A Targeted and Adjuvanted Nanocarrier Lowers the Effective Dose of Liposomal Amphotericin B and Enhances Adaptive Immunity in Murine Cutaneous Leishmaniasis

Background: Amphotericin B (AmB), the most effective drug against leishmaniasis, has serious toxicity. As Leishmania species are obligate intracellular parasites of antigen presenting cells (APC), an immunopotentiating APC-specific AmB nanocarrier would be ideally suited to reduce the drug dosage and regimen requirements in leishmaniasis treatment. Here, we report a nanocarrier that results in effective treatment shortening of cutaneous leishmaniasis in a mouse model, while also enhancing L. major specific T-cell immune responses in the infected host. Methods: We used a Pan-DR-binding epitope (PADRE)-derivatized-dendrimer (PDD), complexed with liposomal amphotericin B (LAmB) in an L. major mouse model and analyzed the therapeutic efficacy of low-dose PDD/ LAmB vs full dose LAmB. Results: PDD was shown to escort LAmB to APCs in vivo, enhanced the drug efficacy by 83% and drug APC targeting by 10-fold and significantly reduced parasite burden and toxicity. Fortuitously, the PDD immunopotentiating effect significantly enhanced parasite-specific T-cell responses in immunocompetent infected mice. Conclusions: PDD reduced the effective dose and toxicity of LAmB and resulted in elicitation of strong parasite specific T-cell responses. A reduced effective therapeutic dose was achieved by selective LAmB delivery to APC, bypassing bystander cells, reducing toxicity and inducing antiparasite immunity

Regulatory T Cells and Hematopoiesis in Bone Marrow Transplantation

CD4+CD25+FoxP3+ regulatory T cells (Treg) possess the capacity to modulate both adaptive and innate immunity. Due to their suppressive nature, Treg cells have been studied and tested in a variety of scenarios in an attempt to ameliorate undesired immune responses. While graft versus host disease (GVHD) has in fact emerged as the first clinical application for human Treg cells (Riley et al. 2009), equally important are issues concerning hematopoietic engraftment and immune reconstitution. Currently, little is known about the effect(s) that regulatory T cells may exert outside the immune system in this context. Based on cytokine effector molecules they can produce we hypothesized that Treg cells could regulate hematopoietic phenomena. The studies portrayed in this dissertation demonstrate that Treg cells can differentially affect the colony forming activity of myeloid and erythroid progenitor cells. In-vitro as well as in-vivo findings demonstrate the ability of Tregs to inhibit and augment the differentiation of primitive and intermediate myeloid (interleukin (IL)-3 driven) and late erythroid (erythropoietin driven) hematopoietic progenitor cells, respectively. The inhibitory and enhancing affects appeared to be mediated by independent pathways, the former requiring cell-cell contact, major histocompatibility complex (MHC) class II expression on marrow cells and involving transforming growth factor beta (TGF-beta), whereas the latter required interleukin (IL)-9 and was not contact dependent. Strikingly, we observed that in addition to regulating hematopoietic activity in normal primary BM cells, Tregs were also capable of suppressing colony forming activity by the myelogenous leukemia cell line NFS-60. Furthermore, studies involving endogenous Treg manipulations in-situ (i.e. depletion of these cells) resulted in elevated overall myeloid colony activity (CFU-IL3) and diminished colony numbers of erythroid precursors (CFU-E) in recipients following BMT. Consistent with these results, it was found that upon co-transplant with limiting numbers of bone marrow cells, exogenously added Treg cells exert in-vivo regulation of myeloid and erythroid CFU activity during the initial weeks post-transplantation. This regulation of hematopoietic activity by freshly generated Tregs upon transplantation led to the elaboration of a second hypothesis; following lethal total body irradiation (TBI) the host microenvironment facilitates regulatory T cell activation/effector function. Substantial evidence has accumulated in support of this hypothesis, for example we demonstrate up-regulation of surface molecules such as GARP and CD150/SLAM, which have been previously reported as indicators of Treg activation following TCR signaling and co-stimulation, occurs in donor (reporter) Treg populations. Acquisition of an activated phenotype and hence of effector/modulatory function is consistent with the previous in-vivo observations, indicating that both recipient and donor Treg cells can influence hematopoietic progenitor cell activity post-transplant. Finally, the present studies may be of great relevance in the emerging field of Treg cell based immunotherapy for prevention and/or treatment of HSCT complications

Novel Scoring Criteria for the Evaluation of Ocular Graft-versus-Host Disease in a Preclinical Allogeneic Hematopoietic Stem Cell Transplantation Animal Model

•Employing an MHC-matched, minor transplantation antigen–mismatched allogeneic model of matched unrelated donor, we developed clinical scoring criterion identifying degrees of ocular pathology at both the ocular surface and adnexa•The association of these clinical changes with the development of immune responses around the ocular adnexa is monitored in real time•We report, for the first time, that these clinical and immune responses occur not only on the ocular surface, but they also heavily involve the lid margin region•Studies facilitate a standardized analysis of the immunologic and pathologic findings in the eye that occurs in animal models of ocular graft-versus-host disease Ocular complications occur after transplantation in 60% to 90% of chronic graft-versus-host disease (GVHD) patients and significantly impair vision-related quality of life. Ocular surface inflammation and dry eye disease are the most common manifestations of ocular GVHD. Ocular GVHD can be viewed as an excellent preclinical model that can be studied to understand the immune pathogenesis of this common and debilitating disease. A limitation of this is that only a few experimental models mimic the ocular complications after hematopoietic stem cell transplantation (HSCT) and have focused on the acute GVHD process. To address this issue, we used a preclinical animal model developed by our group where ocular involvement was preceded by systemic GVHD to gain insight regarding the contributing immune mechanisms. Employing this “matched unrelated donor” model enabled the development of clinical scoring criteria, which readily identified different degrees of ocular pathology at both the ocular surface and adnexa, dependent on the level of conditioning before HSCT. As far as we are aware, we report for the first time that these clinical and immune responses occur not only on the ocular surface, but they also heavily involve the lid margin region. In total, the present study reports a preclinical scoring model that can be applied to animal models as investigators look to further explore GVHD's immunologic effects at the level of the ocular surface and eyelid adnexa compartments. We speculate that future studies will use this clinical scoring index in combination with what is recognized histologically and correlated with serum biomarkers identified in chronic/ocular GVHD

