Nonlymphoid Fas ligand in peptide-induced peripheral lymphocyte deletion

Peripheral lymphocyte deletion is required for reduction of lymphocyte numbers after expansion in response to antigen. Peripheral deletion is mediated in part by the activation of apoptosis by engagement of the death receptor, Fas (CD95), by its ligand, Fas ligand (FasL; CD95L), among other mechanisms. Here we used T cell receptor (TCR) transgenic animals to examine the role of inducible expression of nonlymphoid FasL in response to peptide antigen. Antigenic challenge of TCR transgenic mice resulted in increased expression of FasL in a number of nonlymphoid tissues including the epithelium of the small intestine. Similar results were obtained in an adoptive transfer system in which TCR transgenic T cells were transferred into recipient animals. The functional relevance of nonlymphoid FasL in peripheral deletion is supported by the observation that FasL-deficient gld animals showed a significantly reduced rate of clearance of transferred antigen-specific lymphocytes, although the lymphocytes themselves were wild type for FasL. These observations were supported further by studies in a transgenic mouse model where lacZ was expressed under the control of the proximal promoter of the FasL gene. Using these transgenic mice, we observed induced activity of the FasL promoter in intestinal epithelial cells throughout the crypts and villi, where we also observed infiltration of activated T cells. These data demonstrate that nonlymphoid FasL is expressed in response to peripheral T cell activation and participates in the regulation of T cells that infiltrate peripheral tissues.     

Voltage-gated Ca2+ channel Ca(V)1.3 subunit expressed in the hair cell epithelium of the sacculus of the trout Oncorhynchus mykiss: cloning and comparison across vertebrate classes

Full-length sequence (>6.5 kb) has been determined for the Ca(V)1.3 pore-forming subunit of the voltage-gated Ca(2+) channel from the saccular hair cells of the rainbow trout (Oncorhynchus mykiss). Primary structure was obtained from overlapping PCR and cloned fragments, amplified by primers based on teleost, avian, and mammalian sources. Trout saccular Ca(V)1.3 was localized to hair cells, as evidenced by its isolation from an epithelial layer in which the hair cell is the only intact cell type. The predicted amino acid sequence of the trout hair cell Ca(V)1.3 is approximately 70% identical to the sequences of avian and mammalian Ca(V)1.3 subunits and shows L-type characteristics. The trout hair cell Ca(V)1.3 expresses a 26-aa insert in the I-II cytoplasmic loop (exon 9a) and a 10-aa insert in the IVS2-IVS3 cytoplasmic loop (exon 30a), neither of which is appreciably represented in trout brain. The exon 9a insert also occurs in hair cell organs of chick and rat, and appears as an exon in human genomic Ca(V)1.3 sequence (but not in the Ca(V)1.3 coding sequence expressed in human brain or pancreas). The exon 30a insert, although expressed in hair cells of chick as well as trout, does not appear in comparable rat or human tissues. Further, the IIIS2 region shows a splice choice (exon 22a) that is associated with the hair cell organs of trout, chick, and rat, but is not found in human genomic sequence. The elucidation of the primary structure of the voltage-gated Ca(2+) channel Ca(V)1.3 subunit from hair cells of the teleost, representing the lowest of the vertebrate classes, suggests a generality of sensory mechanism for Ca(V)1.3 across hair cell systems. In particular, the exon 9a insert of this channel appears to be the molecular feature most consistently associated with hair cells from fish to mammal, consonant with the hypothesis that the latter region may be a signature for the hair cell.     

Cerebrospinal fluid brain-derived proteins in the diagnosis of Alzheimer's disease and Creutzfeldt-Jakob disease

The differential diagnosis of dementia can be difficult in the early stages of disease, and with the emergence of new therapeutic agents for Alzheimer's disease (AD) there is an increasing need for reliable and accurate diagnostic tests. The concept of brain-specific proteins was first proposed in the 1960s and, since that time, methods have developed to measure these proteins in the cerebrospinal fluid (CSF). The concentration of individual brain-specific proteins can be altered in disease, and these changes are thought to reflect the underlying pathology. CSF tau protein and amyloid peptide A beta 42 concentrations are altered in AD and have been proposed as early diagnostic tests for this disease. The data from a number of studies suggest that these proteins may be of value, but are less specific than previously thought and further studies with neuropathological confirmation are required before these tests can be introduced into clinical practice. The detection of 14-3-3 in the CSF is an accurate test for sporadic Creutzfeldt-Jakob disease (CJD) and this accuracy has lead the World Health Organization to revise the clinical criteria for probable sporadic CJD to include a positive CSF 14-3-3. However, CSF 14-3-3 is less useful in the diagnosis of variant CJD, where studies are underway investigating the value of other CSF proteins.     

