Pre-existing allo-antibodies (allo-Abs), that preclude transplant due to the risk of hyperacute r... more Pre-existing allo-antibodies (allo-Abs), that preclude transplant due to the risk of hyperacute rejection, lead to prolonged wait times and high mortality rates. Current desensitization approaches are ineffective as they do not adequately deplete allo-specific B cells and plasma cells (PCs). We hypothesize that stringent depletion of these cells is required to eliminate pre-existing allo-Abs. We leverage the exquisite ability of CAR T cells to eliminate target cells to desensitize transplant candidates. We constructed CARs targeting murine CD19 or BCMA, which cover the entire B cell-PC continuum. We first evaluated the function of CAR T cells against B cells and PCs in vitro. C57BL/6 mice were sensitized with BALB/c skin grafts. After skin rejection, sensitized mice received total body irradiation followed by treatment with either control T cells, CART-19 T cells, or a combination of CART-19 and CART-BCMA T cells (combo-CART). Allo-Abs, total Ig, and B cells were measured over 13 we...
Introduction: In heart transplantation, there is a critical need for development of serum biomark... more Introduction: In heart transplantation, there is a critical need for development of serum biomarkers to monitor for donor organ rejection/ injury, which remains a major cause of morbidity and morta...
Background. Immunologic rejection is a major barrier to successful long-term outcomes in clinical... more Background. Immunologic rejection is a major barrier to successful long-term outcomes in clinical transplantation. The importance of B lymphocytes-and their secretory products, alloantibodies-in the pathogenesis of allograft rejection is accepted. Furthermore, it is now clear that the dominant regulator of peripheral B-cell homeostasis and tolerance is the B-Lymphocyte Stimulator (BLyS), also referred to as the B-cell activating factor (BAFF). Recently, a novel class of clinical immunotherapeutic agents specific for BLyS, and its family of cytokines, has emerged for the treatment of B-cell-mediated diseases. In this study, we demonstrate the potential utility of BLyS-directed immunotherapy in preventing allograft rejection using a murine islet transplantation model. Methods. A transient period of mature peripheral B-cell depletion was induced by means of in vivo BLyS neutralization using a murine analog of the monoclonal antibody, Benlysta. Subsequently, fully major histocompatibility complexmismatched islets were transplanted into naïve diabetic mice followed by a short course of rapamycin. Results. After BLyS neutralization, indefinite islet allograft survival was achieved. Induction therapy with rapamycin was necessary, but not sufficient, for the achievement of this long-term graft survival. The tolerant state was associated with (1) abrogation of the donor-specific antibody response, (2) transient preponderance of immature/transitional B cells in all lymphoid organs, (3) impaired CD4 T-cell activation during the period of B-cell depletion, and (4) presence of a "regulatory" cytokine milieu. Conclusions. In vivo BLyS neutralization effectively induces humoral tolerance and promotes long-term islet allograft survival in mice. Therefore, B-lymphocyte-directed immunotherapy targeting the homeostatic regulator, BLyS, may be effective in promoting transplantation tolerance.
Direct measurement of the precursor frequency of alloreactive CD4+ T cells has been impossible du... more Direct measurement of the precursor frequency of alloreactive CD4+ T cells has been impossible due to the lack of a specific means of determining the absolute number of daughter cells generated with each division in a repertoire of stimulated T cells. Responder lymphocytes were fluorescently labeled and adoptively transferred into irradiated allogeneic stimulator mice or incubated in vitro with irradiated stimulator splenocytes. After a 65- to 70-hr stimulation period, responder cells were analyzed by flow cytometry. The precursor frequency of dividing CD4+ T cells was determined both in vivo and in vitro. The observed number of alloreactive daughter cells generated with each round of division was used to calculate the frequency of alloantigen-specific CD4+ T cells. A novel method for the direct calculation of the frequency of alloreactive CD4+ T cells is described. This technique allows the determination of changes in the frequency of alloreactive T cells that might underlie tolerance to alloantigens.
