In vitro drug causality assessment in Stevens-Johnson syndrome - alternatives for lymphocyte transformation test

BACKGROUND Patients with Stevens-Johnson syndrome (SJS) or toxic epidermal necrolysis (TEN) are often exposed simultaneously to a few potentially culprit drugs. However, both the standard lymphocyte transformation tests (LTT) with proliferation as the assay end-point as well as skin tests, if done, are often negative. OBJECTIVE As provocation tests are considered too dangerous, there is an urgent need to identify the relevant drug in SJS/TEN and to improve sensitivity of tests able to identify the causative drug. METHODS Fifteen patients with SJS/TEN with the ALDEN score ≥ 6 and 18 drug-exposed controls were included. Peripheral blood mononuclear cells (PBMC) were isolated and cultured under defined conditions with drugs. LTT was compared to the following end-points: cytokine levels in cell culture supernatant, number of granzyme B secreting cells by ELISpot and intracellular staining for granulysin and IFNγ in CD3(+) CD4(+), CD3(+) CD8(+) and NKp46(+) cells. To further enhance sensitivity, the effect of IL-7/IL-15 pre-incubation of PBMC was evaluated. RESULTS Lymphocyte transformation tests was positive in only 4/15 patients (sensitivity 27%, CI: 8-55%). Similarly, with granzyme B-ELISpot culprit drugs were positive in 5/15 patients (sensitivity 33%, CI: 12-62%). The expression of granulysin was significantly induced in NKp46(+) and CD3(+) CD4(+) cells (sensitivity 40%, CI: 16-68% and 53%, CI: 27-79% respectively). Cytokine production could be demonstrated in 38%, CI: 14-68% and 43%, CI: 18-71% of patients for IL-2 and IL-5, respectively, and in 55%, CI: 23-83% for IFNγ. Pre-incubation with IL-7/IL-15 enhanced drug-specific response only in a few patients. Specificities of tested assays were in the range of 95 (CI: 80-99%)-100% (CI: 90-100%). CONCLUSIONS AND CLINICAL RELEVANCE Granulysin expression in CD3(+) CD4(+) , Granzyme B-ELISpot and IFNγ production considered together provided a sensitivity of 80% (CI: 52-96%) and specificity of 95% (80-99%). Thus, this study demonstrated that combining different assays may be a feasible approach to identify the causative drug of SJS/TEN reactions; however, confirmation on another group of patients is necessary.

Allocentric visual cues influence mental transformation of bodies

Identifying a human body stimulus involves mentally rotating an embodied spatial representation of one's body (motoric embodiment) and projecting it onto the stimulus (spatial embodiment). Interactions between these two processes (spatial and motoric embodiment) may thus reveal cues about the underlying reference frames. The allocentric visual reference frame, and hence the perceived orientation of the body relative to gravity, was modulated using the York Tumbling Room, a fully furnished cubic room with strong directional cues that can be rotated around a participant's roll axis. Sixteen participants were seated upright (relative to gravity) in the Tumbling Room and made judgments about body and hand stimuli that were presented in the frontal plane at orientations of 0°, 90°, 180° (upside down), or 270° relative to them. Body stimuli have an intrinsic visual polarity relative to the environment whereas hands do not. Simultaneously the room was oriented 0°, 90°, 180° (upside down), or 270° relative to gravity resulting in sixteen combinations of orientations. Body stimuli were more accurately identified when room and body stimuli were aligned. However, such congruency did not facilitate identifying hand stimuli. We conclude that static allocentric visual cues can affect embodiment and hence performance in an egocentric mental transformation task. Reaction times to identify either hands or bodies showed no dependence on room orientation.

Correlating the Ancient Maya and Modern European Calendars with High-Precision AMS 14C Dating

The reasons for the development and collapse of Maya civilization remain controversial and historical events carved on stone monuments throughout this region provide a remarkable source of data about the rise and fall of these complex polities. Use of these records depends on correlating the Maya and European calendars so that they can be compared with climate and environmental datasets. Correlation constants can vary up to 1000 years and remain controversial. We report a series of high-resolution AMS C-14 dates on a wooden lintel collected from the Classic Period city of Tikal bearing Maya calendar dates. The radiocarbon dates were calibrated using a Bayesian statistical model and indicate that the dates were carved on the lintel between AD 658-696. This strongly supports the Goodman-Martinez-Thompson (GMT) correlation and the hypothesis that climate change played an important role in the development and demise of this complex civilization.

Downregulation of Rap1GAP contributes to Ras transformation.

