Homeostasis and the age-associated defect of CD4 T cells

Survival and homeostatic division of naive CD4 T cells is regulated by the cellular and non-cellular milieu and together these processes ensure that a population of naive CD4 T cells persists into old age. However, the naive CD4 T cells from aged animals show reduced IL-2 production, proliferation, helper function and effector generation and memory function. We explore here whether the age-related defects in naive CD4 T cells are due to the aged environment from which they come or to intrinsic defects that are caused by homeostasis and their long lifespan.     

Relative carriage rates of nuclear dehydrogenating clostridia in two populations of different colorectal cancer risk

Carriage of nuclear dehydrogenating clostridia has been associated with colon cancer and implicated in its aetiology. This study has compared the carriage of these organisms in a British population at high risk for the development of colon cancer with a low risk Nigerian population. Clostridia were found in all of the stools from both populations. Nuclear dehydrogenating clostridia were only found in the stools of the British subjects (32%). These results support the suggestion that the carriage rate of nuclear dehydrogenating clostridia in a population is related to the risk of colon cancer.     

Quantitation of corneal neovascularization using computerized image analysis

We have developed a method for quantitating corneal neovascularization, induced in anesthetized rats by silver nitrate/potassium nitrate cauterization, using a LeMont OASYS video input image analyzer. Corneal vessels are visualized by perfusing the upper half of deeply anesthetized animals with a mixture of 10% india ink, 11% gelatin in lactated Ringer's solution. The eyes are then rapidly cooled using a stream of compressed dichlorodifluoromethane (Freon) to solidify the gelatin mixture. Magnified images of flat preparations of the corneas are acquired using a television camera attached to a dissecting microscope. The images are electronically converted to digital form and the digitized data are stored in the image analyzer. The area of the cornea and blood vessels are independently determined by analyzing the digitized data as discrete values of varying shades of gray (gray-scale analysis). The area and gray scale of the injury and its distance from the corneoscleral limbus can also be measured to determine variability of location and intensity of the injury in different animals. This technique allows the area occupied by new blood vessels during studies on corneal neovascularization in rats to be rapidly quantitated.     

Androgen receptor YAC transgenic mice carrying CAG 45 alleles show trinucleotide repeat instability

X-linked spinal and bulbar muscular atrophy (SBMA) is caused by a CAG repeat expansion in the first exon of the androgen receptor (AR) gene. Disease-associated alleles (37-66 CAGs) change in length when transmitted from parents to offspring, with a significantly greater tendency to shift size when inherited paternally. As transgenic mice carrying human AR cDNAs with 45 and 66 CAG repeats do not display repeat instability, we attempted to model trinucleotide repeat instability by generating transgenic mice with yeast artificial chromosomes (YACs) carrying AR CAG repeat expansions in their genomic context. Studies of independent lines of AR YAC transgenic mice with CAG 45 alleles reveal intergenerational instability at an overall rate of approximately 10%. We also find that the 45 CAG repeat tracts are significantly more unstable with maternal transmission and as the transmitting mother ages. Of all the CAG/CTG repeat transgenic mice produced to date the AR YAC CAG 45 mice are unstable with the smallest trinucleotide repeat mutations, suggesting that the length threshold for repeat instability in the mouse may be lowered by including the appropriate flanking human DNA sequences. By sequence-tagged site content analysis and long range mapping we determined that one unstable transgenic line has integrated an approximately 70 kb segment of the AR locus due to fragmentation of the AR YAC. Identification of the cis - acting elements that permit CAG tract instability and the trans -acting factors that modulate repeat instability in the AR YAC CAG 45 mice may provide insights into the molecular basis of trinucleotide repeat instability in humans.      

Heterogeneity of hepatocyte antigen expression in rat liver carcinogenesis: concordance between neoplastic nodules and tumours.

