Milagros Lagman

Cyclosporine induces cancer progression by a cell-autonomous mechanism

Malignancy is a common and dreaded complication following organ transplantation,,,. The high incidence of neoplasm and its aggressive progression, which are associated with immunosuppressive therapy, are thought to be due to the resulting impairment of the organ recipients immune-surveillance system,,,,. Here we report a mechanism for the heightened malignancy that is independent of host immunity. We show that cyclosporine (cyclosporin A), an immunosuppressant that has had a major impact on improving patient outcome following organ transplantation,, induces phenotypic changes, including invasiveness of non-transformed cells, by a cell-autonomous mechanism. Our studies show that cyclosporine treatment of adenocarcinoma cells results in striking morphological alterations, including membrane ruffling and numerous pseudopodial protrusions, increased cell motility, and anchorage-independent (invasive) growth. These changes are prevented by treatment with monoclonal antibodies directed at transforming growth factor-β (TGF-β). In vivo, cyclosporine enhances tumour growth in immunodeficient SCID–beige mice; anti-TGF-β monoclonal antibodies but not control antibodies prevent the cyclosporine-induced increase in the number of metastases. Our findings suggest that immunosuppressants like cyclosporine can promote cancer progression by a direct cellular effect that is independent of its effect on the hosts immune cells, and that cyclosporine-induced TGF-β production is involved in this.

In vivo expression of transforming growth factor-beta1 in humans: stimulation by cyclosporine.

BACKGROUND Transforming growth factor-beta1 (TGF-beta1) is an immunoregulatory and fibrogenic cytokine. In an earlier in vitro study, we demonstrated that cyclosporine (CsA) increases TGF-beta1 transcription rate in human T lymphocytes. Herein, we explored whether CsA augments the in vivo expression of TGF-beta1 in humans. METHODS The inherent difficulty in studying the in vivo effect of CsA in humans was circumvented by investigating stable end-stage renal disease patients who were preconditioned with CsA before their living donor renal transplantation. Sera and peripheral blood mononuclear cells were obtained from CsA-preconditioned patients and quantified for TGF-beta1 expression at the mRNA (by competitive polymerase chain reaction) and protein (sandwich enzyme-linked immunosorbent assay) levels. RESULTS Our studies demonstrated a significant increase in TGF-beta1 expression after CsA therapy. The stimulatory effect was unique to TGF-beta1, and CsA did not increase interleukin (IL)-10, IL-6, IL-2, or tumor necrosis factor-alpha expression. CONCLUSIONS Our first-time demonstration of a TGF-beta1-selective in vivo stimulatory effect of CsA in humans: (1) advances a TGF-beta1-centered hypothesis for the beneficial (immunosuppression) and detrimental (fibrosis, hypertension) effects of CsA use, and (2) broadens the mechanism of immunosuppressive action of CsA to include heightened expression of an endogenous immunosuppressive cytokine.Background.Transforming growth factor-β1(TGF-β1) is an immunoregulatory and fibrogenic cytokine. In an earlier in vitro study, we demonstrated that cyclosporine (CsA) increases TGF-β1transcription rate in human T lymphocytes. Herein, we explored whether CsA augments the in vivo expression of TGF-β1i

Molecular executors of cell death--differential intrarenal expression of Fas ligand, Fas, granzyme B, and perforin during acute and/or chronic rejection of human renal allografts.

Two distinct cytolytic pathways have been characterized: one in which the interaction between the Fas antigen and its ligand results in apoptosis, and another in which the pore forming protein perforin and the serine protease granzyme B contribute to DNA fragmentation and cell death. We investigated intrarenal expression of these molecular executors of cell death in light of the potential participation of cytolytically active cellular elements in the antiallograft repertory. Reverse transcriptase-polymerase chain reaction was used to identify intrarenal expression of Fas antigen, Fas ligand, granzyme B and perforin in eighty human renal allograft biopsies; mRNA display was correlated with the Banff histological diagnosis of renal allografts. Our studies demonstrate that: (1) intrarenal expression of Fas ligand mRNA and of granzyme B mRNA are correlates of acute but not chronic rejection; (2) Fas ligand mRNA is not detectable in allografts in the absence of rejection; (3) intrarenal coexpression of members of each lytic pathway (Fas ligand and Fas, granzyme B, and perforin) and that of both pathways (e.g., Fas ligand and granzyme B) are correlates of acute rejection; and (4) a direct correlation exists between the histological severity of acute rejection and intrarenal coexpression of mRNA encoding Fas ligand, Fas, granzyme B, and perforin. Our studies identify, for the first time, the differential expression of the two major lytic pathways in acute and chronic allograft rejection and suggest that specific therapy directed at the cytotoxic attack molecules might be efficacious in the prevention and/or treatment of acute rejection.

