Allogeneic Rejection Research Articles

Rapamycin-Based Expansion of Natural CD8+Tregs for Cell Therapy

Introduction We identified a subset of natural regulatory CD8+ T cells in peripheral blood of healthy volunteers characterized by the low expression of CD45RC surface molecule. These CD8+Tregs can inhibit allogeneic T cell proliferation in vitro through secretion of IFNg, IL-10, TGFbeta and IL-34. To date, there is no clinical study using CD8+Tregs as a cell-based therapy. Considering their potential for cellular therapy, we set up a process to expand these CD8+Tregs in a short term culture. Materials and Methods Tregs were expanded for 14 days with anti-CD3/CD28 mAbs or allogeneic APCs in presence of 1000U/ml IL-2, 10ng/ml IL-15, and with or without cyclosporine A (45ng/ml), rapamycin (45ng/ml), methylprednisolone (500pg/ml), tacrolimus (2ng/ml) or mycophenolate mofetil (1μg/ml). Suppressive function of CD8+Tregs was assessed in vitro on syngeneic CD4+CD25-T cells stimulated by alloAPCs, and in vivo into NSG mice infused with PBMCs and grafted or not with allogeneic human skin for allograft survival and xenogeneic GVHD studies. Results We demonstrated that natural CD8+Tregs can be efficiently expanded until 2000 fold in 14 days, when stimulated with either allogeneic APCs or anti-CD3 and anti-CD28 mAbs. High dose of IL-2 and IL-15 were required to maintain CD8+Tregs survival. After expansion, CD8+Tregs suppressive capacity was preserved and even increased in vitro, in accordance with their higher expression of immunoregulatory cytokines, while they displayed no cytotoxic activity. Furthermore, transfer of ex-vivo expanded CD8+Tregs allowed to delay GVHD development and allogeneic skin graft rejection in humanized mice models. As usually administered to patients after transplantation, we analyzed the impact of culture medium supplementation with immunosuppressive drugs during expansion. Interestingly, addition of rapamycin improved both CD8+Tregs expansion yield and suppressive activity by 16 and 1.6 fold respectively. Conclusions We demonstrated that natural CD8+Tregs can be greatly expanded while improved in their suppressive function particularly when cultured in presence of rapamycin, proving their relevance for cell therapy.

NK Cells Regulate CD8 T Cell-Mediated Allogeneic Rejection in Immunocompetent Recipients across an MHC Class I Mismatched Barrier

Non-T natural killer (NK) cells are a population of granular lymphoid cells TcR- CD3− CD122+ (NK1.1+ in C57BL/6 mice and DX5+ in Balb/c mice). These innate lymphoid cells mature in the bone marrow and are responsible for killing viral-infected and transformed cells in a non-MHC restricted manner, without the need of prior sensitization due to a recognition mechanism based on the array of germline-encoded inhibitory and activating receptors expressed on the cell surface of NK cells [1]. NK cells modulate CD8 T cell expansion during homeostatic proliferation in response to lymphopenic conditions by killing activated T cells and competing for IL-15 with the proliferating CD8 effector T cells [2]. This NK cell behavior may respond to an adaptive evolutionary mechanism to prevent aggressive anti-viral immune responses during the acute phase of infection and thus attenuate immunopathology [3]. We set up an experimental mouse model of alloreactivity across an MHC class I barrier to evaluate the role of NK cells in CD8 T cell-mediated rejection of bm1 skin allografts in naïve B6 recipients. In this transplant setting, host CD8 T cells are the main effectors T cells involved in the rejection of class I MHC mismatched skin allografts preferentially through the direct pathway of antigen presentation with limited help from CD4 T cells primed by the indirect pathway of antigen presentation. As opposed to previous studies done in lymphopenic immunodeficient recipients [2], depletion of NK cells was performed in immunocompetent C57BL/6 recipients with or without CD8 T depletion, the former displaying homeostatic proliferation in response to lymphopenia due to antibody-mediated elimination of CD8 T cells. We observed that depletion of NK cells in both CD8-repleted and CD8-depleted naïve C57BL/6 immunocompetent mice accelerated significantly bm1 graft rejection compared to non-NK-depleted controls (p<0.0005) (Figure 1). Our data claims for precaution in solid organ transplantation under inducing protocols of extensive depletion of leukocytes, such as ATG or alentuzumab. The immunotherapeutic interventions that include the removal of NK cells may accelerate graft rejection and promote aggressive CD8 T cell cytotoxic responses, which could be refractory to current immunosuppression. It also suggests that enhancing NK-cell functional activity combined with CD8 T cell depletion under tolerogenic regimen that tolerizes CD4 T cells at the time of transplantation may contribute to allogeneic donor-specific tolerance induction by elimination of the direct pathway of antigen presentation and by regulating the recovery of CD8 T cells.Bibliographic