Despite the well-recognized concordance of chimerism with spontaneous acceptance of rat liver all... more Despite the well-recognized concordance of chimerism with spontaneous acceptance of rat liver allografts, the active role and the identity of chimeric cells mediating liver allograft tolerance are unknown. Because resting B cells are endowed with a tolerogenic antigen-presenting capacity, we assessed whether donor B cells propagated from the grafted liver may be responsible for liver allograft tolerance. Dark Agouti or Lewis rats were grafted with Lewis or Dark Agouti livers as a tolerogenic or a rejection combination, respectively. We followed the kinetics of donor B cells in recipients by flow cytometry, and we examined the fate of liver allografts depleted of passenger B cells in either B cell-sufficient or -deficient recipients. B-cell depletion was achieved by treatment of animals with polyclonal goat anti-rat IgM antibody from birth. During the first 3 days after liver allografting, donor B cells rapidly migrated from graft-infiltrating cells and appeared in systemic circulation in both the tolerogenic and rejection combinations. However, systemic chimerism was detectable in the tolerogenic combination by day 14, whereas it was undetectable in the rejection combination by day 7. In graft-infiltrating cells, a significant expansion of chimeric IgM+ (newly formed) B cells was observed on day 5 in the tolerogenic, but not in the rejection, combination. However, depletion of B cells from liver grafts and the absence of antibodies failed to alter the outcome of liver allograft survival in the tolerogenic or immunogenic combination. Although intragraft chimeric B cells proliferated in tolerogenic liver allografts, their clonal expansion does not seem to be essential for the promotion of liver allograft tolerance.
We found that an induction immunotherapy regimen consisting of rabbit anti-thymocyte globulin (Th... more We found that an induction immunotherapy regimen consisting of rabbit anti-thymocyte globulin (Thymoglobulin) and the monoclonal antibody to CD20 rituximab (Rituxan) promoted long-term islet allograft survival in cynomolgus macaques maintained on rapamycin monotherapy. B lymphocyte reconstitution after rituximab-mediated depletion was characterized by a preponderance of immature and transitional cells, whose persistence was associated with long-term islet allograft survival. Development of donor-specific alloantibodies was abrogated only in the setting of continued rapamycin monotherapy.
patic ICAM-1 mRNA expression and neutrophil infiltration were also reduced. (meanϮSE, nϭ3-6 anima... more patic ICAM-1 mRNA expression and neutrophil infiltration were also reduced. (meanϮSE, nϭ3-6 animals per group, p Ͻ 0.05). HO-1 protein expression was induced in the AdiNOS-treated, but not AdLacZ-treated, donor liver grafts. Administration of L-NIL (30 mg/kg iv) or SnPP (10 M/kg iv) partially abrogated the protection afforded by AdiNOS (data not shown). Conclusion: These results demonstrate that AdiNOS pretreatment decreases hepatic transplant preservation injury. With severe I/R injury due to prolonged cold storage, AdiNOS delivery also improves transplant survival, indicating the clinical relevance of this treatment modality. Hepatic HO-1 induction by iNOS transduction may account in part for the protective effect of donor pre-treatment with AdiNOS. Two-week animal survival after liver transplantation.
Nonobese diabetic (NOD) mice spontaneously develop an acute onset of hyperglycemia reminiscent of... more Nonobese diabetic (NOD) mice spontaneously develop an acute onset of hyperglycemia reminiscent of human type I diabetes. The disease is the end result of a mononuclear cell infiltration of pancreatic islets (insulitis), culminating in the selective destruction of islet P-cells by autoreactive T-cells. NOD mice also exhibit defects in B-cell tolerance as manifested by the presence of autoantibodies against islet cell autoantigens. Based on the potential ability of B-cells to act as antigen presenting cells, we hypothesized that autoreactive B-cells of NOD mice may be necessary for the activation of islet reactive CD4 + T-cells. In the present study, we utilized an anti-u antibody to induce in vivo depletion of B-cells and found that B-cell depletion completely abrogates the development of insulitis and sialitis in NOD mice. In contrast, control IgG-treated NOD mice developed insulitis and sialitis by 5 weeks of age. Additionally, the discontinuation of anti-u chain antibody treatment led to the full restoration of the Bcell pool and the reappearance of insulitis and sialitis. Thus, we conclude that B-cells are a requisite cell population for the genesis of the inflammatory lesions of the islets of Langerhans. This finding suggests that autoreactive B-cells may act as the antigen presenting cells necessary for the initial activation of (S-cell-reactive CD4 + T-cells implicated in the pathogenesis of autoimmune diabetes. Diabetes 46:941-946, 1997
