A novel transcriptional repression domain mediates p21(WAF1/CIP1) induction of p300 transactivation.

The transcriptional coactivators p300 and CREB binding protein (CBP) are important regulators of the cell cycle, differentiation, and tumorigenesis. Both p300 and CBP are targeted by viral oncoproteins, are mutated in certain forms of cancer, are phosphorylated in a cell cycle-dependent manner, interact with transcription factors such as p53 and E2F, and can be found complexed with cyclinE-Cdk2 in vivo. Moreover, p300-deficient cells show defects in proliferation. Here we demonstrate that transcriptional activation by both p300 and CBP is stimulated by coexpression of the cyclin-dependent kinase inhibitor p21(WAF/CIP1). Significantly this stimulation is independent of both the inherent histone acetyltransferase (HAT) activity of p300 and CBP and of the previously reported carboxyl-terminal binding site for cyclinE-Cdk2. Rather, we describe a previously uncharacterized transcriptional repression domain (CRD1) within p300. p300 transactivation is stimulated through derepression of CRD1 by p21. Significantly p21 regulation of CRD1 is dependent on the nature of the core promoter. We suggest that CRD1 provides a novel mechanism through which p300 and CBP can switch activities between the promoters of genes that stimulate growth and those that enhance cell cycle arrest.

Transcriptional cross talk between NF-kappaB and p53.

Many cellular stimuli result in the induction of both the tumor suppressor p53 and NF-kappaB. In contrast to activation of p53, which is associated with the induction of apoptosis, stimulation of NF-kappaB has been shown to promote resistance to programmed cell death. These observations suggest that a regulatory mechanism must exist to integrate these opposing outcomes and coordinate this critical cellular decision-making event. Here we show that both p53 and NF-kappaB inhibit each other's ability to stimulate gene expression and that this process is controlled by the relative levels of each transcription factor. Expression of either wild-type p53 or the RelA(p65) NF-kappaB subunit suppresses stimulation of transcription by the other factor from a reporter plasmid in vivo. Moreover, endogenous, tumor necrosis factor alpha-activated NF-kappaB will inhibit endogenous wild-type p53 transactivation. Following exposure to UV light, however, the converse is observed, with p53 downregulating NF-kappaB-mediated transcriptional activation. Both p53 and RelA(p65) interact with the transcriptional coactivator proteins p300 and CREB-binding protein (CBP), and we demonstrate that these results are consistent with competition for a limiting pool of p300/CBP complexes in vivo. These observations have many implications for regulation of the transcriptional decision-making mechanisms that govern cellular processes such as apoptosis. Furthermore, they suggest a previously unrealized mechanism through which dysregulated NF-kappaB can contribute to tumorigenesis and disease.

Identification of localized anti-host responses in the graft mesenteric lymph node and Peyer's patches after rat small bowel transplantation.

This study determined the activation status of recipient and donor lymphocyte populations in the graft mesenteric lymph node (MLN) and Peyer's patches (PP) after allogeneic, heterotopic rat small bowel transplantation without immunosuppression. Untransplanted and isografted animals served as controls. The activation status of lymphocyte subsets was determined by flow cytometric evaluation of lymphoblastoid transformation (forward light scatter; FSc). The proportion of activated lymphocytes in the MLN and PP of allografted animals progressively increased. There was also an early transient activation of MLN lymphocytes in isografted animals which probably resulted from surgery-related inflammation. Activated alpha/beta TCR+ and CD4+ cells were detected in the MLN as early as day 3, whereas there was little activation of CD8+ cells. Interestingly, donor lymphocytes became more activated than recipient lymphocytes. Allografting also led to activation of graft-derived PP alpha/beta TCR+ and CD8+ cells, yet there was no detectable activation of recipient-derived lymphocytes. In summary, this study has identified activated donor lymphocytes in the graft MLN and PP after allogeneic small bowel transplantation. Although rejection predominates without immunosuppression, the presence of an underlying anti-recipient response within the small bowel allograft may contribute to graft damage via the localized release of cytokines and inflammatory mediators.

Identification of migratory graft and host cell populations after allogeneic rat small bowel transplantation.

This study used flow cytometry to identify graft cells in the recipient peripheral blood and spleen and host cells infiltrating the graft mesenteric lymph node and Peyer's patches after heterotopic rat small bowel transplantation. Transplantation had little effect on the overall cell subset composition of these compartments and no changes appeared characteristic or indicative of developing rejection, suggesting that physiological control of cell migration remained unaltered. A small and transient population of graft cells was detected in the peripheral blood and spleen of the recipient which disappeared after 5 and 3 days respectively. Graft-derived cells in the peripheral blood comprised predominantly CD4+ cells on day 1 with B cells predominating on day 5. Graft cells infiltrating the spleen were predominantly B cells. Host cells infiltrated the graft mesenteric lymph nodes and Peyer's patches to a lesser extent than previously reported using immunohistochemical analysis. For both tissues, infiltrating host-derived cells initially comprised mainly CD4+ cells. On day 4 approximately equal proportions of CD4+ and B cells were present in the mesenteric lymph node, whereas B cells were predominant in the host cell infiltrate of the graft Peyer's patches. In summary, these findings indicate that the cell subset composition of recipient and graft lymphoid compartments does not change after small bowel transplantation, even in the presence of a substantial recipient cell infiltration. The reasons for the apparent discrepancies in the degree of host cell infiltration when assessed using immunohistochemical and flow cytometric techniques are currently uncertain, but may result from the localised release of soluble MHC class I in graft tissues as a consequence of infiltrating host cell activation or localised cell destruction.

