Gitte S. Jensen

Regulation of urokinase plasminogen activator/plasmin- mediated invasion of melanoma cells by the integrin vitronectin receptor αVβ3

The integrin vitronectin receptor αvβ3 is a mediator of cellular migration and invasion and has been identified as a marker of progression in malignant melanoma. Using a human melanoma model, we have previously shown that this receptor was coordinately expressed with the receptor for the urokinase plasminogen activator (uPAR). In our present study, the link between these receptors was further investigated by assessing the effect of αvβ3 ligation on uPAR transcription and function. Using the reverse transcription‐polymerase chain reaction, we found that receptor ligation by immobilized monoclonal antibodies (MAbs) induced a rapid increase (up to 4.5 fold) in uPAR mRNA levels, which was maximal 4 hr after cell attachment. An increase was also noted in plasminogen activator inhibitor type‐1 (PAI‐1) mRNA levels (2.7‐fold), but none was noted in uPA levels. In addition, ligation of αvβ3 resulted in a significant increase in cell surface‐associated plasmin levels, which coincided with a 2‐ to 3‐fold increase in cell invasion as measured in the Matrigel invasion assay. This increase in invasion could in turn be abolished by antibodies directed to uPA and uPAR and by the plasmin inhibitors ϵ‐aminocaproic acid and aprotinin. Furthermore, ligation of the integrin αvβ3 triggered a rapid increase of up to 12‐fold in total cellular PKC activity, and this coincided with the redistribution of PKCβ, but not PKCα, from the cytosol to the membrane. Treatment of the cells with the PKCβ‐specific inhibitor LY379196 blocked uPAR and PAI‐1 mRNA induction and reduced the increase in cell invasion due to αvβ3 ligation, confirming the involvement of this isoform in the response. The results provide evidence that the vitronectin receptor can enhance invasion by regulating the uPAR/uPA/plasmin system of proteolysis and implicate PKCβ as an intermediate in the activation pathway.

Experimental discordant hepatic xenotransplantation in the recipient with liver failure: implications for clinical bridging trials

BACKGROUND Clinical xenotransplantation might start with bridge-to-bridge trials. Situations where hyperacute rejection is avoided would provide opportunities for the initiation of bridging trials. Patients with liver failure have a diminished capacity to initiate antibody and complement-induced injury of xenogeneic endothelium. Hyperacute rejection of a liver xenograft manifests as a coagulopathy. We examined the ability of a recipient with liver failure to hyperacutely reject a liver xenograft in the dog-to-pig model in the immediate postoperative period. STUDY DESIGN Liver failure in pigs was induced with galactosamine. Canine livers were transplanted into pigs with liver failure and into healthy pigs. The postoperative course was monitored for 1 hour for histologic changes in the xenograft, changes in platelet counts, and whole blood clotting with Sonoclot analysis. In vitro assays with pig serum and canine hepatic sinusoidal endothelial cells were used to assess the effect of liver failure on serum cytotoxicity and xenoreactive antibody levels. RESULTS All untreated pig recipients of liver xenografts died from a coagulopathy. Recipients with liver failure manifested no signs of coagulopathy, and had minimal change in platelet counts or Sonoclot (Sienco Inc., Morrison, CO) tracings. Liver xenograft biopsies from recipients with liver failure showed no evidence of the tissue injury that characterized the biopsies of control recipients. Serum from pigs was less cytotoxic to the canine hepatic sinusoidal endothelium after induction of liver failure. The xenoreactive antibody levels and repertoire were similar in the pig serum before and after liver failure was induced. CH50 (total complement) levels were diminished in pigs after the induction of liver failure. CONCLUSIONS Liver xenotransplantation used in bridging trials in recipients with liver failure might not face the barrier of hyperacute rejection.

Apoptosis induced in human colorectal carcinoma by anti-Fas antibody.