The Journal of Thoracic and Cardiovascular Surgery, 1993
Permanent tolerance to an experimental cardiac allograft can be achieved by pretransplantation in... more Permanent tolerance to an experimental cardiac allograft can be achieved by pretransplantation intrathymic inoculation of donor-specific lymphoid cells. We studied the effects of intrathymic inoculation of xenogeneic cells and intravenous cobra venom factor in a rodent model of cardiac xenotransplantation. Lewis rats underwent intraabdominal heterotopic heart transplantation with Syrian hamster donors. In untreated animals, mean graft survival time was 3 days. Five rats had 1 ml of antilymphocyte serum administered intraperitoneally. One day later, 2.5 x 10(7) hamster spleen cells were inoculated into the thymus under direct vision. Twenty-one days after antilymphocyte serum was given, heterotopic heart transplantation with a hamster donor was carried out. In all cases, rejection was accelerated and occurred between 20 minutes and 1 day after transplantation. Mean graft survival time was 5.2 hours (p < 0.0001 versus control). Six animals treated with antilymphocyte serum and intrathymic xenogeneic cells had 0.5 ml of cobra venom factor, a complement antagonist, administered intravenously 3 hours before transplantation and every other day thereafter. Mean graft survival was 3 days, which was not different from the response of naive animals. Animals treated with antilymphocyte serum only had no prolongation of graft survival (mean survival time 3 days, p = not significant). Animals treated with cobra venom factor alone (n = 5) before transplantation and on alternate days subsequently had mild graft prolongation with a mean survival time of 4 days (p = 0.0133). In contrast to experimental allograft models, intrathymic inoculation of xenogeneic cells produces hyperacute rejection in these naturally concordant species. The administration of cobra venom factor abrogates the hyperacute response, but the combination of cobra venom factor and intrathymic inoculation does not produce long-term graft survival.
The Journal of Thoracic and Cardiovascular Surgery, 2018
Objective: In heart transplantation, there is a critical need for development of biomarkers to no... more Objective: In heart transplantation, there is a critical need for development of biomarkers to noninvasively monitor cardiac allografts for immunologic rejection or injury. Exosomes are tissue-specific nanovesicles released into circulation by many cell types. Their profiles are dynamic, reflecting conditional changes imposed on their tissue counterparts. We proposed that a transplanted heart releases donor-specific exosomes into the recipient's circulation that are conditionally altered during immunologic rejection. We investigated this novel concept in a rodent heterotopic heart transplantation model. Materials and Methods: Full major histocompatibility mismatch (BALB/c [H2-K d ] into C57BL/6 [H2-K b ]) heterotopic heart transplantation was performed in 2 study arms: Rejection (n ¼ 64) and Maintenance (n ¼ 28). In the Rejection arm, immunocompetent recipients fully rejected the donor heart, whereas in the Maintenance arm, immunodeficient recipients (C57BL/6 Prkdc SCID) accepted the allograft. Recipient plasma exosomes were isolated and a donor heart-specific exosome signal was characterized on the nanoparticle detector for time-specific profile changes using anti-H2-K d antibody quantum dot. Results: In the Maintenance arm, allografts were viable throughout follow-up of 30 days, with histology confirming absence of rejection or injury. Time course analysis (days 1, 2, 3, 4, 5, 7, 9, 11, 15, and 30) showed that total plasma exosome concentration (P ¼ .157) and donor heart exosome signal (P ¼ .538) was similar between time points. In the Rejection arm, allografts were universally rejected (median, day 11). Total plasma exosome quantity and size distribution were similar between follow-up time points (P ¼ .278). Donor heart exosome signals peaked on day 1, but significantly decreased by day 2 (P ¼ 2 3 10 À4) and day 3 (P ¼ 3.3 3 10 À6), when histology showed grade 0R rejection. The receiver operating characteristic curve for a binary separation of the 2 study arms (Maintenance vs Rejection) demonstrated that a donor heart exosome signal threshold<0.3146 was 91.4% sensitive and 95.8% specific for diagnosis of early acute rejection. Conclusions: Transplant heart exosome profiling enables noninvasive monitoring of early acute rejection with high accuracy. Translation of this concept to clinical settings might enable development of a novel biomarker platform for allograft monitoring in transplantation diagnostics.
