immunodeficiency virus (HIV) infection and AIDS. Five populations representing various stages of disease were studied: controls, 2 AIDS groups with <50/mm3 CD4 cells, and 2 groups of HIV-positive subjects-1 with stable CD4 T cells (median, 545/mm3) and 1 with >100/mm3 CD4 cell decline in 1 year. Relatively stable levels of tumor necrosis factor (TNF)-alpha, soluble TNF receptor (R)II, soluble interleukin-2R, neopterin, and beta2-microglobulin (beta2M) were documented over 5-8 weeks in patients with AIDS and for 1-4 years in the other groups. beta2M was generally the most stable marker. Interferon-gamma levels, however, fluctuated substantially. Individuals, whether normal or HIV-positive, maintain characteristic plasma levels of cytokines and immune activation markers. Thus, documented changes, in excess of the variability observed in this study, are likely to be significant indicators of change in disease status or effects of therapy.
Adult↗
Overexpression of human hepatic lipase and ApoE in transgenic rabbits attenuates response to dietary cholesterol and alters lipoprotein subclass distributions.
The effect of the expression of human hepatic lipase (HL) or human apoE on plasma lipoproteins in transgenic rabbits in response to dietary cholesterol was compared with the response of nontransgenic control rabbits. Supplementation of a chow diet with 0.3% cholesterol and 3.0% soybean oil for 10 weeks resulted in markedly increased levels of plasma cholesterol and VLDL and IDL in control rabbits as expected. Expression of either HL or apoE reduced plasma cholesterol response by 75% and 60%, respectively. The HL transgenic rabbits had substantial reductions in medium and small VLDL and IDL fractions but not in larger VLDL. LDL levels were also reduced, with a shift from larger, more buoyant to smaller, denser particles. In contrast, apoE transgenic rabbits had a marked reduction in the levels of large VLDLs, with a selective accumulation of IDLs and large buoyant LDLs. Combined expression of apoE and HL led to dramatic reductions of total cholesterol (85% versus controls) and of total VLDL+IDL+LDL (87% versus controls). HDL subclasses were remodeled by the expression of either transgene and accompanied by a decrease in HDL cholesterol compared with controls. HL expression reduced all subclasses except for HDL2b and HDL2a, and expression of apoE reduced large HDL1 and HDL2b. Extreme HDL reductions (92% versus controls) were observed in the combined HL+apoE transgenic rabbits. These results demonstrate that human HL and apoE have complementary and synergistic functions in plasma cholesterol and lipoprotein metabolism.
Animals↗
Cytoskeletal changes induced by GRAF, the GTPase regulator associated with focal adhesion kinase, are mediated by Rho.
Graf, the GTPase regulator associated with focal adhesion kinase was previously shown to have GAP activity for &Rgr; A and Cdc42 in vitro (Hildebrand et al 1996 Mol. Cell Biol. 16: 3169-3178). In this study we sought to determine whether Graf acted at the level of Cdc42, Rho, or both in vivo and whether Graf was a signal terminator or transducer for these proteins. Microinjection of Graf cDNA into subconfluent Swiss 3T3 cells (in the presence of serum) has marked effects on cell shape and actin localization. Graf expression causes clearing of stress fibers followed by formation of long actin based filopodial-like extensions. Similar phenotypes were observed following injection of the Rho-inhibitor, C3 into these cells. The Graf response was dependent on GAP activity, since injection of Graf cDNA containing point mutations in the GAP domain (R236Q or N351V) which block enzymatic activity, does not confer this phenotype. Injection of Graf into Swiss 3T3 cells in which Rho has been down-regulated by serum starvation has no effect on cell morphology. Using this system, we demonstrate that Graf blocks sphingosine-1-phosphate (SPP) stimulated (Rho-mediated) stress fiber formation. Conversely, Graf expression does not inhibit bradykinin stimulated (Cdc42-mediated) filopodial extensions. These data indicate that Graf is a GAP for Rho in vivo. To further substantiate these results we examined the effect of Graf over-expression on Rho-mediated neurite retraction in nerve growth factor (NGF)-differentiated PC12 cells. In PC12 cells, which express relatively high levels of endogenous Graf, overexpression of Graf (but not Graf containing the R236Q mutation) enhances SPP-induced neurite retraction. These data indicate the possibility that Graf may be an effector for Rho in certain cell types.
3T3 Cells↗
Rapid growth of microscopic rectal cancer as a determinant of response to preoperative radiation therapy.