AbstractBackground: Apoptosis or programmed cell death has been shown to play an important role in the progression from polyps to carcinomas. Fas/APO-1 is a cell surface protein that can induce apoptosis in a variety of cell types upon specific antibody binding. In this study seven human colorectal carcinoma (HCRC) cell lines of varying differentiation were analyzed for cell surface Fas expression, Fas-mediated apoptosis, and correlation of apoptosis withbcl-2 expression. Methods and Results: Using flow cytometry, all seven lines expressed varying amounts of cell surface Fas antigen. Exposure to anti-Fas antibody induced cell death in all the cell lines, albeit to varying degrees. The rate of apoptosis was quantitated using flow cytometry with propidium iodide staining of nuclear DNA. The poorly differentiated cell lines had a significantly decreased (p<0.05) anti-Fas sensitivity as compared with the well-differentiated lines. Measurement ofbcl-2 expression by flow cytometry showed an inverse correlation with anti-Fas sensitivity. Conclusions: This study confirms that HCRC cell lines express Fas antigen and, more importantly, provides the first evidence that exposure to anti-Fas antibody can induce apoptosis. Fas-mediated apoptosis in HCRC cell lines may be regulated bybcl-2 and may correlate with the degree of differentiation.

Original scientific articleExperimental discordant hepatic xenotransplantation in the recipient with liver failure: implications for clinical bridging trials1

BACKGROUND Clinical xenotransplantation might start with bridge-to-bridge trials. Situations where hyperacute rejection is avoided would provide opportunities for the initiation of bridging trials. Patients with liver failure have a diminished capacity to initiate antibody and complement-induced injury of xenogeneic endothelium. Hyperacute rejection of a liver xenograft manifests as a coagulopathy. We examined the ability of a recipient with liver failure to hyperacutely reject a liver xenograft in the dog-to-pig model in the immediate postoperative period. STUDY DESIGN Liver failure in pigs was induced with galactosamine. Canine livers were transplanted into pigs with liver failure and into healthy pigs. The postoperative course was monitored for 1 hour for histologic changes in the xenograft, changes in platelet counts, and whole blood clotting with Sonoclot analysis. In vitro assays with pig serum and canine hepatic sinusoidal endothelial cells were used to assess the effect of liver failure on serum cytotoxicity and xenoreactive antibody levels. RESULTS All untreated pig recipients of liver xenografts died from a coagulopathy. Recipients with liver failure manifested no signs of coagulopathy, and had minimal change in platelet counts or Sonoclot (Sienco Inc., Morrison, CO) tracings. Liver xenograft biopsies from recipients with liver failure showed no evidence of the tissue injury that characterized the biopsies of control recipients. Serum from pigs was less cytotoxic to the canine hepatic sinusoidal endothelium after induction of liver failure. The xenoreactive antibody levels and repertoire were similar in the pig serum before and after liver failure was induced. CH50 (total complement) levels were diminished in pigs after the induction of liver failure. CONCLUSIONS Liver xenotransplantation used in bridging trials in recipients with liver failure might not face the barrier of hyperacute rejection.

Circulating B-Ceils in Follicular Non-Hodgkin’s Lymphoma Show Variant Expression of L-Selectin Epitopes

The L-selectin receptor on normal lymphocytes is involved in homing to lymph nodes via interaction with high endothelial venules [1,2]. Lymphoma cell expression of L-selectin and recognition of corresponding ligand on high endothelial venule cells may influence the dissemination of lymphomatous B cells into nodal and extranodal sites [3–9]. Pals et al [7] found significant differences in L-selectin expression by malignant cells between nodal and gastrointestinal non-Hodgkin’s lymphomas, with frequent (84/116 cases, or 72%) expression in nodal biopsies compared to 24% of cases with gastrointestinal tumors. Moller et al [8,9] found low or absent expression of L-selectin on lymphoma regardless of site of involvement. Discordance in these results may be partly explained by the use of an immunohistochemical assay to evaluate L-selectin expression on freshly frozen tissue rather than flow cytometric analysis of fresh tumor cells. The presence of circulating malignant cells with a t(14;18) translocation detected by PCR has been demonstrated in 15 out of 22 (68%) of patients [10], including patients in apparent clinical remission [11]. We have analyzed peripheral blood B cells from patients with follicular NHL and normal donors, and demonstrated that the L-selectin on the surface of circulating B cells in follicular non-Hodgkin’s lymphoma is different from L-selectin expressed on normal blood B cells.