Proapoptotic Bax is hyperexpressed in isolated human islets compared with antiapoptotic Bcl-2

Background. Apoptosis is a well-documented pathway for islet cell death. One potential mechanism is overexpression of death-promoting Bax compared with antiapoptotic Bcl-2 in islets. Methods. We isolated islets from 10 human pancreata and measured the expression of Bax mRNA and Bcl-2 mRNA by real-time quantitative polymerase chain reaction; islet and pancreas expression of Bax, Bcl-2, activated caspase-3, and cleaved poly (ADP-ribose) polymerase were also assessed by immunohistochemistry. Islet cell apoptosis was evaluated by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assay and by flow cytometry. Results. The mean (±SE) level of Bax mRNA was 336±79 copies per nanogram of total RNA, and the level of Bcl-2 mRNA was 36±10 (P =0.001). A positive correlation existed between islet expression of Bax mRNA and Bcl-2 mRNA (P =0.001). The islet Bax to Bcl-2 ratio was 10.8±1.3 and 1.71±0.3 for the spleens (P =0.0001). Bax mRNA (P =0.04), but not Bcl-2 mRNA, was expressed at a higher level in islets compared with spleens. Human islets contained large numbers of cells expressing Bax protein, whereas only infrequent islet cells expressed Bcl-2 protein, activated caspase-3, and poly (ADP-ribose) polymerase. The apoptotic index was 5% by TUNEL assay, and the percentage of apoptotic islet cells was 9.7±2.5% by flow cytometry. Sections of human pancreas before islet isolation showed islet staining for Bax but not Bcl-2. Conclusions. Our finding that isolated human islets express Bax at a higher level compared with Bcl-2 suggests a molecular mechanism for islet cell death by apoptosis. We hypothesize that reducing islet expression of Bax, or regulating its activation, will help preserve islet cell mass after islet transplantation.

Enforced c-REL deficiency prolongs survival of islet allografts1.

Background. The NF-&kgr;B/Rel family of transcription factors regulates biologic processes ranging from apoptosis to inflammation and innate immunity. Whether c-Rel, a lymphoid-predominant member of the NF-&kgr;B/Rel family, is essential for transplantation immunity is not known. Methods. We explored the role of c-Rel in the anti-allograft repertory using mice with targeted disruption of the c-Rel gene (c-Rel-/-) as recipients of H-2 mismatched islet allografts. Allogeneic DBA/2 (H-2d) islets were transplanted into the renal subcapsular space of diabetic c-Rel-/- C57BL/6 (H-2b) mice or the c-Rel +/+ C57BL/6 wild-type mice. Islet graft survival, cellular traffic into the islet grafts and their phenotype, and intragraft expression of cytokines and cytotoxic attack molecules were determined at the protein (by immunohistochemistry) and mRNA (by real-time quantitative polymerase chain reaction) levels. Results. We found superior islet graft survival in the c-Rel-/- recipients compared to c-Rel+/+ C57BL/6 recipients. Splenocytes from c-Rel-/- mice proliferated poorly compared to splenocytes from the c-Rel+/+ mice on stimulation with anti-CD3 mAbs or Con A. Peri-islet infiltration composed of T lymphocytes and macrophages was found in both c-Rel+/+ recipients and c-Rel-/- recipients, but intra-islet infiltration was observed only in c-Rel+/+ recipients. Immunohistologic and molecular studies showed impaired T helper-type 1 immunity and decreased intragraft expression of cytotoxic attack molecules perforin and granzyme B in c-Rel-/- recipients as compared to wild-type recipients. Conclusions. Our results demonstrate that c-Rel is essential for robust rejection of islet allografts and support the idea that strategies that impair c-Rel function may be of value for constraining alloimmunity and facilitating survival of allogafts.