Pre-existing allo-antibodies (allo-Abs), that preclude transplant due to the risk of hyperacute r... more Pre-existing allo-antibodies (allo-Abs), that preclude transplant due to the risk of hyperacute rejection, lead to prolonged wait times and high mortality rates. Current desensitization approaches are ineffective as they do not adequately deplete allo-specific B cells and plasma cells (PCs). We hypothesize that stringent depletion of these cells is required to eliminate pre-existing allo-Abs. We leverage the exquisite ability of CAR T cells to eliminate target cells to desensitize transplant candidates. We constructed CARs targeting murine CD19 or BCMA, which cover the entire B cell-PC continuum. We first evaluated the function of CAR T cells against B cells and PCs in vitro. C57BL/6 mice were sensitized with BALB/c skin grafts. After skin rejection, sensitized mice received total body irradiation followed by treatment with either control T cells, CART-19 T cells, or a combination of CART-19 and CART-BCMA T cells (combo-CART). Allo-Abs, total Ig, and B cells were measured over 13 we...
Introduction: In heart transplantation, there is a critical need for development of serum biomark... more Introduction: In heart transplantation, there is a critical need for development of serum biomarkers to monitor for donor organ rejection/ injury, which remains a major cause of morbidity and morta...
Background. Immunologic rejection is a major barrier to successful long-term outcomes in clinical... more Background. Immunologic rejection is a major barrier to successful long-term outcomes in clinical transplantation. The importance of B lymphocytes-and their secretory products, alloantibodies-in the pathogenesis of allograft rejection is accepted. Furthermore, it is now clear that the dominant regulator of peripheral B-cell homeostasis and tolerance is the B-Lymphocyte Stimulator (BLyS), also referred to as the B-cell activating factor (BAFF). Recently, a novel class of clinical immunotherapeutic agents specific for BLyS, and its family of cytokines, has emerged for the treatment of B-cell-mediated diseases. In this study, we demonstrate the potential utility of BLyS-directed immunotherapy in preventing allograft rejection using a murine islet transplantation model. Methods. A transient period of mature peripheral B-cell depletion was induced by means of in vivo BLyS neutralization using a murine analog of the monoclonal antibody, Benlysta. Subsequently, fully major histocompatibility complexmismatched islets were transplanted into naïve diabetic mice followed by a short course of rapamycin. Results. After BLyS neutralization, indefinite islet allograft survival was achieved. Induction therapy with rapamycin was necessary, but not sufficient, for the achievement of this long-term graft survival. The tolerant state was associated with (1) abrogation of the donor-specific antibody response, (2) transient preponderance of immature/transitional B cells in all lymphoid organs, (3) impaired CD4 T-cell activation during the period of B-cell depletion, and (4) presence of a "regulatory" cytokine milieu. Conclusions. In vivo BLyS neutralization effectively induces humoral tolerance and promotes long-term islet allograft survival in mice. Therefore, B-lymphocyte-directed immunotherapy targeting the homeostatic regulator, BLyS, may be effective in promoting transplantation tolerance.
Direct measurement of the precursor frequency of alloreactive CD4+ T cells has been impossible du... more Direct measurement of the precursor frequency of alloreactive CD4+ T cells has been impossible due to the lack of a specific means of determining the absolute number of daughter cells generated with each division in a repertoire of stimulated T cells. Responder lymphocytes were fluorescently labeled and adoptively transferred into irradiated allogeneic stimulator mice or incubated in vitro with irradiated stimulator splenocytes. After a 65- to 70-hr stimulation period, responder cells were analyzed by flow cytometry. The precursor frequency of dividing CD4+ T cells was determined both in vivo and in vitro. The observed number of alloreactive daughter cells generated with each round of division was used to calculate the frequency of alloantigen-specific CD4+ T cells. A novel method for the direct calculation of the frequency of alloreactive CD4+ T cells is described. This technique allows the determination of changes in the frequency of alloreactive T cells that might underlie tolerance to alloantigens.