Although abundant in well-differentiated rat thyroid cells, Rap1GAP expression was extinguished in a subset of human thyroid tumor-derived cell lines. Intriguingly, Rap1GAP was downregulated selectively in tumor cell lines that had acquired a mesenchymal morphology. Restoring Rap1GAP expression to these cells inhibited cell migration and invasion, effects that were correlated with the inhibition of Rap1 and Rac1 activity. The reexpression of Rap1GAP also inhibited DNA synthesis and anchorage-independent proliferation. Conversely, eliminating Rap1GAP expression in rat thyroid cells induced a transient increase in cell number. Strikingly, Rap1GAP expression was abolished by Ras transformation. The downregulation of Rap1GAP by Ras required the activation of the Raf/MEK/extracellular signal-regulated kinase cascade and was correlated with the induction of mesenchymal morphology and migratory behavior. Remarkably, the acute expression of oncogenic Ras was sufficient to downregulate Rap1GAP expression in rat thyroid cells, identifying Rap1GAP as a novel target of oncogenic Ras. Collectively, these data implicate Rap1GAP as a putative tumor/invasion suppressor in the thyroid. In support of that notion, Rap1GAP was highly expressed in normal human thyroid cells and downregulated in primary thyroid tumors.

MODELING REACTIVE GAS UPTAKE, TRANSPORT, AND TRANSFORMATION IN AGGREGATED SOILS

Gas diffusion research in soils covers, to a large extent, the transport behavior of practically insoluble gases. We extend the mathematical description of gas transport to include reactive gaseous components that hydrolyze in water such as SO2 and CO2. The path between the free atmosphere and the microporous niches is modeled by assuming penetration through gas-filled macropores, air-water phase transfer, and diffusion and speciation in the liquid phase. For hydrolyzable gases, the rate of mass transfer into and the total absorption capacity of the soil solution may be high. Both the capacity and the transfer rate are influenced by the soil-solution pH; for high pH, they become extremely high for SO2. The soil absorption of such gases is also influenced by soil structure. Well-aerated, near-neutral soils are a potentially important sink for SO2.

FoxM1B regulates NEDD4-1 expression, leading to cellular transformation and full malignant phenotype in immortalized human astrocytes.

Our recent studies have shown that the FoxM1B transcription factor is overexpressed in human glioma tissues and that the level of its expression correlates directly with glioma grade. However, whether FoxM1B plays a role in the early development of glioma (i.e., in transformation) is unknown. In this study, we found that the FoxM1B molecule causes cellular transformation and tumor formation in normal human astrocytes (NHA) immortalized by p53 and pRB inhibition. Moreover, brain tumors that arose from intracranial injection of FoxM1B-expressing immortalized NHAs displayed glioblastoma multiforme (GBM) phenotypes, suggesting that FoxM1B overexpression in immortalized NHAs not only transforms the cells but also leads to GBM formation. Mechanistically, our results showed that overexpression of FoxM1B upregulated NEDD4-1, an E3 ligase that mediates the degradation and downregulation of phosphatase and tensin homologue (PTEN) in multiple cell lines. Decreased PTEN in turn resulted in the hyperactivation of Akt, which led to phosphorylation and cytoplasmic retention of FoxO3a. Blocking Akt activation with phosphoinositide 3-kinase/Akt inhibitors inhibited the FoxM1B-induced transformation of immortalized NHAs. Furthermore, overexpression of FoxM1B in immortalized NHAs increased the expression of survivin, cyclin D1, and cyclin E, which are important molecules for tumor growth. Collectively, these results indicate that overexpression of FoxM1B, in cooperation with p53 and pRB inhibition in NHA cells, promotes astrocyte transformation and GBM formation through multiple mechanisms.

Hebbian analysis of the transformation of medial entorhinal grid-cell inputs to hippocampal place fields.

The discovery of grid cells in the medial entorhinal cortex (MEC) permits the characterization of hippocampal computation in much greater detail than previously possible. The present study addresses how an integrate-and-fire unit driven by grid-cell spike trains may transform the multipeaked, spatial firing pattern of grid cells into the single-peaked activity that is typical of hippocampal place cells. Previous studies have shown that in the absence of network interactions, this transformation can succeed only if the place cell receives inputs from grids with overlapping vertices at the location of the place cell's firing field. In our simulations, the selection of these inputs was accomplished by fast Hebbian plasticity alone. The resulting nonlinear process was acutely sensitive to small input variations. Simulations differing only in the exact spike timing of grid cells produced different field locations for the same place cells. Place fields became concentrated in areas that correlated with the initial trajectory of the animal; the introduction of feedback inhibitory cells reduced this bias. These results suggest distinct roles for plasticity of the perforant path synapses and for competition via feedback inhibition in the formation of place fields in a novel environment. Furthermore, they imply that variability in MEC spiking patterns or in the rat's trajectory is sufficient for generating a distinct population code in a novel environment and suggest that recalling this code in a familiar environment involves additional inputs and/or a different mode of operation of the network.

