Amplification of fluorescent in situ hybridisation signals in formalin fixed paraffin wax embedded sections of colon tumour using biotinylated tyramide.

Fluorescent in situ hybridisation (FISH) is a powerful tool for the evaluation of chromosomal alterations in formalin fixed paraffin wax embedded sections of colorectal cancer. However, initial experiments using a two-step detection system for digoxigenin labelled chromosome specific centromeric probes resulted in a complete lack of hybridisation signal from a number of colorectal tumour sections. This was due to high levels of background autofluorescence observed in this tissue, which masked any relatively weak hybridisations present. To overcome this problem, a biotinylated tyramide mediated amplification system was incorporated into the FISH detection protocol. This involves the use of horseradish peroxidase to activate the biotinylated tyramide, resulting in the deposition of a large number of biotin molecules at the site of bound peroxidase, which corresponds directly to the location of hybridised probe. Final detection was by means of a streptavidin-FITC conjugate. Using this technique, a panel of 11 colorectal tumour samples studied to date have shown strong, specific hybridisation signals to the nucleus of tumour cells. Amplification of FISH signals by biotinylated tyramide has the potential to improve weak hybridisation signals in cells from numerous sources, using a variety of probe types, including single copy gene probes as well as centromere specific probes.     

Electrodermal Lability and Visual Information Processing

Individual differences in electrodermal lability have been related to performance in vigilance and reaction time tasks. The goal of the present study was to employ an "additive factors" approach to identify the stages of information processing that might underlie these effects. Nineteen labile and 17 stabile subjects performed a choice reaction time task in which a visual imperative stimulus was presented under two conditions of intensity (presumed to affect the speed of pre-processing operations) X three conditions of degradation (which influences later encoding processes related to feature extraction). Measures of both reaction time and movement time were obtained. The major findings were: (a) labile subjects had faster reaction times than stabiles, and (b) lability interacted significantly with stimulus degradation. Labiles also tended (p less than .10) to have faster movement times. This pattern indicates that labiles and stabiles differ in the performance of later encoding operations, and possibly in the speed of motor processes as well. However, they do not appear to differ in the early pre-processing of the simple physical attributes of a stimulus.     

Concurrent de novo interstitial deletion of band 2p22 and reciprocal translocation (3;7)(p21;q22).

A child is described with a de novo interstitial deletion of band 2p22 and a reciprocal translocation (3;7)(p21;q22). The child has mild developmental delay, coloboma of the right eye, and Hirschsprung's disease. The clinical and cytogenetic findings are described.     

Receptor-binding function of type 1 pili effects bladder colonization by a clinical isolate of Escherichia coli.

The role of type 1 pili in promoting bladder colonization was examined by constructing two mutant strains of a clinical Escherichia coli isolate. One mutant was isogenic to the parental strain save for a lesion in a gene required for pilus receptor binding; the other mutant was isogenic save for a lesion in the gene encoding the pilus structural subunit. Using mixed infections of the parental and mutant strains in an ascending rat cystitis model, we found that type 1-piliated mutants that lacked the receptor-binding function were as ineffective in bladder colonization as were mutants lacking the entire organelle.     

Tissue engineering of arteries by directed remodeling of intact arterial segments

Traditional approaches to generating tissue-engineered arteries in vitro rely on expansion of cells in culture to seed appropriate scaffolds. In most envisioned applications, small autologous blood vessels would be harvested and used as a source for these cells. We propose that small autologous arteries, not the cells derived from them, may be an attractive starting point for engineered arteries. This approach capitalizes on the ability of intact arteries to grow and remodel in response to chronic changes in their mechanical environment. Carotid arteries from juvenile (approximately 30-kg) pigs were stretched longitudinally in an ex vivo perfusion system over 9 days. This resulted in a 40% increase in artery length at physiological longitudinal stress and a 20 +/- 3% increase when unstressed. Control arteries were perfused for 9 days ex vivo at their physiological loaded length. Control and elongated arteries displayed native appearance (macroscopic and histological), excellent viability (cellularity and mitochondrial activity), normal vasoactivity, and similar mechanical properties (ultimate stress and ultimate strain) as compared with freshly harvested arteries. Growth, as opposed to just redistribution of existing mass, contributed to elongation as evidenced by an increase in artery weight. Results on elongation of arteries from neonatal and adolescent pigs are also presented and discussed.     

