CD95 apoptosis resistance in certain cells can be overcome by noncanonical activation of caspase-8.

CD95 apoptosis resistance of tumor cells is often acquired through mutations in the death domain (DD) of one of the CD95 alleles. Furthermore, Type I cancer cells are resistant to induction of apoptosis by soluble CD95 ligand (CD95L), which does not induce efficient formation of the death-inducing signaling complex (DISC). Here, we report that tumor cells expressing a CD95 allele that lacks a functional DD, splenocytes from heterozygous lpr(cg) mice, which express one mutated CD95 allele, and Type I tumor cells stimulated with soluble CD95L can all die through CD95 when protein synthesis or nuclear factor kappa B is inhibited. This noncanonical form of CD95-mediated apoptosis is dependent on the enzymatic activity of procaspase-8 but does not involve fully processed active caspase-8 subunits. Our data suggest that it is possible to overcome the CD95 apoptosis resistance of many tumor cells that do not efficiently form a DISC through noncanonical activation of the caspase-8 proenzyme.

pQCT provides better prediction of canine femur breaking load than does DXA.

Our study was designed to examine the validity of dual energy X-ray absorptiometry (DXA) and peripheral quantitative computed tomography (pQCT) measurements as predictors of whole bone breaking strength in beagle femora. DXA was used to determine the bone mineral content, bone area, and 'areal' bone mineral density. PQCT was used to determine the cross-sectional moments of inertia, volumetric densities of the bone, and to calculate bone strength indices based on bone geometry and density. A three-point bending mechanical test was used to determine maximal load. Three variables from the pQCT data set explained 88% of the variance in maximal load, with the volumetric bone mineral density explaining 32% of the variance. The addition of the volumetric cortical density increased the adjusted r(2) to 0.601 (p=0.001) and the addition of an index created by multiplying volumetric cortical bone density by the maximum cross-sectional moment of inertia made further significant (p<0.001) improvements to an adjusted r(2) of 0.877. In comparison, when only the DXA variables were considered in a multiple regression model, areal bone mineral density was the only variable entered and explained only 51% (p<0.001) of the variance in maximal load. These results suggest that pQCT can better predict maximal load in whole beagle femora since pQCT provides information on the bone's architecture in addition to its volumetric density.

Effectiveness of endoscopy in patients admitted to the intensive care unit with upper GI hemorrhage.

BACKGROUND: Esophagogastroduodenoscopy (EGD) is generally indicated for the management of patients admitted to intensive care units (ICUs) with upper gastrointestinal (GI) hemorrhage but its impact in community practice has not been measured. Thus, the effectiveness of 3 EGD factors, viz., accurate initial diagnosis, performance within 24 hours of admission (early EGD), and appropriate intervention, was examined. METHODS: Records of 214 patients admitted to the ICU of 10 metropolitan hospitals with upper GI hemorrhage were reviewed. Unadjusted and severity-adjusted associations of the 3 EGD factors with length of hospital stay, length of ICU stay, readmission to ICU, recurrent bleeding, surgery, and death were evaluated. RESULTS: Inaccurate diagnosis occurred in 10% of patients at initial EGD and was associated with significant increases in risk of recurrent bleeding (70% vs. 11%, p < 0.001), rate of surgery (20% vs. 4%, p < 0.05), length of hospital stay (median 7.5 vs. 5 days, p < 0.005), length of ICU stay (median 4 vs. 2 days, p < 0.005), and rate of readmission to ICU (20% vs. 0.6%, p < 0.001). These associations persisted after adjusting for severity of illness. Early EGD performed in 82% of patients was associated with significant severity-adjusted reductions in hospital (-33%: 95% CI [-45%, -18%]) and ICU (-20%: 95% CI [-24%, -3%]) stay. Appropriate intervention at initial EGD, performed in 84% of patients, was associated with reductions in severity-adjusted length of ICU stay (-18%: 95% CI [-32%, 0%]) and rate of recurrent bleeding (odds ratio = 0.37, 95% CI [0.13, 1.06]). CONCLUSIONS: Early, accurate EGD with appropriate therapeutic intervention is effective as practiced in the community and is associated with improved outcomes for patients with upper GI hemorrhage admitted to the ICU. Inaccurate diagnosis at initial EGD is uncommon but has a significant adverse association with all outcome measures.

Identification of a complex that binds to the CD154 3' untranslated region: implications for a role in message stability during T cell activation.