PURPOSE: To quantify the dose-time fractionation factors in preoperative radiation therapy for microscopic pelvic deposits of rectal cancer. This provides a biologic basis for understanding and improving the results of adjuvant therapies for this disease. METHODS: The reduction in incidence of pelvic relapses as a function of radiation dose and overall treatment time was determined from the literature. The displacement of dose-response curves to higher doses reflects the growth during radiation treatment of subclinical pelvic deposits which are beyond the future surgical margins. RESULTS: Dose-response curves are steep if the effect of overall duration of radiation therapy is accounted for. The time-related displacement of these steep dose-response curves is consistent with a median doubling time for malignant clonogenic cells of about 4 or 5 days, much faster than the growth rate of the average primary tumor at diagnosis. This rapid growth is evident within the first few days of irradiation, implying that the natural growth rate of these microscopic deposits if fast, and/or that an acceleration of growth follows initiation of radiation injury with a very short lag time. CONCLUSION: Subclinical pelvic deposits of rectal cancer grow rapidly during preoperative radiation therapy with an adverse influence on the rate of pelvic tumor control from protracting the duration of adjuvant treatment. Low doses only offer clinically relevant reduction in risk of pelvic relapses if the overall radiation treatment time is short. For a given overall treatment duration there is a relatively steep dose-response curve, predicting that significant improvements in tumor control are possible.
Dose Fractionation, Radiation↗
Statistical issues for HIV surrogate endpoints: point/counterpoint. An NIAID workshop.
This paper summarizes the proceedings of an NIAID-sponsored workshop on statistical issues for HIV surrogate endpoints. The workshop brought together statisticians and clinicians in an attempt to shed light on some unresolved issues in the use of HIV laboratory markers (such as HIV RNA and CD4+ cell counts) in the design and analysis of clinical studies and in patient management. Utilizing a debate format, the workshop explored a series of specific questions dealing with the relationship between markers and clinical endpoints, and the choice of endpoints and methods of analysis in clinical studies. This paper provides the position statements from the two debaters on each issue. Consensus conclusions, based on the presentations and discussion, are outlined. While not providing final answers, we hope that these discussions have helped clarify a number of issues, and will stimulate further consideration of some of the highlighted problems. These issues will be critical in the proper assessment and use of future therapies for HIV disease.
Biomarkers↗
Does the covariance structure matter in longitudinal modelling for the prediction of future CD4 counts?
We investigate the importance of the assumed covariance structure for longitudinal modelling of CD4 counts. We examine how individual predictions of future CD4 counts are affected by the covariance structure. We consider four covariance structures: one based on an integrated Ornstein-Uhlenbeck stochastic process; one based on Brownian motion, and two derived from standard linear and quadratic random-effects models. Using data from the Multicenter AIDS Cohort Study and from a simulation study, we show that there is a noticeable deterioration in the coverage rate of confidence intervals if we assume the wrong covariance. There is also a loss in efficiency. The quadratic random-effects model is found to be the best in terms of correctly calibrated prediction intervals, but is substantially less efficient than the others. Incorrectly specifying the covariance structure as linear random effects gives too narrow prediction intervals with poor coverage rates. Fitting using the model based on the integrated Ornstein-Uhlenbeck stochastic process is the preferred one of the four considered because of its efficiency and robustness properties. We also use the difference between the future predicted and observed CD4 counts to assess an appropriate transformation of CD4 counts; a fourth root, cube root and square root all appear reasonable choices.
Acquired Immunodeficiency Syndrome↗
Overexpression and accumulation of apolipoprotein E as a cause of hypertriglyceridemia.
The molecular mechanisms of hypertriglyceridemia (HTG), a common lipid metabolic disorder in humans, often of genetic origin, are not well understood. In studying the effect of apolipoprotein (apo) E on the metabolism of triglyceride-rich lipoproteins, we found that expressing high plasma levels of human apoE3 in transgenic mice lacking endogenous mouse apoE caused HTG. These transgenic animals had 3-fold higher plasma triglyceride levels, higher very low density lipoproteins (VLDL), and lower high density lipoproteins than did nontransgenics. Removing one or both low density lipoprotein receptor alleles in the apoE3-overexpressing mice caused severe HTG (8-11-fold over nontransgenics) and increased VLDL and decreased low and high density lipoproteins, and apoE3-enriched VLDL were markedly depleted in apoC-II. At least two mechanisms could explain HTG associated with apoE3 overexpression: stimulated VLDL triglyceride production and impaired VLDL lipolysis. The apoE3 mice with HTG had a 50% increase in hepatic VLDL triglyceride production. Furthermore, overexpression of apoE (E2, E3, or E4) in cultured hepatocytes (McA-RH7777 cells) correlated positively with secretion of VLDL into the medium. However, apoE3 overexpression-associated HTG was only partially explained by VLDL overproduction, as lipoprotein lipase-mediated VLDL lipolysis was also decreased 20-86% depending on apoE3 levels, most likely by displacing or masking apoC-II on the particles. In human subjects, HTG correlated positively with increased VLDL triglyceride and plasma and VLDL apoE levels. However, plasma and VLDL apoE correlated negatively with VLDL apoC-II levels and lipoprotein lipase-mediated VLDL lipolysis. Thus, optimal expression of apoE is crucial for normal metabolism of triglyceride-rich lipoproteins, and overexpression and/or accumulation of apoE may contribute to HTG by stimulating VLDL triglyceride production and by impairing VLDL lipolysis. The apoE3-overexpressing mice will be useful for studying the pathophysiology of this disorder.