Chromosomal control of the dominant suppression of {\it ras\/}-mediated transformation

The role of tumor suppressor function in the multistep process of carcinogenesis was studied in the human teratocarcinoma cell line PA-1. Early passage PA-1 cells ($<$P100) are preneoplastic while late passage ($>$P100) PA-1 cells are spontaneously transformed. Previous work demonstrated a causal role for the N-ras oncogene in the neoplastic transformation of this cell line and the gene was cloned. A clonal cell line established at passage 40 has been shown to suppress the neoplastic transformation potential of the PA-1 N-ras oncogene in gene transfer experiments. This phenotype has been termed SRT+ for suppression of ras transformation. A clonal cell line established at passage 63 is neoplastically transformed by the N-ras in similar gene transfer experiments and is regarded as srt$-$. Somatic cell hybrids were formed between the SRT+ cell and two different N-ras transformed srt$-$ cells. The results indicate that five of the seven independent hybrid clones, and all 14 subclones, failed to form tumors in the nude mouse tumor assay. Chromosomal analysis of rare neoplastic segregants which arose from suppressed hybrid populations demonstrate that the general loss of chromosomes correlates with the reemergence of neoplastic transformation. Karyotype analyses demonstrate a statistically correlative loss of chromosomes 1, 4, 19, and to a lesser extent 11, 14, and 16. DNA hybridization analysis demonstrates a single copy of the intact N-ras oncogene in parental cells, suppressed hybrids, and neoplastically transformed hybrids. These results indicate that functional ras transformation suppression is a trans-dominant trait which may be controlled by sequences residing on particular chromosomes in the human genome. Furthermore, the suppression of ras transformation results from a unique step in the multistep process of carcinogenesis that is different from the induction of immortality. Thus, the neoplastic process of the PA-1 cell line involves at least three steps: (1) induction of immortality, (2) activation of the N-ras oncogene, and (3) loss of tumor suppressor function. ^

The transformation of a health care system: An ecology perspective

Beginning in the early 1980s, the health care system experienced momentous realignments. Fundamental changes in structures of traditional health care organizations, shifts in authority and relationships of professionals and institutions, and the increasing influence of managed care contributed to a relatively stable industry entering into a state of turbulence. The dynamics of these changes are recurring themes in the health services literature. The purpose of this dissertation was to examine the content of this literature over a defined time period and within the perspective of a theory of organizational change. ^ Using a theoretical framework based upon the organizational theory known as Organizational Ecology, secondary data from the period between 1983 and 1994 was reviewed. Analysis of the literature identified through a defined search methodology was focused upon determining the manner in which the literature characterized changes that were described. Using a model constructed from fundamentals of Organizational Ecology with which to structure an assessment of content, literature was summarized for the manner and extent of change in specific organizational forms and for the changes in emphasis by the environmental dynamics directing changes in the population of organizations. Although it was not the intent of the analysis to substantiate causal relationships between environmental resources selected as the determinants of organizational change and the observed changes in organizational forms, the structured review of content of the literature established a strong basis for inferring such a relationship. ^ The results of the integrative review of the literature and the power of the appraisal achieved through the theoretical framework constructed for the analysis indicate that there is considerable value in such an approach. An historical perspective on changes which have transformed the health care system developed within a defined organizational theory provide a unique insight into these changes and indicate the need for further development of such an analytical model. ^

EPSTEIN-BARR VIRUS TRANSFORMATION OF B LYMPHOCYTE SUBPOPULATIONS

Epstein-Barr virus is a herpes virus distinguished by its remarkable specificity for the B lymphocyte of humans and certain other primates. Although the transformation process is very efficient, is has become clear that only a fraction of B lymphocytes is susceptible. Therefore the question may be raised if transformation is related to B cell stage of activation. B cells were purified from peripheral blood mononuclear cells by the removal of monocytes using elutriation and sheep red blood cell rosetting to remove T cells. Retesting B cells were purified using discontinuous Percoll gradients. Activation of resting cells for 24 hours with anti-mu or Staphylococcus aureus Cowan I (SAC) resulted in transition of susceptible cells into the G(,1) phase of the cell cycle as shown by an increase in cell size, an increase in uridine incorporation and an increase in sensitivity to B cell growth factor (BCGF). Entry into S phase was achieved by extending the period of activation to 48-96 hr as shown by an increase in thymidine incorporation. By this criterion, SAC activated cells entered S phase on day 2 and anti-mu treated cells on day 3. Control (G(,0)) cells and cells activated for varying lengths of time (G(,1), G(,1) plus S) were exposed to EBV and plated in a limiting dilution assay to determine the frequency of EBV-transformable cells. Control cells and cells activated for 24 hr had a precursor frequency of 1% to 2%. With continued activation, however, precursor frequency decreased as a function of the duration of activation. The decrease in frequency of transformable cells correlated with the entry of the population into S phase. The transformation frequency in the SAC-treated population was reduced twenty-fold on day 4, whereas in the anti-mu treated population it was reduced ten-fold. Treating cells with BCGF in conjunction with low concentrations of anti-mu decreased the transformation frequency to levels lower than anti-mu alone, further suggesting that entry into S phase is accompanied by a reduction in transformability. These results indicate that resting B cells are highly susceptible to transformation and that with in vitro activation into the cell cycle B cells become progressively insensitive to EBV. ^