Despite the well-recognized concordance of chimerism with spontaneous acceptance of rat liver all... more Despite the well-recognized concordance of chimerism with spontaneous acceptance of rat liver allografts, the active role and the identity of chimeric cells mediating liver allograft tolerance are unknown. Because resting B cells are endowed with a tolerogenic antigen-presenting capacity, we assessed whether donor B cells propagated from the grafted liver may be responsible for liver allograft tolerance. Dark Agouti or Lewis rats were grafted with Lewis or Dark Agouti livers as a tolerogenic or a rejection combination, respectively. We followed the kinetics of donor B cells in recipients by flow cytometry, and we examined the fate of liver allografts depleted of passenger B cells in either B cell-sufficient or -deficient recipients. B-cell depletion was achieved by treatment of animals with polyclonal goat anti-rat IgM antibody from birth. During the first 3 days after liver allografting, donor B cells rapidly migrated from graft-infiltrating cells and appeared in systemic circulation in both the tolerogenic and rejection combinations. However, systemic chimerism was detectable in the tolerogenic combination by day 14, whereas it was undetectable in the rejection combination by day 7. In graft-infiltrating cells, a significant expansion of chimeric IgM+ (newly formed) B cells was observed on day 5 in the tolerogenic, but not in the rejection, combination. However, depletion of B cells from liver grafts and the absence of antibodies failed to alter the outcome of liver allograft survival in the tolerogenic or immunogenic combination. Although intragraft chimeric B cells proliferated in tolerogenic liver allografts, their clonal expansion does not seem to be essential for the promotion of liver allograft tolerance.
We found that an induction immunotherapy regimen consisting of rabbit anti-thymocyte globulin (Th... more We found that an induction immunotherapy regimen consisting of rabbit anti-thymocyte globulin (Thymoglobulin) and the monoclonal antibody to CD20 rituximab (Rituxan) promoted long-term islet allograft survival in cynomolgus macaques maintained on rapamycin monotherapy. B lymphocyte reconstitution after rituximab-mediated depletion was characterized by a preponderance of immature and transitional cells, whose persistence was associated with long-term islet allograft survival. Development of donor-specific alloantibodies was abrogated only in the setting of continued rapamycin monotherapy.
patic ICAM-1 mRNA expression and neutrophil infiltration were also reduced. (meanϮSE, nϭ3-6 anima... more patic ICAM-1 mRNA expression and neutrophil infiltration were also reduced. (meanϮSE, nϭ3-6 animals per group, p Ͻ 0.05). HO-1 protein expression was induced in the AdiNOS-treated, but not AdLacZ-treated, donor liver grafts. Administration of L-NIL (30 mg/kg iv) or SnPP (10 M/kg iv) partially abrogated the protection afforded by AdiNOS (data not shown). Conclusion: These results demonstrate that AdiNOS pretreatment decreases hepatic transplant preservation injury. With severe I/R injury due to prolonged cold storage, AdiNOS delivery also improves transplant survival, indicating the clinical relevance of this treatment modality. Hepatic HO-1 induction by iNOS transduction may account in part for the protective effect of donor pre-treatment with AdiNOS. Two-week animal survival after liver transplantation.
Nonobese diabetic (NOD) mice spontaneously develop an acute onset of hyperglycemia reminiscent of... more Nonobese diabetic (NOD) mice spontaneously develop an acute onset of hyperglycemia reminiscent of human type I diabetes. The disease is the end result of a mononuclear cell infiltration of pancreatic islets (insulitis), culminating in the selective destruction of islet P-cells by autoreactive T-cells. NOD mice also exhibit defects in B-cell tolerance as manifested by the presence of autoantibodies against islet cell autoantigens. Based on the potential ability of B-cells to act as antigen presenting cells, we hypothesized that autoreactive B-cells of NOD mice may be necessary for the activation of islet reactive CD4 + T-cells. In the present study, we utilized an anti-u antibody to induce in vivo depletion of B-cells and found that B-cell depletion completely abrogates the development of insulitis and sialitis in NOD mice. In contrast, control IgG-treated NOD mice developed insulitis and sialitis by 5 weeks of age. Additionally, the discontinuation of anti-u chain antibody treatment led to the full restoration of the Bcell pool and the reappearance of insulitis and sialitis. Thus, we conclude that B-cells are a requisite cell population for the genesis of the inflammatory lesions of the islets of Langerhans. This finding suggests that autoreactive B-cells may act as the antigen presenting cells necessary for the initial activation of (S-cell-reactive CD4 + T-cells implicated in the pathogenesis of autoimmune diabetes. Diabetes 46:941-946, 1997