CD154 expression is regulated throughout a time course of CD3-dependent T cell activation by differential mRNA decay. To understand the molecular basis of the "stability" phase of this pathway, experiments were conducted to identify sequences and specific complexes important in this regulation. Gel retardation assays using extracts from both Jurkat T cells and CD3-activated CD4(+) T cells revealed a major complex (complex I) that bound a 65-bp highly CU-rich region of the CD154 3' untranslated region. The specificity of the CU-rich element for complex-I formation was confirmed by disruption of this complex by oligo(dCT) competition. Formation of complex I strongly correlated with CD154 mRNA stability across a time course of T cell activation. UV cross-linking identified a major oligo(dCT)-sensitive species at approximately 90 kDa that showed induced and increased expression in extracts from 24- and 48-hr anti-CD3-activated T cells, respectively. This protein was absent in equivalent extracts from resting or 2-h-activated T cells. Using an in vitro decay assay, we found that a CD154-specific transcript was more rapidly degraded in 2-h-activated extract and stabilized in the 24- and 48-h extracts compared to extracts from resting T cells. Disruption of complex I resulted in the rapid decay of a CD154-specific transcript demonstrating a functional role for complex I in mRNA stabilization in vitro. These studies support a model of posttranscriptional regulation of CD154 expression being controlled in part by the interaction of a poly(CU)-binding complex with a specific sequence in the 3' untranslated region.

A transcriptional defect underlies B lymphocyte dysfunction in a patient diagnosed with non-X-linked hyper-IgM syndrome.

To establish the underlying cause of hyper-IgM syndrome in one female patient, B cell function was examined in response to CD40- and IL-4-mediated pathways. When CD40-induced functional responses were measured in unfractionated B cells, CD80 up-regulation, de novo Cmu-Cgamma recombination, and Igamma transcription were all found to be relatively unaffected. However, CD40- and IL-4-mediated CD23 up-regulation and VDJ-Cgamma transcription were clearly diminished compared to control cells. IL-4-induced CD23 expression was measurably reduced in the CD20- population as well. These results suggested that the patient's defect is positioned downstream of CD40 contact and affects both CD40- and IL-4 signal transduction pathways. Further analysis of B cell function in CD19+ B cells revealed a clear B cell defect with respect to Igamma and mature VDJ-Cgamma transcription and IgG expression. However, under the same conditions Iepsilon transcription was relatively normal. Partial restoration of B cell function occurred if PBMC or CD19+ B cells were cultured in vitro in the presence of CD154 plus IL-4. Because addition of IL-4 to cocultures containing activated T cells failed to induce B cells to undergo differentiation, the ability of the patient's B cells to acquire a responsive phenotype correlated with receiving a sustained signal through CD40. These findings support a model in which the patient expresses an intrinsic defect that is manifested in the failure of specific genes to become transcriptionally active in response to either CD154 or IL-4 and results in a functionally unresponsive B cell phenotype.

A polymorphic CD40 ligand (CD154) molecule mediates CD40-dependent signalling but interferes with the ability of soluble CD40 to functionally block CD154:CD40 interactions.

We report the characterization of a naturally occurring polymorphism in CD40 ligand (CD40L, CD154) expressed by activated T cells from a young female patient. This polymorphism encodes a nonconservative Gly --> Arg substitution in amino acid 219 in the extracellular, CD40 binding domain of the molecule. Studies carried out with 293 epithelial cells ectopically expressing the polymorphic protein (CD154/G219R) revealed reduced levels of binding to different anti-CD154 monoclonal antibodies (mAb) and CD40-immunoglobulin (CD40-Ig). However, recognition of the polymorphic and wild-type CD154 molecules by a polyclonal antiserum was comparable, suggesting that the polymorphism affects the ability of the protein to interact with CD40 but does not significantly alter its surface expression. To determine if reduced cross-linking of CD40 mediated decreased functional effects, three CD40-dependent properties were measured. We found that pathways leading to the induction of surface CD23, CD80, and Igamma transcription were activated in response to CD154/G219R signalling. However, the decrease in affinity for CD40 by the mutated CD154 affected the ability of CD40-Ig to efficiently interfere with the binding and effectively block induced CD80 expression. In contrast, we found that the 5c8 mAb, which recognized the polymorphic molecule to a similar extent as wild-type CD154, effectively blocked the interaction between CD154/G219R and CD40 as measured by CD80 expression. These findings suggest that naturally occurring polymorphisms in the CD154 molecule may affect the ability of CD40-mediated functions to be blocked by soluble CD40 or anti-CD154 mAb in the therapeutic treatment of disease and graft rejection.

Regulation of CD154 (CD40 ligand) mRNA stability during T cell activation.