Animals↗
Prognostic significance of plasma markers of immune activation, HIV viral load and CD4 T-cell measurements.
OBJECTIVE: To evaluate the prognostic significance for AIDS occurrence of plasma levels of immune activation markers in comparison with and in conjunction with HIV viral load and CD4 T-cell measurements. DESIGN: A retrospective analysis was conducted of three plasma activation markers, the soluble tumor necrosis factor (TNF) receptor II (TNF-RII), neopterin and soluble interleukin-2 receptor levels, and of CD4 T-cell levels and plasma HIV viral load. SUBJECTS: The participants were 659 men taking part in the University of California Los Angeles Multicenter AIDS Cohort Study who were HIV-seropositive but AIDS-free in 1985. MAIN OUTCOME MEASURE: Clinically defined AIDS within 3 years. Failure time statistical regression models for the time to development of AIDS were used to assess prognostic capacity of the parameters alone and in combination. RESULTS: All the markers had prognostic capability. The levels of the three plasma activation markers correlated well with each other (median r = 0.61). They related less well with HIV RNA plasma levels (median r = 0.50) and least well with CD4 cell levels (median r = 0.36). Furthermore, plasma marker levels were shown to be able to stratify patients for prognosis within all the major categories of CD4 T-cell and HIV RNA levels. CONCLUSIONS: Plasma levels of soluble TNF-RII and other soluble markers of immune activation have prognostic capabilities which are different from HIV and CD4 T-cell levels. Combination of a single plasma activation marker measurement (such as soluble TNF-RII) with CD4 T-cell levels improved the prognostic capability of each. A new graphic technique for presenting prognostic capability indicated that plasma soluble TNF-RII and CD4 cell levels are better prognostic factors than HIV plasma level with CD4 cells < 200 x 10(6)/l. Inexpensive tests for one of the plasma activation markers, such as soluble TNF-RII or neopterin, can be useful for evaluations of HIV disease course, especially when expensive equipment, technical expertise and funding required for flow cytometry and for HIV load measurements are not readily available.
Antigens, CD↗
Accelerated changes (inflection points) in levels of serum immune activation markers and CD4+ and CD8+ T cells prior to AIDS onset.
The trajectories of change in CD4+ and CD8+ lymphocytes and serum neopterin and beta2-microglobulin (beta2M) levels were determined in 158 HIV-seropositive individuals during 5.5 years before a clinical AIDS diagnosis. Each patient was evaluated separately using a two-piece regression model with seven possible change points to identify any adverse change (inflection point) in the slopes of each immunologic marker of HIV infection. Two categories of subjects were distinguished for each marker--those with statistically significant inflection points and those who demonstrated a steady progression of changes to AIDS. Fifty-nine percent had an inflection point for CD4+ T cells. The frequency of inflection points for CD8+ was 49%, for serum neopterin -48% and for beta2M -38%. Inflection points were found over a 4-year span. Three distinctive categories of inflection points were observed on the basis of their independent occurrence: one was in CD4+ T cells, another in CD8+ T cells, and a third in the serum markers of immune activation. The inflection point for CD4+ usually occurred prior to those for CD8+ T cells (p=.0002). The HIV-positive persons with inflection points were diagnosed with AIDS when immunologic parameters were significantly more abnormal than in those with steady progression (p < .0003). Thus, these two groups differed in the course of immune changes and in the levels of immune abnormalities associated with the occurrence of clinical AIDS.
Acquired Immunodeficiency Syndrome↗
Increased expression of apolipoprotein E in transgenic rabbits results in reduced levels of very low density lipoproteins and an accumulation of low density lipoproteins in plasma.
