Clinical trial management and remote data entry on the Internet based on XML case report forms.

The Internet and the World Wide Web have recently been introduced into the management of some aspects of clinical trials such as remote randomisation and data entry and the distribution of information on trial progress. A few larger trials use electronic Case Report Forms (eCRFs) built with HTML. This article describes a more flexible approach using the Extensible Markup Language XML.

Syndactyly of Ft/+ mice correlates with an imbalance in bmp4 and fgf8 expression.

The most obvious phenotype of Ft/+ mice is a syndactyly of fore limbs characterised by a fusion of the tips of digits 1 to 4. The tempospatial expression of genes involved in limb development revealed that patterning of Ft/+ limb buds is not affected by the mutation. However, an upregulation of Bmp4 in the anterior-distal region of the limb bud at d12.0 of embryonic development is accompanied by a loss of Fgf8 expression in the distal part of the AER. Downstream target genes of Bmp action such as Msx1 and 2 are upregulated. This induction of the signalling cascade indicates ectopic expression of functional Bmp4. Nevertheless, analysis of physical parameters of bones from adult mice revealed a reduction of the bone mass of the autopod. The data suggest a negative effect of Bmp4 on Fgf8 expression and a positive influence on the induction of bone elements.

Id genes are direct targets of bone morphogenetic protein induction in embryonic stem cells.

Bone morphogenetic proteins (BMPs) are morphogenetic signaling molecules essential for embryonic patterning. To obtain molecular insight into the influence of BMPs on morphogenesis, we searched for new genes directly activated by BMP signaling. In vitro cultured mouse embryonic stem (ES) cells were used, cultivated in chemically defined growth medium (CDM). CDM-cultured ES cells responded very selectively to stimulation by various mesoderm inducers (BMP2/4, activin A, and basic fibroblast growth factor). BMP2/4 rapidly induced transcript levels of the homeobox genes Msx-1 and Msx-2 and the proto-oncogene JunB, whereas c-jun transcripts displayed delayed albeit prolonged increase. Using differential display cDNA cloning, six direct BMP target genes were identified. These include Id3, which showed strong mRNA induction, and the moderately induced Cyr61, DEK, and eIF4AII genes, as well as a gene encoding a GC-binding protein. Besides Id3, also the Id1 and Id2 genes were activated by BMP4 in both ES cells and a range of different cell lines. Id genes encode negative regulators of basic helix-loop-helix transcription factors. In vivo we observed local ectopic expression of Id3 and Msx-2 mRNAs in Ft/+ embryos at overlapping regions of ectopic Bmp4 misexpression. We therefore propose that the Msx and Id genes are direct target genes of embryonic BMP4 signaling in vivo.

The expression pattern of nodal and lefty in the mouse mutant Ft suggests a function in the establishment of handedness.

We have investigated the expression of left/right (L/R) asymmetry markers, nodal and lefty, in the situs inversus mouse mutant Fused toes (Ft). Both genes exhibited bilateral expression in the lateral plate mesoderm (LPM) at developmental stages whereas in wildtype embryos these genes were found to be expressed exclusively in the left LPM. Inspection of tail location and primitive heart tube looping, structures known to be handed in their orientation, documented a random orientation of these structures. Crossing of the Ft mutation into a different genetic background resulted in a strong reduction of this random orientation. Although the major fraction of these individuals still displayed nodal and lefty on both sides of the LPM, expression was almost always found to be weaker in the right LPM. These results suggest that the establishment of asymmetry is independent of nodal or lefty signals. However, handed asymmetry, which means consistent L/R differences, such as the dextral looping of the primitive heart tube or the right-oriented tail, is directed by differences in the L/R expression pattern of these two genes.

Evidence for genetic control of Sonic hedgehog by Gli3 in mouse limb development.

Sonic hedgehog (Shh) expression in the developing limb is associated with the zone of polarising activity (ZPA), and both are restricted to the posterior part of the limb bud. We show that the expression patterns of Shh and Gli3, a member of the Gli-family believed to function in transcriptional control, appear to be mutually exclusive in limb buds of mouse embryos. In the polydactyly mouse mutant extra toes (Xt), possessing a null mutation of Gli3, Shh is additionally expressed in the anterior region of the limb bud. The transcript of Ptc, the putative receptor for Shh protein, can be detected anteriorly as well. Other genes known to be involved in limb outgrowth and patterning, like Fibroblast growth factor (Fgf), Bone morphogenetic protein (Bmp), and Hoxd are misexpressed in relation to the ectopic Shh expression domain in Xt limb buds. This data suggest that Gli3 is a regulator of Shh expression in mouse limb development.

Kinetics of inhibition of replication of vesicular stomatitis virus in blood mononuclear cells of horses after in vitro and in vivo treatment with recombinant equine interferon-beta 1.

Recombinant equine interferon-beta 1 (reqIFN-beta 1) induces an antiviral state in blood mononuclear cells (BMC) of horses. Maximal protection against replication of vesicular stomatitis virus is achieved 6 hours after treatment with IFN in vitro and in vivo. Duration of the protective effect depends on the dose of IFN in vitro and in vivo. Availability of reqIFN-beta 1 in cultures of BMC for up to 48 hours does not prolong the antiviral state. The protective effect on BMC after treatment with IFN has similar duration in vivo and in vitro. Monitoring of the effect of IFN in vivo is, thus, simplified because the antiviral state may be recorded by testing cells twice (ie, before and 6 hours after application of interferon). All further tests may be performed in vitro. Multiple administration of reqIFN-beta 1 do not prolong duration of the protective phases after each administration. Duration of the antiviral state depends only on the dose of reqIFN-beta 1.