Novel Scoring Criteria for the Evaluation of Ocular Graft-versus-Host Disease in a Preclinical Allogeneic Hematopoietic Stem Cell Transplantation Animal Model

Ocular complications occur after transplantation in 60% to 90% of chronic graft-versus-host disease (GVHD) patients and significantly impair vision-related quality of life. Ocular surface inflammation and dry eye disease are the most common manifestations of ocular GVHD. Ocular GVHD can be viewed as an excellent preclinical model that can be studied to understand the immune pathogenesis of this common and debilitating disease. A limitation of this is that only a few experimental models mimic the ocular complications after hematopoietic stem cell transplantation (HSCT) and have focused on the acute GVHD process. To address this issue, we used a preclinical animal model developed by our group where ocular involvement was preceded by systemic GVHD to gain insight regarding the contributing immune mechanisms. Employing this "matched unrelated donor" model enabled the development of clinical scoring criteria, which readily identified different degrees of ocular pathology at both the ocular surface and adnexa, dependent on the level of conditioning before HSCT. As far as we are aware, we report for the first time that these clinical and immune responses occur not only on the ocular surface, but they also heavily involve the lid margin region. In total, the present study reports a preclinical scoring model that can be applied to animal models as investigators look to further explore GVHD's immunologic effects at the level of the ocular surface and eyelid adnexa compartments. We speculate that future studies will use this clinical scoring index in combination with what is recognized histologically and correlated with serum biomarkers identified in chronic/ocular GVHD

Immunological Disruption of Antiangiogenic Signals by Recruited Allospecific T Cells Leads to Corneal Allograft Rejection

Corneal transplantation is the most common solid organ transplantation. The immunologically privileged nature of the cornea results in high success rates. However, T cell-mediated rejection is the most common cause of corneal graft failure. Using antiangiogenesis treatment to prevent corneal neovascularization, which revokes immune privilege, prevents corneal allograft rejection. Endostatin is an antiangiogenic factor that maintains corneal avascularity. In this study, we directly test the role of antiangiogenic and immunological signals in corneal allograft survival, specifically the potential correlation of endostatin production and T cell recruitment. We report that 75% of the corneal allografts of BALB/c mice rejected after postoperative day (POD) 20, whereas all syngeneic grafts survived through POD60. This correlates with endogenous endostatin, which increased and remained high in syngeneic grafts but decreased after POD10 in allografts. Immunostaining demonstrated that early recruitment of allospecific T cells into allografts around POD10 correlated with decreased endostatin production. In Rag(-/-) mice, both allogeneic and syngeneic corneal grafts survived; endostatin remained high throughout. However, after T cell transfer, the allografts eventually rejected, and endostatin decreased. Furthermore, exogenous endostatin treatment delayed allograft rejection and promoted survival secondary to angiogenesis inhibition. Our results suggest that endostatin plays an important role in corneal allograft survival by inhibiting neovascularization and that early recruitment of allospecific T cells into the grafts promotes destruction of endostatin-producing cells, resulting in corneal neovascularization, massive infiltration of effector T cells, and ultimately graft rejection. Therefore, combined antiangiogenesis and immune suppression will be more effective in maintaining corneal allograft survival

A Targeted and Adjuvanted Nanocarrier Lowers the Effective Dose of Liposomal Amphotericin B and Enhances Adaptive Immunity in Murine Cutaneous Leishmaniasis

Background.  Amphotericin B (AmB), the most effective drug against leishmaniasis, has serious toxicity. As Leishmania species are obligate intracellular parasites of antigen presenting cells (APC), an immunopotentiating APC-specific AmB nanocarrier would be ideally suited to reduce the drug dosage and regimen requirements in leishmaniasis treatment. Here, we report a nanocarrier that results in effective treatment shortening of cutaneous leishmaniasis in a mouse model, while also enhancing L. major specific T-cell immune responses in the infected host. Methods.  We used a Pan-DR-binding epitope (PADRE)-derivatized-dendrimer (PDD), complexed with liposomal amphotericin B (LAmB) in an L. major mouse model and analyzed the therapeutic efficacy of low-dose PDD/LAmB vs full dose LAmB. Results.  PDD was shown to escort LAmB to APCs in vivo, enhanced the drug efficacy by 83% and drug APC targeting by 10-fold and significantly reduced parasite burden and toxicity. Fortuitously, the PDD immunopotentiating effect significantly enhanced parasite-specific T-cell responses in immunocompetent infected mice. Conclusions.  PDD reduced the effective dose and toxicity of LAmB and resulted in elicitation of strong parasite specific T-cell responses. A reduced effective therapeutic dose was achieved by selective LAmB delivery to APC, bypassing bystander cells, reducing toxicity and inducing antiparasite immunity