Passive catheter tracking using MRI: comparison of conventional and magnetization-prepared FLASH

PURPOSE: To compare a magnetization-prepared gradient-echo (GRE) sequence with a conventional GRE sequence for visualizing contrast agent-filled catheters. MATERIALS AND METHODS: Passive visualization of endovascular catheters using MRI was compared between two imaging sequences: 1) inversion recovery (IR)-fast low angle shot (FLASH), and 2) conventional FLASH. Two-dimensional projection images of the catheters filled with 4% diluted contrast agent in a phantom and the aorta of swine were obtained with each sequence with a temporal resolution of two frames per second. We compared background suppression and catheter visibility using the catheter-to-background signal ratio and the ratings of two radiologists. RESULTS: In the phantom, IR-FLASH allowed for a 200% increase in catheter-to-background ratio (p < 0.01) and improved depiction of catheters over conventional FLASH. In swine, the IR-FLASH images showed a statistically significant improvement of 80% (p < 0.001) over conventional FLASH in all comparisons of the catheter-to-background signal ratio, and an improvement of 160% (p < 0.05) in comparison with the radiologists' observations. CONCLUSION: This study shows that IR-FLASH is a better technique for passive tracking of contrast agent-filled catheters than conventional FLASH.     

The effect of bone remodeling inhibition by zoledronic acid in an animal model of cartilage matrix damage

OBJECTIVE: The purpose of this work was to test the effect of inhibition of bone remodeling, through the use of the bisphosphonate, zoledronic acid, on cartilage matrix damage in an animal model of cartilage matrix damage. DESIGN: New Zealand white rabbits were divided into four groups for treatment purposes: (1) untreated controls; (2) injected into one knee joint with the cartilage matrix degradation enzyme, chymopapain; (3) injected into one knee joint with chymopapain and also given subcutaneous injections of the bisphosphonate, zoledronic acid, three times per week until sacrifice at either day 28 or 56 post-chymopapain-injection; (4) received only the zoledronic acid injections. At sacrifice, the knee joints were examined grossly and histologically, and biochemically for proteoglycan content. Urine samples were analysed, at intervals, for levels of collagen cross-links which are biochemical markers of cartilage and bone. RESULTS: Animals receiving both intraarticular chymopapain injections and subcutaneous zoledronic acid injections displayed a significantly lower degree of grossly and histologically detectable cartilage degeneration on the tibial articular surfaces (the articular surface displaying the greatest degree of degeneration) than did animals only receiving the chymopapain injections. In addition, urinary levels of collagen cross-links for bone and cartilage were significantly higher in those animals only receiving chymopapain injections. CONCLUSION: The bone resorption observed after chymopapain injection into the rabbit knee joint can be inhibited through the use of the bisphosphonate, zoledronic acid. Furthermore, zoledronic acid does not increase the level of cartilage degeneration and appears to provide some level of chondroprotection in this model.     

Discriminating between cell surface and intracellular plasminogen-binding proteins: heterogeneity in profibrinolytic plasminogen-binding proteins on monocytoid cells

When plasminogen binds to cell surfaces, its activation is markedly enhanced compared to soluble plasminogen. Although several distinct molecules may contribute to plasminogen binding to a given cell type, the subset of plasminogen receptors responsible for enhancing plasminogen activation expose a carboxyl-terminal lysine on the cell surface and are sensitive to proteolysis by carboxypeptidase B (CpB). To distinguish this subset of plasminogen receptors from plasminogen-binding proteins that are not profibrinolytic, we treated intact U937 monocytoid cells and peripheral blood monocytes with CpB to remove exposed carboxyl-terminal lysines, and subjected the membrane proteins to two-dimensional gel electrophoresis followed by ligand blotting with 125I-plasminogen. Western blotting was performed with antibodies against previously characterized candidate plasminogen receptors to identify plasminogen-binding proteins on the two-dimensional ligand blots. Densitometry of autoradiograms of the 125I-plasminogen ligand blots of U937 cell membranes revealed that membrane-associated alpha-enolase, actin and annexin II showed minimal changes in 125I-plasminogen binding following CpB treatment of intact cells, suggesting that these proteins are not accessible to CpB on the U937 cell surface and most likely do not serve as profibrinolytic plasminogen receptors on U937 cells. In contrast, densitometry of autoradiograms of 125I-plasminogen ligand blots of monocyte membranes revealed that 125I-plasminogen binding to alpha-enolase was reduced 71% by treatment of intact cells with CpB, while binding to annexin II was reduced 14%. Thus, a portion of membrane-associated alpha-enolase and annexin II expose carboxyl terminal lysines that are accessible to CpB on the peripheral blood monocyte surface, suggesting that these molecules may serve as profibrinolytic plasminogen receptors on monocytes. Our data suggest that U937 cells and peripheral blood monocytes have distinct sets of molecules that constitute the population of cell surface profibrinolytic plasminogen-binding proteins. Furthermore, our data suggest that while several plasminogen-binding proteins with carboxyl terminal lysines are associated with cell membranes, only a small subset of these proteins expose a carboxyl terminal lysine that is accessible to CpB on the cell surface.     