Transgenic rabbits expressing human apo E3 were generated to investigate mechanisms by which apo E modulates plasma lipoprotein metabolism. Compared with nontransgenic littermates expressing approximately 3 mg/dl of endogenous rabbit apo E, male transgenic rabbits expressing approximately 13 mg/dl of human apo E had a 35% decrease in total plasma triglycerides that was due to a reduction in VLDL levels and an absence of large VLDL. With its greater content of apo E, transgenic VLDL had an increased binding affinity for the LDL receptor in vitro, and injected chylomicrons were cleared more rapidly by the liver in transgenic rabbits. In contrast to triglyceride changes, transgenic rabbits had a 70% increase in plasma cholesterol levels due to an accumulation of LDL and apo E-rich HDL. Transgenic and control LDL had the same binding affinity for the LDL receptor. Both transgenic and control rabbits had similar LDL receptor levels, but intravenously injected human LDL were cleared more slowly in transgenic rabbits than in controls. Changes in lipoprotein lipolysis did not contribute to the accumulation of LDL or the reduction in VLDL levels. These observations suggest that the increased content of apo E3 on triglyceride-rich remnant lipoproteins in transgenic rabbits confers a greater affinity for cell surface receptors, thereby increasing remnant clearance from plasma. The apo E-rich large remnants appear to compete more effectively than LDL for receptor-mediated binding and clearance, resulting in delayed clearance and the accumulation of LDL in plasma.
Animals↗
Characterization of graf, the GTPase-activating protein for rho associated with focal adhesion kinase. Phosphorylation and possible regulation by mitogen-activated protein kinase.
Graf is a GTPase-activating protein for Rho that interacts with focal adhesion kinase and co-localizes with the actin cytoskeleton (Hildebrand, J. D., Taylor, J. M. and Parsons, J. T. (1996) Mol. Cell. Biol. 16, 3169-3178). We examined the expression and regulation of Graf as a prelude to understanding the role of Graf in mediating signal transduction in vivo. We demonstrated that Graf is a ubiquitously expressed 95-kDa protein with high levels observed in heart and brain and cells derived from these tissues. Stimulation of PC12 cells with epidermal growth factor or nerve growth factor induced a phosphatase-reversible mobility shift upon gel electrophoresis, indicative of phosphorylation. In vitro, purified mitogen-activated protein (MAP) kinase catalyzed the phosphorylation of Graf on serine 510, suggesting that Graf phosphorylation may be mediated through MAP kinase signaling. In addition, the mutation of serine 510 to alanine inhibited the epidermal growth factor-induced mobility shift of mutant Graf protein in vivo, consistent with serine 510 being the site of in vivo phosphorylation. Based on these data we suggest that phosphorylation of Graf by MAP kinase or related kinases may be a mechanism by which growth factor signaling modulates Rho-mediated cytoskeletal changes in PC12 and perhaps other cells.
Amino Acid Sequence↗
Atypical splicing of the latency-associated transcripts of herpes simplex type 1.
We previously have shown that two latency-associated transcripts (LATs) of herpes simplex type 1 (HSV-1) are probably lariats, produced during splicing. By RNaseH digestion analysis, we now show that the major branchpoint of the 2.0-kb LAT was within 46 nt 5' of the splice acceptor site. A more detailed mapping by primer extension revealed the branchpoint as an adenosine 29 nt 5' of the splice acceptor site. Introduction of two branchpoint sequences with good matches to the consensus at position -25 had no effect on the splicing efficiency but reduced the accumulation of the 2.0-kb LATs at least 90-fold. The second focus of our studies was the 1.5-kb LAT. It was not detected by Northern analyses in either productively infected or transfected cultured cells or even in cells of neuronal origin. However, it was detected in the trigeminal ganglia of mice experimentally infected with HSV-1 after 10 days. Moreover, its abundance relative to that of the 2.0-kb species increased 4-fold from 10 to 30 days after infection, consistent with an interpretation that the 1.5-kb species, once formed, was more stable than the 2.0-kb species.
Animals↗
Overexpression of hepatic lipase in transgenic mice decreases apolipoprotein B-containing and high density lipoproteins. Evidence that hepatic lipase acts as a ligand for lipoprotein uptake.