The Journal of Thoracic and Cardiovascular Surgery, 1993
Permanent tolerance to an experimental cardiac allograft can be achieved by pretransplantation in... more Permanent tolerance to an experimental cardiac allograft can be achieved by pretransplantation intrathymic inoculation of donor-specific lymphoid cells. We studied the effects of intrathymic inoculation of xenogeneic cells and intravenous cobra venom factor in a rodent model of cardiac xenotransplantation. Lewis rats underwent intraabdominal heterotopic heart transplantation with Syrian hamster donors. In untreated animals, mean graft survival time was 3 days. Five rats had 1 ml of antilymphocyte serum administered intraperitoneally. One day later, 2.5 x 10(7) hamster spleen cells were inoculated into the thymus under direct vision. Twenty-one days after antilymphocyte serum was given, heterotopic heart transplantation with a hamster donor was carried out. In all cases, rejection was accelerated and occurred between 20 minutes and 1 day after transplantation. Mean graft survival time was 5.2 hours (p < 0.0001 versus control). Six animals treated with antilymphocyte serum and intrathymic xenogeneic cells had 0.5 ml of cobra venom factor, a complement antagonist, administered intravenously 3 hours before transplantation and every other day thereafter. Mean graft survival was 3 days, which was not different from the response of naive animals. Animals treated with antilymphocyte serum only had no prolongation of graft survival (mean survival time 3 days, p = not significant). Animals treated with cobra venom factor alone (n = 5) before transplantation and on alternate days subsequently had mild graft prolongation with a mean survival time of 4 days (p = 0.0133). In contrast to experimental allograft models, intrathymic inoculation of xenogeneic cells produces hyperacute rejection in these naturally concordant species. The administration of cobra venom factor abrogates the hyperacute response, but the combination of cobra venom factor and intrathymic inoculation does not produce long-term graft survival.
The Journal of Thoracic and Cardiovascular Surgery, 2018
Objective: In heart transplantation, there is a critical need for development of biomarkers to no... more Objective: In heart transplantation, there is a critical need for development of biomarkers to noninvasively monitor cardiac allografts for immunologic rejection or injury. Exosomes are tissue-specific nanovesicles released into circulation by many cell types. Their profiles are dynamic, reflecting conditional changes imposed on their tissue counterparts. We proposed that a transplanted heart releases donor-specific exosomes into the recipient's circulation that are conditionally altered during immunologic rejection. We investigated this novel concept in a rodent heterotopic heart transplantation model. Materials and Methods: Full major histocompatibility mismatch (BALB/c [H2-K d ] into C57BL/6 [H2-K b ]) heterotopic heart transplantation was performed in 2 study arms: Rejection (n ¼ 64) and Maintenance (n ¼ 28). In the Rejection arm, immunocompetent recipients fully rejected the donor heart, whereas in the Maintenance arm, immunodeficient recipients (C57BL/6 Prkdc SCID) accepted the allograft. Recipient plasma exosomes were isolated and a donor heart-specific exosome signal was characterized on the nanoparticle detector for time-specific profile changes using anti-H2-K d antibody quantum dot. Results: In the Maintenance arm, allografts were viable throughout follow-up of 30 days, with histology confirming absence of rejection or injury. Time course analysis (days 1, 2, 3, 4, 5, 7, 9, 11, 15, and 30) showed that total plasma exosome concentration (P ¼ .157) and donor heart exosome signal (P ¼ .538) was similar between time points. In the Rejection arm, allografts were universally rejected (median, day 11). Total plasma exosome quantity and size distribution were similar between follow-up time points (P ¼ .278). Donor heart exosome signals peaked on day 1, but significantly decreased by day 2 (P ¼ 2 3 10 À4) and day 3 (P ¼ 3.3 3 10 À6), when histology showed grade 0R rejection. The receiver operating characteristic curve for a binary separation of the 2 study arms (Maintenance vs Rejection) demonstrated that a donor heart exosome signal threshold<0.3146 was 91.4% sensitive and 95.8% specific for diagnosis of early acute rejection. Conclusions: Transplant heart exosome profiling enables noninvasive monitoring of early acute rejection with high accuracy. Translation of this concept to clinical settings might enable development of a novel biomarker platform for allograft monitoring in transplantation diagnostics.
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Papers by Susan Rostami