Nuclear iASPP may facilitate prostate cancer progression.

One of the major challenges in prostate cancer (PCa) research is the identification of key players that control the progression of primary cancers to invasive and metastatic disease. The majority of metastatic PCa express wild-type p53, whereas loss of p63 expression, a p53 family member, is a common event. Here we identify inhibitor of apoptosis-stimulating protein of p53 (iASPP), a common cellular regulator of p53 and p63, as an important player of PCa progression. Detailed analysis of the prostate epithelium of iASPP transgenic mice, iASPP(Δ8/Δ8) mice, revealed that iASPP deficiency resulted in a reduction in the number of p63 expressing basal epithelial cells compared with that seen in wild-type mice. Nuclear and cytoplasmic iASPP expression was greater in PCa samples compared with benign epithelium. Importantly nuclear iASPP associated with p53 accumulation in vitro and in vivo. A pair of isogenic primary and metastatic PCa cell lines revealed that nuclear iASPP is enriched in the highly metastatic PCa cells. Nuclear iASPP is often detected in PCa cells located at the invasive leading edge in vivo. Increased iASPP expression associated with metastatic disease and PCa-specific death in a clinical cohort with long-term follow-up. These results suggest that iASPP function is required to maintain the expression of p63 in normal basal prostate epithelium, and nuclear iASPP may inactivate p53 function and facilitate PCa progression. Thus iASPP expression may act as a predictive marker of PCa progression.

Cross-species reactivity of a panel of antibodies with monkey and porcine tissue.

The continuing shortage of organs available for transplantation limits the number of patients able to benefit from this highly successful form of therapy. Interest in alternative sources of organs has now turned towards the pig because of its physiological similarity to human. There is a requirement therefore for reagents not only for research purposes but possibly for studying xenotransplants in the clinical situation in the future. In this study, we have concentrated on determining the cross-species reactivity of a large panel of antibodies directed against human leukocyte markers, testing peripheral blood leukocytes and also including renal tissue to determine non-leukocyte cross-reactivity. A total of 63 out of 127 antibodies cross-reacted with cynomolgus monkey cells. Twenty of these antibodies stained similar populations of leukocytes to human, whereas the remaining 43 reacted with clearly different populations. The majority of antibodies (108/127) were unreactive with porcine leukocytes, reflecting the evolutionary differences between pig and man. Of the 19 antibodies cross-reactive with porcine cells, seven reacted with similar proportions of leukocytes to human, whereas the remaining 12 antibodies stained entirely different populations. The most interesting, and potentially most useful, antibodies were four that reacted with human, cynomolgus monkey and porcine tissue in a similar manner, suggesting that the epitopes recognized are present on similar molecules. These antibodies were directed against CD29 (MEM1O1A, K20) and CD18 (BU87, 7E4), the common beta1- and beta2-integrin subunits respectively. This study demonstrates that there are antigens common to cynomolgus monkey, pig and man that react with currently available antibodies. Nevertheless, when determining cross-species reactivity of human antibodies, it is important to consider the possibility that there may be additional non-leukocyte reactivity in other tissues.

Nested polymerase chain reaction with sequence-specific primers typing for HLA-A, -B, and -C alleles: detection of microchimerism in DR-matched individuals.

It is widely accepted that donor leukocytes survive within the recipient periphery after blood transfusion or solid organ transplantation. The significance of this microchimerism remains unclear, partially because of the insecurity of assays used to detect the donor-derived material. The techniques used to detect donor-derived DNA within recipient peripheral blood rely largely on major histocompatibility complex class II polymorphism. We and others have shown that the sensitivity of polymerase chain reaction with sequence-specific primers (PCR-SSP) typing for HLA class II alleles can be increased 100-fold by the addition of a primary amplification step (nested PCR-SSP). We have now extended this technique to encompass typing for HLA class I alleles, thereby adding flexibility to microchimerism testing by enabling testing of recipients HLA-DR matched with their donors. However, the high level of sensitivity achieved with the technique (1:100,000) leads to a concomitant decrease in the specificity that results in the amplification of unexpected products, a phenomenon we encountered in the development of our nested PCR-SSP typing system for HLA class II alleles. We describe here how it is possible to compensate for these anomalies by including multiple testing of a pretransfusion sample that acts as a specificity control, establishing a rigorous baseline for subsequent analysis.

Apoptosis in ischemia/reperfusion injury of human renal allografts.

BACKGROUND: Ischemia/reperfusion injury of human renal allografts has a number of clinically significant consequences. A number of mechanisms of ischemia/ reperfusion injury have been elucidated, and there is evidence that apoptosis may be a contributing factor. METHODS: To examine immediate posttransplant events, fixed tissue sections from paraffin-embedded wedge biopsy specimens taken before and after reperfusion of human renal allografts were stained using terminal deoxytransferase-mediated dUTP nick-end labeling to detect the DNA fragmentation characteristic of apoptosis. Thirty-six pairs of pre- and postreperfusion biopsy specimens were examined, 11 from living-related donor renal transplants and 25 from cadaveric donor transplants. RESULTS: Quantitation of the terminal deoxytransferase-mediated dUTP nick-end labeling signal showed that significantly more apoptosis occurred in postreperfusion compared with prereperfusion biopsy specimens from cadaveric donor transplants, but a similar difference was not observed in living-related donor renal transplants. Furthermore, significantly more apoptosis was observed in postreperfusion biopsy specimens from cadaveric compared with living-related renal transplants. Postreperfusion biopsy specimens from kidneys that were cold preserved longer than 30 hr had a higher mean apoptosis score than those stored for less than 24 hr, but the result was not statistically significant. CONCLUSIONS: Thus, apoptosis occurs predominantly as a result of reperfusion after cold preservation of cadaveric donor renal allografts and provides additional information regarding the extent of ischemia/ reperfusion injury in an organ. The clinical value of this information remains to be determined.

Detection of microchimerism after allogeneic blood transfusion using nested polymerase chain reaction amplification with sequence-specific primers (PCR-SSP): a cautionary tale.

In bone marrow transplantation, the detection of chimerism is an important adjunct to the repertoire of tests available for determining acceptance of the graft. In solid organ transplantation, there is currently intense interest in the role that chimerism plays in both short- and long-term host reactivity to the graft. Allogeneic blood transfusion has been associated with a subtle immunosuppressive effect in renal transplantation and chimerism is implicated as a possible mechanism for this effect. To assess the survival of allogeneic cells after blood transfusion or transplantation, we have developed a technique based on molecular typing for HLA class II alleles, which enables the detection of donor-derived cells in patients receiving blood transfusions. While developing this technology, we investigated why we and others observe false amplification. Sequencing of false products has shown that they arise from amplification of both pseudogenes and non-pseudogenes present in the DNA under test. Elucidation of this phenomenon allows the amplification of these false products to be predicted in any given combination and hence avoided by the judicious selection of primers. Validation has been achieved by following donor alleles after transfusion of blood containing defined numbers of leukocytes expressing selected mismatched antigens.