To determine the mechanisms by which human hepatic lipase (HL) contributes to the metabolism of apolipoprotein (apo) B-containing lipoproteins and high density lipoproteins (HDL) in vivo, we developed and characterized HL transgenic mice. HL was localized by immunohistochemistry to the liver and to the adrenal cortex. In hemizygous (hHLTg+/0) and homozygous (hHLTg+/+) mice, postheparin plasma HL activity increased by 25- and 50-fold and plasma cholesterol levels decreased by 80% and 85%, respectively. In mice fed a high fat, high cholesterol diet to increase endogenous apoB-containing lipoproteins, plasma cholesterol decreased 33% (hHLTg+/0) and 75% (hHLTg+/+). Both apoB-containing remnant lipoproteins and HDL were reduced. To extend this observation, the HL transgene was expressed in human apoB transgenic (huBTg) and apoE-deficient (apoE-/-) mice, both of which have high plasma levels of apoB-containing lipoproteins. (Note that the huBTg mice that were used in these studies were all hemizygous for the human apoB gene.) In both the huBTg,hHLTg+/0 mice and the apoE-/-,hHLTg+/0 mice, plasma cholesterol decreased by 50%. This decrease was reflected in both the apoB-containing and the HDL fractions. To determine if HL catalytic activity is required for these decreases, we expressed catalytically inactive HL (HL-CAT) in apoE-/- mice. The postheparin plasma HL activities were similar in the apoE-/- and the apoE-/-,HL-CAT+/0 mice, reflecting the activity of the endogenous mouse HL and confirming that the HL-CAT was catalytically inactive. However, the postheparin plasma HL activity was 20-fold higher in the apoE-/-,hHLTg+/0 mice, indicating expression of the active human HL. Immunoblotting demonstrated high levels of human HL in postheparin plasma of both apoE-/-,hHLTg+/0 and apoE-/-,HL-CAT+/0 mice. Plasma cholesterol and apoB-containing lipoprotein levels were approximately 60% lower in apoE-/-,HL-CAT+/0 mice than in apoE-/- mice. However, the HDL were only minimally reduced. Thus, the catalytic activity of HL is critical for its effects on HDL but not for its effects on apoB-containing lipoproteins. These results provide evidence that HL can act as a ligand to remove apoB-containing lipoproteins from plasma.
Animals↗
Modular domains of focal adhesion-associated proteins.
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Amino Acid Sequence↗
An evaluation of a measure of the proportion of the treatment effect explained by a surrogate marker.
Time-dependent markers, such as CD4 and viral load, are potential surrogate markers in AIDS clinical trials. A critical issue with surrogate markers is whether changes in these markers explain the beneficial effect of treatment on the real end point of the clinical trial. A statistic to measure the proportion of the treatment effect explained by the surrogate is p(FGS) = 1 - gamma/alpha, where alpha is the treatment effect coefficient in a Cox model and gamma is the treatment effect coefficient from a time-dependent Cox model adjusted for the marker. In this article we evaluate the statistical properties of p(FGS). Using a Monte Carlo study we show that the statistic is not well calibrated, because it can fall outside the range zero to one, even in very large samples. In the simulation study we consider situations where the time-dependent marker is measured with error at a fixed number of times. We show that a method of fitting a time-dependent Cox model involving smoothing the marker reduces the bias in the estimate of p(FGS) compared with the standard method of using the current or last observed marker value. We also show that the estimate of p(FGS) has considerable variability and can have wide confidence intervals. We conclude that p(FGS) is only likely to be useful in large trials with a strong treatment effect. The methods are illustrated using CD4 counts from an AIDS clinical trial of zidovidine versus placebo.
Acquired Immunodeficiency Syndrome↗
A non-parametric method of reconstructing single-dose survival curves from multi-fraction experiments.
PURPOSE: A method of estimating the single-dose curve from designed multifraction experiments is described and applied to three datasets. MATERIALS AND METHODS: The method, which is non-parametric and based on standard statistical regression techniques, can be used for functional endpoints which are either continuous or binary. The datasets are concerned with wound healing on mice, myelopathy in guinea pigs and spermatogenesis in mice. The results are compared with the results from fitting the linear quadratic model. The statistical methods of Bootstrapping and residual plots are illustrated. RESULTS: The method is based in part on an assumed statistical model, however, exact knowledge of the correct statistical model is not necessary to obtain an estimate of the shape of the single-dose survival curve. We find no good evidence from the reconstructed single-dose survival curve of an "induced repair" phenomena at low doses for the wound healing and spermatogenesis experiments. For the myelopathy experiment the data are consistent with the LQ model with a low alpha-beta ratio down to doses of at least 1.5 Gy per fraction. CONCLUSIONS: A robust statistical method of estimating the shape of the single-dose survival curve is demonstrated using standard statistical software on three datasets.
Animals↗