Hyperexpression of Foxp3 and IDO during acute rejection of islet allografts

Background. We investigated the hypothesis that Foxp3+ cells are an integral component of antiallograft immunity but are dominated by pathogenic effectors. Methods. Wild-type H-2b C57BL/6 (B6) mice or B6 mice with a targeted disruption of c-Rel gene (c-Rel−/−) were used as recipients of islet grafts from allogeneic DBA/2 (H-2d) mice or syngeneic B6 mice. We developed kinetic quantitative polymerase chain reaction assays and measured intragraft expression of mRNA for Foxp3, IDO, cytolytic molecules, proinflammatory cytokines, and chemokines/receptors. Results. Intraislet levels of mRNA for Foxp3, IDO, CD3, CD25, tumor necrosis factor-&agr;, RANTES, IP-10, and CXCR3 were highest in DBA/2 islet allografts from WT B6 recipients compared to DBA/2 islet allografts from c-Rel−/− B6 recipients or syngeneic B6 islet grafts from WT B6 mice. The ratio of granzyme B or IFN-gamma to Foxp3 was higher with the DBA/2 islet allografts from the WT B6 recipients compared to DBA/2 islet allografts from c-Rel−/− B6 recipients or B6 islet grafts from WT B6 recipients. Conclusions. Foxp3+ cells are an integral component of acute rejection of allografts but may be dominated by pathogenic effectors.

Dendritic cells armed with anti-CD3 mAbs reduce pulmonary metastases, prolong survival, and engender antitumor effector cells demonstrable by adoptive transfer.

AbstractBackground: Dendritic cells (DCs) pulsed with tumor cells or peptides are effective antitumor agents in a number of tumor models. In light of our earlier demonstration that T-cell signaling via the CD3 proteins induces cytolytic activity and constrains tumor progression, we equipped DCs pulsed with tumor cells with anti-CD3 mAbs and tested their antitumor efficacy in a murine renal cell cancer pulmonary metastasis model. Methods: We investigated the antitumor efficacy of DCs pulsed with whole irradiated tumor cells (DC/R) or DCs pulsed with irradiated tumor cells and armed with anti-CD3 mAbs (DC/R/anti-CD3 mAbs). Experimental end points included the number of pulmonary metastases and survival of tumor-inoculated mice. Results: Our studies demonstrate that arming tumor-pulsed DCs with anti-CD3 mAbs results in a superior outcome compared to that from tumor-pulsed DCs alone in terms of reduction in the number of pulmonary metastases and survival times. Furthermore, adoptive transfer experiments revealed that the splenocytes from DC/R/anti-CD3 mAbs-treated mice are superior to splenocytes from DC/R-treated mice in reducing renal cancer pulmonary metastases in severe combined immunodeficient (SCID) beige mice. Conclusion: Our data suggest that the therapeutic efficacy of DCs pulsed with tumor cells can be augmented by arming them with anti-CD3 mAbs. DC-based treatment regimens that currently are being pursued in clinical trials might be improved by equipping such cells with anti-CD3 mAbs.

DEVELOPMENT OF A NEW STRATEGY FOR MINIMIZING ISLET CELL MASS IN TYPE 1 DIABETIC RECIPIENTS: DIANNEXIN REDUCES PROAPOPTOTIC BID, PROMOTES ANTI-INFLAMMATORY INTERLEUKIN-10 AND IMPROVES EARLY GRAFT FUNCTION IN THE MARGINAL ISLET MASS TRANSPLANTATION MODEL: 331