The CD154 protein (CD40 ligand), which is critical to the regulation of both humoral and cellular immune responses, is expressed transiently on the surface of activated CD4+ T cells. To determine whether control of mRNA stability contributes to the highly regulated expression of CD154 during T cell activation, CD4+ T cells were isolated from human peripheral blood and stimulated for various lengths of time with plate-bound anti-CD3 mAb. At early times after anti-CD3 activation, the CD154 message was found to be very unstable, however, the stability measurably increased after 24-48 h of activation. Similar analyses of TNF-alpha and c-myc mRNA decay throughout a time course of T cell activation revealed patterns of regulation that were distinct from CD154. Similar to the effect on TNF-alpha mRNA, stimulation of T cells with PMA + ionomycin greatly increased the stability of CD154 message. However, CD154 message stability was only modestly increased in T cells coactivated with anti-CD3 and anti-CD28 at 5 h and not increased by costimulation at 24 h. Finally, an analysis of both mRNA and surface protein expression over a time course of T cell activation with anti-CD3 revealed a rapid induction of expression early after activation. This induction was followed by a more gradual decrease in expression over the next 48 h. Together, these data support a role for posttranscriptional regulation in the control and overall expression of CD154 in activated T cells.

CD40 ligand exerts differential effects on the expression of I gamma transcripts in subclones of an IgM+ human B cell lymphoma line.

The CD40:CD40 ligand (CD40L) interaction plays a critical role in T cell-dependent isotype switching. To elucidate the role of CD40 signaling in the activation of gamma germline transcription and as an extension, in targeting Cgamma regions for isotype switching, an IgM+ Burkitt lymphoma cell line (Ramos 2G6) was assayed for the up-regulation of germline gamma transcripts after CD40L stimulation. Independent Ramos 2G6 subclones that either expressed (Igamma+) or did not express (Igamma-) basal levels of Igamma transcripts were assessed for their transcriptional response to CD40L signaling by contact with either a Jurkat T cell line (D1.1) or a transfected CD40L-expressing epithelial cell line (293/CD40L) in the presence or absence of IL-4. Both Igamma- and Igamma+ Ramos 2G6 subclones cultured with IL-4 and CD40L markedly up-regulated germline transcription predominantly from the gamma1, gamma2, and gamma3 subclasses over levels obtained with IL-4 alone. In addition, these two signals were required to obtain de novo switch recombination. However, incubation with CD40L alone resulted in a substantial increase in germline transcription only in the Igamma+ and not the Igamma- subclones. Observed basal transcription at the gamma1 locus also correlated with the ability of not only the gamma1 locus, but also the gamma2 and gamma3 loci, to up-regulate germline transcripts in response to CD40 signaling. These data are consistent with CD40:CD40L contact up-regulating germline transcription only after the B cell has received a signal that alters the transcriptional state of the heavy chain locus.

Ray casting and the enclosing-net algorithm for extracting shapes from volume data.

One of the most useful applications of virtual reality is to let doctors view the inside of the human body non-invasively and in real time. In this paper, we first survey the area of virtual reality and volume-visualization techniques. We discuss our ray casting implementation for viewing the medical data which is available as a set of slices from NMR and CT scans. Next we present a new method, the enclosing-net algorithm, for extracting iso-surfaces from volume data. A simple implementation of our technique is described. Since the topology of the extracted surface is well defined and non-ambiguous, the enclosing-net algorithm eliminates a major problem of surface extraction techniques. In addition, the number of polygons representing the surface can be controlled to obtain a finer shape. Therefore real-time interaction is feasible on both low-end and high-end graphics machines, making the enclosing-net algorithm suitable for virtual reality experiments.

Feto-placental steroid metabolism in growth retarded human fetuses.

The goal of the study was the determination of the relative roles of the placenta and the fetus in causing low serum estriol (E3) levels in women bearing fetuses with intrauterine growth retardation (IUGR). Umbilical venous levels of E3 and dehydroepiandrosterone sulfate (DHAS) were measured in 31 samples from fetuses with IUGR, 21 of whom were vaginally delivered and 10 who were delivered by cesarean section. In addition, estrone (E1) and estradiol (E2) were measured in 11 of the samples. The results were compared with 11 samples from cesarean section delivered control term infants and 54 samples from vaginally delivered control infants. The vaginally delivered IUGR group had a significantly lower mean umbilical venous DHAS level than did their control group (2128 +/- 158 ng/ml SEM versus 2645 +/- 130, p less than 0.05). Both the vaginally delivered and cesarean section delivered IUGR infants had umbilical venous E3 levels significantly lower than in their control groups (70 +/- 10 ng/ml SEM versus 144 +/- 10, p less than 0.001, and 46 +/- 11 ng/ml SEM versus 136 +/- 23, p less than .01, respectively). Umbilical venous E1 and E2 levels were not different from the control values. E1, E2, E3, and DHAS were measured in eight maternal venous samples obtained from mothers bearing fetuses with IUGR. In comparison with 11 control mothers, only E3 was significantly different (10.7 +/- 3.0 ng/ml SEM in mothers with IUGR fetuses versus 25.0 +/- 4.9 in control mothers p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of 6-thioguanine-resistant mutations by rat-liver homogenate (S9)-activated promutagens in human embryonic skin fibroblasts.