Breast cancer risk and drinking water contaminated by wastewater: a case control study

Background  

Drinking water contaminated by wastewater is a potential source of exposure to mammary carcinogens and endocrine disrupting compounds from commercial products and excreted natural and pharmaceutical hormones. These contaminants are hypothesized to increase breast cancer risk. Cape Cod, Massachusetts, has a history of wastewater contamination in many, but not all, of its public water supplies; and the region has a history of higher breast cancer incidence that is unexplained by the population's age, in-migration, mammography use, or established breast cancer risk factors. We conducted a case-control study to investigate whether exposure to drinking water contaminated by wastewater increases the risk of breast cancer.     

California Verbal Learning Test indicators of suboptimal performance in a sample of head-injury litigants

Cutoff scores suggested by Millis, Putnam, Adams, and Ricker (1995) for detecting suboptimal performance on indices from the California Verbal Learning Test (CVLT) were evaluated using data from 193 compensation-seeking participants. All participants claimed to have suffered a blow to the head in an accident causing subsequent deterioration in cognitive function. The participants were divided into those with negligible or possible mild brain injuries and those with clear evidence of moderate to severe brain injuries. In addition to the CVLT, all participants were administered the Computerized Assessment of Response Bias (CARB), a two-alternative forced choice test of recognition memory that is used to detect feigned cognitive impairment. For all CVLT indices, the distributions of outcome (valid vs. suboptimal performance) was unrelated to age and brain injury severity, and only weakly associated with education. However, a significantly higher proportion of males than females obtained scores in the suboptimal performance range. The CVLT indices were not fully redundant with each other with respect to binary participant classifications; substantial disagreement between pairwise classifications was found among those participants who obtained at least one score in the suboptimal performance range. CVLT index classifications were also found to be non-redundant with classifications based on CARB scores. The CVLT may thus add useful data over and above that obtained from symptom validity testing. However, the data suggest that the use of the strategy where any one or more below-cutoff CVLT scores are considered a positive indicator of suboptimal performance may be associated with a higher than acceptable false-positive error rate.     

Phosphatidylinositol 3-kinase inhibition by LY294002 radiosensitizes human cervical cancer cell lines.

PURPOSE: The phosphatidylinositol 3-kinase (PI3K) catalytic subunit is amplified in cervical cancers, implicating PI3K in cervical carcinogenesis. We evaluated the radiosensitizing effect of PI3K inhibition by LY294002 on clonogenic survival, growth characteristics, and gene expression in cervical cancer cell lines (HeLa and CaSki). EXPERIMENTAL DESIGN: Cervical cancer cells were treated separately and concurrently with the PI3K inhibitor LY294002 (10 micromol/L) and radiation (2 Gy) with serial analysis of cell count, apoptosis, and flow cytometry. PI3K inhibition was assessed by protein analysis of phosphorylated Akt. Clonogenic assays were done with varying doses of radiation and LY294002 and varied time points of administration of LY294002 proximate to the radiation dose. Surviving fractions and dose modification factors (DMF) were calculated. Each experiment was done in triplicate and analyzed using ANOVA regression analysis and Dunnett's t Test. Microarray gene expression analysis was done on the HeLa cell line. RESULTS: PI3K inhibition with LY294002 alone did not decrease cell survival. However, treatment with LY294002 significantly radiosensitized HeLa and CaSki cell lines with DMFs (1 log cell kill) of 1.95 and 1.37, respectively. Compared with post-irradiation, pretreatment produced more radiosensitization (P < 0.0001). DMFs were 2.2, 2.0, 2.0, and 1.2 for LY294002 added at 6, 2, and 0.5 hours before irradiation and 6 hours after irradiation, respectively. LY294002 pretreatment in irradiated HeLa cells led to altered gene expression. CONCLUSIONS: Although LY294002 alone did not produce cytotoxic effects, PI3K inhibition with LY294002 produced significant radiosensitization, showed significant time-dependent effects, increased apoptosis, and altered gene expression. These findings support future investigation of PI3K inhibitors in combination with radiation therapy for carcinoma of the cervix.