Estrogen effects on object memory and cholinergic receptors in young and old female mice.

We investigated whether object recognition memory is modulated by estrogen in young (5 month) and aged (24 month) female C57Bl/6J mice, and if cholinergic muscarinic receptors might contribute to this response. Mice that were ovariectomized, or ovariectomized plus estradiol-treated three weeks before behavioral testing or quantitative autoradiography were compared to intact mice. Memory for a previously encountered object deteriorated significantly between 3 and 6h after initial exposure, regardless of animal age. In both young and aged mice, estradiol-treated mice showed significantly greater recall than did ovariectomized mice. In both age groups, the apparent number of [(3)H]pirenzepine/M(1)-like and [(3)H]AFDX384/M(2)-like muscarinic receptor binding sites was reduced in the basal forebrain as well as its projection areas following ovariectomy, but this decrease was not alleviated by estrogen. Aging poorly affected object memory, but reduced muscarinic binding in some cortical subregions and in the caudate nucleus. These findings suggest that estrogen effects on memory in C57Bl/6J mice are not due to changes in the number of muscarinic receptors.     

T cell immunodominance and maintenance of memory regulated by unexpectedly cross-reactive pathogens

We show here that T cell cross-reactivity between heterologous viruses influences the immunodominance of virus-specific CD8(+) T cells by two mechanisms. First, T cells specific for cross-reactive epitopes dominate acute responses to viral infections; second, within the memory pool, T cells specific for cross-reactive epitopes are maintained while those specific for non-cross-reactive epitopes are selectively lost. These findings suggest an immunological paradigm in which viral infections shape the available T cell repertoire, causing alterations in the hierarchies of both the primary and memory CD8(+) T cell responses elicited by subsequent viral infections. Thus, immunodominance is a function of the host's previous exposure to unrelated pathogens, and this may have an impact on protective immunity and immunopathology.     

CYFIP2 is highly abundant in CD4+ cells from multiple sclerosis patients and is involved in T cell adhesion

DNA microarray profiling of CD4(+) and CD8(+) cells from non-treated relapsing and remitting multiple sclerosis (MS) patients determined that the cytoplasmic binding partner of fragile X protein (CYFIP2, also called PIR121) was increased significantly compared to healthy controls. Western analysis confirmed that CYFIP2 protein was increased approximately fourfold in CD4(+) cells from MS compared to inflammatory bowel disorder (IBD) patients or healthy controls. Because CYFIP2 acts as part of a tetrameric complex that regulates WAVE1 activation we hypothesized that high levels of CYFIP2 facilitate T cell adhesion, which is elevated in MS patients. Several findings indicated that increased levels of CYFIP2 facilitated adhesion. First, adenoviral-mediated overexpression of CYFIP2 in Jurkat cells increased fibronectin-mediated adhesion. Secondly, CYFIP2 knock-down experiments using antisense oligodeoxynucleotides reduced fibronectin-mediated binding in Jurkat and CD4(+) cells. Thirdly, inhibition of Rac-1, a physical partner with CYFIP2 and regulator of WAVE1 activity, reduced fibronectin-mediated adhesion in Jurkat and CD4(+) cells. Finally, inhibition of Rac-1 or reduction of CYFIP2 protein decreased fibronectin-mediated adhesion in CD4(+) cells from MS patients to levels similar to controls. These studies suggest that overabundance of CYFIP2 protein facilitates increased adhesion properties of T cells from MS patients.