Most normal human fibroblasts grown in culture do not metabolize promutagens/procarcinogens. Thus screening assays employing normal human fibroblasts have only been successful for direct-acting chemical mutagens and various radiations. In this report we describe a mutation assay (HGPRT locus) employing a normal human embryonic skin fibroblast and a rat-liver homogenate (S9) mixture. 3 model promutagens, benzo[a]pyrene (B[a]P), 3-methylcholanthrene (3MC), and dimethylnitrosamine (DMN) have been utilized in these studies. In addition to discussing conditions for optimizing the response of this assay, our results indicate that at constant amount of S9 protein concentration, there exists a linear correlation between mutagenicity and dose. At 50% survival, the mutant frequencies induced by B[a]P and 3MC (5 micrograms/ml) are 60 and 30 times the background mutant frequency, respectively. Similarly, at 50% survival, DMN (5 mg/ml) induced 6-TGr mutant frequencies are 25-fold over the background frequency. The increase in cytotoxicity resulting from exposure of cells to these 'activated' chemicals is also a linear dose response. At high S9 concentrations a deactivation or detoxification phenomenon occurs. However, the mutagenic efficiency of S9-activated chemicals when plotted as the number of induced mutations versus log survival is unaffected by the deactivating capacity of S9 proteins. This study demonstrates a quantitative mutation assay using an early passage human culture with an exogenous rat-liver microsomal preparation providing activating enzymes.

Adrenal cortical function in the postmature fetus and newborn infant.

The umbilical venous blood concentrations of cortisol, dehydroepiandrosterone sulfate (DHAS), and unconjugated estriol were compared in 54 normal, 37 postterm, and 22 postmature newborns. Pre- and postadrenocorticotropic hormone (ACTH) stimulation levels of serum cortisol and DHAS were compared in the first 2 to 4 days of life in 19 postterm and 15 postmature infants. Comparison was also made between vaginally an cesarean section delivered postterm and postmature newborns. There were significantly greater cord blood cortisol levels in th postmature [260 +/- 22 ng/ml (+/- S.E.)], than in the normal (193 +/- 11 ng/ml) (P less than 0.01) or postterm (193 +/- 18 ng/ml) (0.01 less than P less than 0.05) vaginally delivered infants. There were no significant differences in the mean cord blood DHAS levels in the three groups (normal, 2645 +/- 130 ng/ml; postterm 2323 +/- 188 ng/ml; postmature, 2310 +/- 224 ng/ml). Cortisol and DHAS responses to ACTH stimulation were the same in the postterm and postmature groups. There was a significantly lower mean umbilical venous unconjugated estriol level in the vaginally delivered postmature group (75 +/- 11 ng/ml) as compared to values in vaginally delivered postterm [120 +/- 14 ng/ml (P = 0.01)] and normal [144 +/- 10 ng/ml (P less than 0.002)] newborns. Stressed postmature infants delivered by cesarean section had higher unconjugated estriol levels (83 +/- 12 ng/ml) than their unstressed, postterm cesarean section controls [40 +/- 9 ng/ml (P less than 0.01)], but levels were still below those from vaginally delivered postterm infants. These findings substantiate normal adrenal function in the postmature fetus and newborn. Lowered umbilical venous unconjugated estriol levels in the postmature infants at birth appear to be a function of limited aromatizing activity of the placenta rather than due to the low levels of fetal adrenal-derived neutral steroid substrate.

Mutation of Chinese hamster cells by near-UV activation of promutagens.

A tissue-culture assay for mutagenesis and cytotoxicity incorporating near ultraviolet (NUV) light activation of polyaromatic hydrocarbons (PAH) has been developed. Cultures of Chinese hamster cells (line CHO) growing in suspension culture were inoculated with benzo[a]pyrene (B[a]P), 7,12-dimethylbenzanthracene (DMBA) of shale-oil retort-water and exposed to light from a high-pressure mercury lamp fitted with a Corning NUV bandpass filter. This light source both permitted activation of PAH and the shale-oil water and preculded detectable damage to DNA. Neither the PAH nor the NUV alone had any effect on cell survival or mutation frequencies but the chemicals plus NUV were extremely effective in producing mutations to 6-thioguanine resistance (hgprt gene).