Single and dual labelling of cytotoxic target cells. Comparison of three radioactive tracers, [35S]methionine, [75Se]selenomethionine, and chromium-51.

[75Se]selenomethionine (75SeM) has been shown to provide several advantages over Na(2)51CrO4 (51Cr) labelling of metabolizing target cells: high labelling efficiency and low spontaneous release of 75SeM-labelled target cells permit improved monitoring of cytotoxicity due to extended effector/target ratios in short- and long-term assays. Unfortunately, 75SeM will soon be difficult to obtain. Therefore we studied the suitability of [35S]methionine (35SM) as a substitute for 75SeM. Furthermore, we explored the potential of dual labelling of suspension target cells applying combinations of 35SM and 51Cr or 75SeM and 51Cr. 35SM is a suitable substitute for 75SeM retaining most of the advantages of 75SeM labelling. Although considerably higher labelling of cells is possible we obtained the most efficient labelling with 100-400 kBq/ml of 35SM or 75SeM resulting in a relatively high uptake (3-15 cpm/cell) and very low spontaneous release (1-2%/h) up to 24 h. This permits short- and long-term cytotoxic assays and the use of low numbers of target cells (1 x 10(3)) providing increased cytotoxic sensitivity with reduced amounts of effector cells. Suitable dual labelling of target cells with 35SM plus 51Cr or 75SeM plus 51Cr documented convincingly identical release kinetics for 35SM and 75SeM but partially discordant ones for 51Cr. Depending on the target cell used dual labelling permits discrimination and monitoring of different cytotoxic or release mechanisms in cellular cytotoxicity.

"Discordant" influence of equine recombinant interferon-beta 1 on the cytotoxic capacity of equine polymorphonuclear neutrophils and peripheral blood mononuclear cells in vitro and in vivo.

The influence of recombinant equine interferon-beta 1 (rEqIFN-beta 1) on mononuclear cells of peripheral blood (PBMC) and polymorphonuclear neutrophilic granulocytes (PMN) was tested under in vitro and ex vivo conditions. Treatment of equine PBMC with IFN in vitro enhanced the antibody-independent cytotoxicity (AICC) and antibody-dependent cytotoxicity (ADCC) while there was no significant effect on the cytotoxic capacity of PMN treated with rEqIFN-beta 1 in vitro. Ex vivo there was an increased capacity of AICC and ADCC upon single or multiple application of rEqIFN-beta 1 in PMN, only. Treatment with rEqIFN-beta 1 thus induced an increased cellular cytotoxicity in vitro and in vivo but in different populations of peripheral blood cells. In vivo rEqIFN-beta 1 causes a pronounced activation of PMN but not of PBMC as cytotoxic effector cells. This might be achieved indirectly, e.g., by cytokines produced by IFN-sensitive cells.

Monitoring of effects induced by recombinant equine interferon-beta 1 in whole blood and separated fractions of peripheral blood of horses.

Interferon is known to induce antiviral mechanisms and to exert immunoregulatory capacities on various cell types. The antiviral capacity of recombinant equine interferon-beta 1 (rEqIFN-beta 1) is most sensitively monitored by indirect quantitation of multiplication of vesicular stomatitis virus (VSV) in blood cells of horses. As few as 0.5 pg rEqIFN-beta 1/ml can be assessed by means of 90% reduction of VSV-replication in whole blood (w.b.) as well as in isolated mononuclear blood cells (MNC) in spite of individual variations. The immunoregulatory influence of 20-50 pg rEqIFN-beta 1/ml is sufficient to cause at least a 50% reduction of mitogen-induced lymphocyte proliferation in MNC, while higher concentrations are needed in w.b. Of the mitogens tested the best stimulation of proliferation on the equine lymphoid cells was obtained with staphylococcal enterotoxin B (SEB). Release of reactive oxygen species (ROS) from phagocytic cells in w.b. or from isolated polymorphonuclear cells (PMN) as monitored by chemiluminescence (CL) does not seem suitable for evaluation of rEqIFN-beta 1-induced immunoregulation as only very high rEqIFN-beta 1-concentrations (10(3)-10(4) pg/ml) result in a minute increase (up to 20%) of CL. Comparative studies on w.b. and isolated leukocyte fractions from identical specimens of individual horses suggest that monitoring of antiviral and distinct immunoregulatory capacities of rEqIFN-beta 1 can be performed on w.b. without loss of information and sensitivity as compared to isolated MNC.

A method for the assay of "difficult" interferons exemplified with recombinant equine interferon-beta 1.

We wished to assay recombinant equine interferon-beta 1 (rEqIFN-beta 1) but could not obtain satisfactory results with previously described methods. Therefore, we developed a yield-reduction assay, using primary horse peripheral blood mononuclear cells (PBMC) with vesicular stomatitis virus (VSV) for challenge, which proved consistently satisfactory and highly sensitive. It is suggested that this method of assay may be useful for IFNs from other animals where problems are encountered.