Introduction: A major unmet challenge is to reduce the islet mass needed to accomplish normoglycemia in type 1 diabetic recipients. Diannexin, a recombinant homodimer of annexin V, is currently undergoing clinical trials for the prevention of ischemia-reperfusion injury (IRI) in solid organ transplantation. We investigated the hypothesis that Diannexin prevents IRI and improves early graft function in a marginal islet cell mass transplantation model. Methods: To test our hypothesis, a marginal mass of BALB/c islets was transplanted under the renal capsule of streptozotocin-diabetic syngeneic recipients. Diannexin (400μg/kg) was administered to islet cell donors immediately prior to pancreas harvest, added to isolation reagents, and infused into recipients at the time of transplantation and repeated daily until day +4. Attainment of normoglycemia was defined as three consecutive blood glucose measurements below 200mg/dL. Results: As illustrated below, Diannexin therapy reduced the median time needed to achieve normoglycemia from 17.0 days among untreated to 3.5 days for Diannexin-treated recipients (P=0.004). In studies designed to understand mechanisms, islets retrieved three days post-transplant from Diannexin-treated mice expressed significantly lower levels of mRNA for the proapoptotic molecule Bid (100 ± 26 copies per ng of total RNA) compared with untreated control grafts (227 ± 46 copies/ng; P=0.03). The levels of mRNA for interleukin-10 was ten-fold higher in Diannexin-treated islet grafts (2.9 ± 0.4 copies/ng) than in control grafts (0.26 ± 0.06 copies/ng). On the other hand , pretreatment of donor mice along with supplementation of islet isolation reagents did not affect islet yield (mean ± SE; 288 ± 21 islets per mouse) relative to untreated controls (284 ± 19; P=0.75). Diannexin also failed to reduce islet cell apoptosis or increase the viability of mouse islets immediately after the isolation process, as determined by dual-parameter flow cytometry analysis. The mean percentage of viable islet cells (annexin V/TO-PRO-3 double-negative) was 74 ± 3% with islets from the control group and 71 ± 4% with islets from the Diannexin group (P=0.40). Furthermore, Diannexin treatment did not reduce the percentage of early apoptotic (annexin V-positive) or late apoptotic/necrotic cells (annexin V/TOPRO-3 double-positive; P>0.05). In studies designed to understand mechanisms, islets retrieved three days post-transplant from Diannexin-treated mice expressed significantly lower levels of mRNA for the proapoptotic molecule Bid (100 ± 26 copies per ng of total RNA) compared with untreated control grafts (227 ± 46 copies/ng; P=0.03). The levels of mRNA for interleukin-10 was ten-fold higher in Diannexin-treated islet grafts (2.9 ± 0.4 copies/ng) than in control grafts (0.26 ± 0.06 copies/ng). On the other hand , pretreatment of donor mice along with supplementation of islet isolation reagents did not affect islet yield (mean ± SE; 288 ± 21 islets per mouse) relative to untreated controls (284 ± 19; P=0.75). Diannexin also failed to reduce islet cell apoptosis or increase the viability of mouse islets immediately after the isolation process, as determined by dual-parameter flow cytometry analysis. The mean percentage of viable islet cells (annexin V/TO-PRO-3 double-negative) was 74 ± 3% with islets from the control group and 71 ± 4% with islets from the Diannexin group (P=0.40). Furthermore, Diannexin treatment did not reduce the percentage of early apoptotic (annexin V-positive) or late apoptotic/necrotic cells (annexin V/TOPRO-3 double-positive; P>0.05). Conclusion: Our study demonstrates that Diannexin is associated with a prompt return to normoglycemia and improves the early function of marginal mass islet grafts. Our mechanistic studies suggest that the protective effects of Diannexin may be associated with a reduced inflammatory response and a decrease in islet cell death by apoptosis following ischemiareperfusion stress. Together our findings suggest that Diannexin may represent a novel and clinically useful strategy to reduce the islet mass required for achieving insulin independence in type 1 diabetic patients. Conclusion: Our study demonstrates that Diannexin is associated with a prompt return to normoglycemia and improves the early function of marginal mass islet grafts. Our mechanistic studies suggest that the protective effects of Diannexin may be associated with a reduced inflammatory response and a decrease in islet cell death by apoptosis following ischemia-reperfusion stress. Together our findings suggest that Diannexin may represent a novel and clinically useful strategy to reduce the islet mass required for achieving insulin independence in type 1 diabetic patients.