Fischer F344 rats were given a cyclical diet of 0.06% 2-acetylaminofluorene (AAF), which progressively induced oval cell proliferation, cirrhosis and hyperplastic (or neoplastic) nodules. Primary liver tumours developed from 7 months after ceasing the diet. Liver samples taken during and after AAF administration and specimens of primary tumours were processed into frozen sections and examined microscopically for morphological changes in cell populations, stained histochemically for gamma-glutamyl transpeptidase (GGTase) and four phosphatases, and stained by the immunoperoxidase technique for the presence of antigens detected by seven anti-liver cell monoclonal antibodies and monoclonal antibodies to six oncoproteins. During and after AAF treatment several of the anti-liver antibodies revealed foci of aberrantly or heterogeneously-stained cells, although anti-oncoprotein antibodies showed no consistent changes. Foci of cells positive for GGTase and heterogeneous for adenosine triphosphatase (ATPase) were also seen. Nodules invariably showed heterogeneous antigenicity, raised GGTase and abnormal ATPase expression. Primary tumours exhibited varying degrees of positivity, negativity and heterogeneity with the anti-liver monoclonal antibodies, and all were positive for GGTase. Comparison between various parameters and different lesions showed the greatest concordance between nodules and tumours, suggesting that nodules are probably the precursors of malignant tumours in this system.     

Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation

The recent improvements in the treatment of cancer by chemo- and radiotherapy have led to a significant increase in the survival rates of patients with malignant disease, but at the expense of distressing side effects. One major problem, especially for younger patients, is that aggressive therapy destroys a significant proportion of the follicular population, which can result in either temporary or permanent infertility. Freeze-banking pieces of ovarian cortex prior to treatment is one strategy for preserving fecundity. When the patient is in remission, fertility could, theoretically, be restored by autografting the thawed tissue at the orthotopic site or by growing isolated follicles to maturity in vitro. Recent studies have found good follicular survival in frozen-thawed human ovarian tissue but to optimize the process an effective cryopreservation method needs to be developed. An essential part of such a technique is to permeate the tissue with a cryoprotectant to minimize ice formation and the extent of this equilibration is an important determinant of post-thaw cellular survival. In the current study, we have investigated the diffusion of four cryoprotective agents into human tissue at both 4 degrees C and 37 degrees C. We have also studied the effect of adding different concentrations of the non penetrating cryoprotective agent, sucrose, to the freezing media using the release of lactate dehydrogenase as a measure of its protective effect. At 4 degrees C propylene glycol and glycerol penetrated the tissue significantly slower than either ethylene glycol or dimethyl sulphoxide. At the higher temperature of 37 degrees C all four cryoprotectants penetrated at a faster rate, however concern about enhanced toxicity prevents the use of these conditions in practice. Thus, the results suggest that the best method of preparing tissue for freezing is exposure for 30 min to 1.5 M solutions of ethylene glycol or dimethyl sulphoxide at 4 degrees C; this achieved a mean tissue concentration that was almost 80% that of the bathing solution. We also report that the addition of low concentrations of sucrose to the freezing medium does not have a significant protective effect against freezing injury.      

Reevaluation of a genetic model for the development of exostosis in hereditary multiple exostosis

EXT1 and EXT2 are genes that have been shown to cause hereditary multiple exostosis (HME), a syndrome marked by the formation of bony growths juxtaposed to the growth plate. These genes are members of a growing family of proteins with glycosyltransferase activity required for the synthesis of heparan sulfate chains. This protein activity is predicted to play a role in the expression of proteoglycans on the cell surface and in the extracellular matrix. We and others have previously suggested that a two-hit mutational model applies to the development of an exostosis where a germline mutation coupled with a somatic mutation results in the loss of EXT1 or EXT2 function and subsequent tumor formation. We report the direct sequencing and loss of heterozygosity (LOH) analysis of 12 exostoses from 10 HME families, 4 solitary exostoses, and their corresponding constitutional DNA. Of the 16 exostoses screened, we find only one solitary case in which two somatic mutations, a deletion and an LOH, are present. This provides limited support for the two-hit hypothesis involving the EXT1 and EXT2 genes for the development of an exostosis. Alternative models are developed based on the functional significance of EXT proteins in heparan sulfate biosynthesis.