Flow cytometric analysis of peripheral blood lymphocyte subset light scatter characteristics as a means of monitoring the development of rat small bowel allograft rejection.

This investigation used flow cytometry to monitor peripheral blood lymphocyte morphology after rat small bowel transplantation. Preliminary studies demonstrated that in vitro activated peripheral blood lymphocytes exhibited increased cell size and granularity as measured by flow cytometric analysis of forward (FSc) and side (SSc) light scatter characteristics. The formation of distinct 'activated' light scatter regions by such lymphoblastoid transformation occurred concomitantly with up-regulated p55IL-2R expression. Heterotopic small bowel transplantation was performed between PVG donor and DA recipient rats without immunosuppression. Animals receiving isografts served as controls. Peripheral blood lymphocyte subsets were identified using appropriate MoAbs, and the light scatter characteristics of each cell subset were determined by backgating strategies. Increased proportions of activated alpha/beta T cell receptor (TCR)-positive cells could be detected in allografted animals as early as day 2 post-transplantation. B cells showed peak activation by day 4, at which time the proportion of activated cells was over two-fold greater than that seen in untransplanted animals--few activated B cells were detected in isografted animals. Resting natural killer (NK) cell light scatter regions only partially overlap with those of resting T and B lymphocytes, but in allografted animals almost the entire NK population fell outside the resting lymphocyte gate by day 2 post-transplantation, an activation state which was maintained until day 4. These findings associate peripheral blood cell subset lymphoblastoid transformation with developing small bowel allograft rejection. Importantly, changes were detected early and prior to the onset of overt rejection. These data suggest that analysis of peripheral blood lymphocyte light scatter properties may provide an insight into in vivo immune status after small bowel transplantation.

Activation antigen expression on peripheral blood neutrophils following rat small bowel transplantation. NKR-P1 is a novel antigen preferentially expressed during allograft rejection.

This study used flow cytometric analyses to monitor activation antigen expression (MHC class II; interleukin-2 receptor, p55IL-2R and 3.2.3/NKR-P1 antigen) on peripheral blood neutrophils following rat small bowel transplantation. The rat 3.2.3 antigen is a member of the NKR-P1 family of natural killer (NK) cell-associated molecules, which are expressed at high levels on NK cells and lymphokine-activated killer cells, and low levels on at least one T cell subset. Peripheral blood neutrophils in normal animals express very low or undetectable levels of NKR-P1. Detectable levels of NKR-P1 were induced as early as day 1 following small bowel transplantation in all allografted animals, whereas expression was only rarely detected in isografted animals. In addition, NKR-P1 density was significantly higher in allografted animals and was maintained as rejection developed. MHC class II and p55IL-2R expression was also induced following transplantation. The mechanisms of induction and functional relevance of NKR-P1 expression on neutrophils remain to be defined. However, the concomitant increased expression of MHC class II and p55IL-2R suggest NKR-P1 to be a neutrophil activation marker and implicate a potential role for NKR-P1+ neutrophils in small bowel allograft rejection. This hypothesis is further supported by the loss of detectable peripheral blood neutrophils only with developing rejection. Flow cytometric analysis of neutrophil activation antigen expression may be useful for monitoring human small bowel transplant recipients.

Effect of red cell lysis protocols on the expression of rat peripheral blood lymphocyte subset and activation antigens.

The effect of ammonium chloride and a commercial red cell lysing agent (Erythrolyse) on cell subset (CD4, CD8, B cell common leucocyte antigen) and activation (p55IL-2R [OX39], MHC class II) antigen expression by freshly isolated and in vitro activated rat peripheral blood lymphocytes was compared. Freshly isolated cells treated with the commercial lysing agent Erythrolyse were 42 +/- 12% p55IL-2R+, whereas p55IL-2R expression was not detected following red cell lysis with ammonium chloride. The expression of cell subset and MHC class II antigens was unaffected by either of the treatment protocols. Density gradient separation of freshly isolated cells had variable effects on antigen expression. In some cases up to a two-fold increase in p55IL-2R expression was observed, whilst in others there was a reduced receptor expression. Similarly, MHC class II antigen expression was either increased or reduced following density gradient separation. IL-2R expression on cells activated in vitro with the mitogen concanavalin A (95 +/- 2% IL-2R+) was unaffected by ammonium chloride lysis. Incubation of freshly isolated lymphocytes with p55IL-2R antibody prior to ammonium chloride treatment did not prevent the reduction in receptor expression. These findings suggest that ammonium chloride lysis of freshly isolated rat peripheral blood prior to IL-2R expression analysis should be avoided.

Epidemiology and diagnosis of echinococcosis in Canada.

TWO SPECIES OF ECHINOCOCCUS OCCUR IN CANADA: (1) E. multilocularis and (2) E. granulosus. E. multilocularis, originating in the Arctic, is spreading southwards and has reached Saskatchewan and the Dakotas. The original hosts are foxes but dogs and cats are alternatives. The larvae occur in field mice as multilocular microcysts containing numerous protoscolices, but in man the cysts are alveolar and sterile and resemble both in histology and growth a cholangiocellular carcinoma of the liver with metastases. Signs and symptoms are chronic and poorly defined. Diagnosis is difficult. Test antigens are not yet satisfactory. E. granulosus has a sylvatic cycle, the adult tapeworms living in wolves and dogs, while the larvae occur only in Cervidae and man. The cysts occur almost exclusively in the lungs as unilocular, macrocystic, relatively benign tumours, although abnormal complications can occur. The Casoni intradermal sensitivity test, its technique and interpretation are discussed.