A semi-quantitative direct contact assay using fluorescein diacetate and L929 mouse fibroblasts in monolayer culture

Summary A semi-quantitative procedure is described for measuring cell viability after short-term exposure to a test substance using a monolayer culture. Test substances are placed in direct contact with cell monolayers for various time intervals. The substances are removed and the monolayers are incubated in the presence of fluorescein diacetate. Monolayers are viewed under a fluorescent microscope and the percentage of fluorescing (viable) cells is estimated. The method is suitable for examining cytotoxic effects at short times of exposure and for discriminating between test substances that give similar, low toxicity endpoints in standard 24-h assays.     

Contributions of semantic and facial information to perception of nonsibilant fricatives.

Most studies have been unable to identify reliable acoustic cues for the recognition of the English nonsibilant fricatives [see text]. The present study was designed to test the extent to which the perception of these fricatives by normal-hearing adults is based on other sources of information, namely, linguistic context and visual information. In Experiment 1, target words beginning with /f/, /theta/, /s/, or [see text] were preceded by either a semantically congruous or incongruous precursor sentence. Results showed an effect of linguistic context on the perception of the distinction between /f/ and /theta/ and on the acoustically more robust distinction between /s/ and [see text]. In Experiment 2, participants identified syllables consisting of the fricatives [see text] paired with the vowels /i, a, u/. Three conditions were contrasted: Stimuli were presented with (a) both auditory and visual information, (b) auditory information alone, or (c) visual information alone. When errors in terms of voicing were ignored in all 3 conditions, results indicated that perception of these fricatives is as good with visual information alone as with both auditory and visual information combined, and better than for auditory information alone. These findings suggest that accurate perception of nonsibilant fricatives derives from a combination of acoustic, linguistic, and visual information.     

TNF-alpha impairs peripheral tolerance towards beta-cells,and local costimulation by B7.1 enhances the effector function of diabetogenic T cells

Maintenance of peripheral tolerance and inactivation of autoreactive T cells is based on a delicate balance between pro-inflammatory and protective cytokines that is poorly understood. We have here addressed how the local expression of the inflammatory cytokine TNF-alpha can impair peripheral tolerance and lead to autoreactivity. After transplantation of pancreata that are immunogenic due to beta-cell expression of B7.1 and TNF-alpha, into thymectomized and euthymic syngeneic mice, we found that only euthymic mice rejected the grafts. This result suggests that under normal circumstances autoreactive T cells are functionally inactivated, and initiation of an autoreactive response requires de-novo generation of T cells. By contrast, thymectomized mice expressing TNF-alpha on the endogenous islets rejected the grafts, showing that expression of TNF-alpha prevents functional silencing of the autoreactive T cells. Thus, this study provides a mechanism by which TNF-alpha and possibly chronic inflammatory responses may promote autoimmune diseases. Furthermore, we have investigated whether B7.1 can enhance T cell responses of already activated T cells leading to islet rejection. By transplantation of wild-type and B7.1-expressing islets into overtly diabetic mice we found that only the wild-type islets could restore normoglycemia, suggesting that costimulation by B7.1 is required in the expansion or effector phase of the response.     

Functional abdominal complaints in pre-school children: parental reports of health-related quality of life

Purpose  

The aim of this study is to assess the influence of functional abdominal complaints (FAC) on health-related quality of life in a group of Dutch pre-school children.     

Neonatal Pattern of VH Gene Utilization in Nonobese Diabetic Mice Does not Correlate with Development of Insulitis

The nonobese diabetic (NOD) mouse model is a model of human autoimmune insulin dependent diabetes, IDDM. The effector cells of the disease have been shown to be T cells, but also B cells seem to contribute. Adult NOD mice have been shown to display a bias in their utilization of immunoglobulin (Ig) variable heavy (V(H)) genes. In this study the analysis of VH gene utilization in NOD mice protected from insulitis by transgenic insertion of a major histocompatibility complex (MHC) class II E(alpha) gene, point out that the bias in V(H) gene expression is not correlated to disease development. The aberrant V(H) gene utilization pattern in mice with the NOD genetic background is instead suggested to be a consequence of a deregulation of the apoptosis inhibiting gene bcl-2. We also investigated if prolonged in vitro survival of NOD lymphocytes is correlated to disease development. The E(alpha) transgenic NOD mice were shown to display a prolonged in vitro survival of spleen T cells, similar to normal NOD mice. These results indicate that defective death mechanisms of T cells may not be primarily involved in the development of autoimmune disease in these mice. However, in contrast to results from other groups, no difference in in vitro survival could be detected for B cells from mice with NOD genetic background compared to C57BL/6 mice.     

Importance of murine study design for testing toxicity of retroviral vectors in support of phase I trials.

Although retroviral vectors are one of the most widely used vehicles for gene transfer, there is no uniformly accepted pre-clinical model defined to assess their safety, in particular their risk related to insertional mutagenesis. In the murine pre-clinical study presented here, 40 test and 10 control mice were transplanted with ex vivo manipulated bone marrow cells to assess the long-term effects of the transduction of hematopoietic cells with the retroviral vector MSCV-MGMT(P140K)wc. Test mice had significant gene marking 8-12 months post-transplantation with an average of 0.93 vector copies per cell and 41.5% of peripheral blood cells expressing the transgene MGMT(P140K), thus confirming persistent vector expression. Unexpectedly, six test mice developed malignant lymphoma. No vector was detected in the tumor cells of five animals with malignancies, indicating that the malignancies were not caused by insertional mutagenesis or MGMT(P140K) expression. Mice from a concurrent study with a different transgene also revealed additional cases of vector-negative lymphomas of host origin. We conclude that the background tumor formation in this mouse model complicates safety determination of retroviral vectors and propose an improved study design that we predict will increase the relevance and accuracy of interpretation of pre-clinical mouse studies.