Combination of MSC spheroids wrapped within autologous composite sheet dually protects against immune rejection and enhances stem cell transplantation efficacy.

Mesenchymal stem cells (MSCs) are widely used in transplantation therapy due to their multilineage differentiation potential, abundance, and immuno-modulating ability. However, the risk of allograft rejection limits their application. Here, we proposed a novel method to facilitate MSC transplantation with enhanced applicability and efficacy. We cultured human adipose-derived MSCs in a 3D culture under in vitro expansion conditions and under conventional 2D adherent culture conditions. MSC spheroids promoted extracellular matrix molecules that stimulate MSC proliferation, and produced more angiogenic cytokines such as vascular endothelial growth factor, hepatocyte growth factor, and fibroblast growth factor than 2D-cultured MSCs. Further, MSC spheroids showed increased IDO expression, increased proportion of M2 macrophages, and decreased macrophage proliferation, compared to 2D-cultured MSCs. Next, we proposed the wrapping of autologous cell sheets from the recipient around in-vitro-grown MSC spheroids to prevent allogenic immune rejection during transplantation. Myoblasts from C57BL/6 mice were used to prepare a stem cell composite sheet containing human-derived MSC spheres. The transplantation of MSC spheroids increased the survival rate and decreased the inflammatory response of the immunocompetent C57BL/6 ischemic mice. Thus, combining 3D-cultured MSC spheroid technology with immune evasion stem cell composite sheet improved the outcome and strengthened the protection against allogenic immune rejection.

Initial Clinical Evaluation of VC-01TM Combination Product—A Stem Cell–Derived Islet Replacement for Type 1 Diabetes (T1D)

VC-01 is a novel cell replacement therapy being developed as a potential long-term diabetes therapy to more effectively control glycemia, reduce hypoglycemia risk and mitigate diabetes-related complications. VC-01 is derived from directed differentiation of human embryonic stem cells to pancreatic progenitor cells, which are loaded into a durable, retrievable delivery device and then implanted subcutaneously. Once implanted and matured, the cells secrete insulin and other islet hormones in response to blood glucose levels. The delivery device is immunoprotective to implanted cells. VC-01 is currently being evaluated in a 24-month open-label, dose-escalating Phase 1/2 study in T1D patients with minimal insulin-producing β-cell function. Cohort 1 has evaluated product at a subtherapeutic dose to assess initial safety and tolerability and establish implantation techniques. To date, 19 subjects have been implanted and evaluated throughout implantation duration until final product explant. Product has been safe and well tolerated; adverse events have been relatively minor and generally related to the surgical procedure. The delivery device has provided protection against the host adaptive immune system with no evidence of allogeneic or autoimmune rejection or sensitization based upon a sensitive panel of immune function tests. No off-target growth of donor cells has been detected. The potential for prolonged cell survival has been demonstrated, as long as 24 months, but has been inconsistent among subjects and primarily limited by a foreign body response to the device component. Positive immunohistochemical staining for pancreatic islet cell markers NKX6-1, insulin, and glucagon has been observed. These preliminary results demonstrate the immune-protective nature of the encapsulation device and safety of this human stem cell-derived product, but also indicate the ongoing requirement for optimization of the host response to device materials. Disclosure R.R. Henry: Consultant; Self; Abbott, Alere Inc., AstraZeneca. Research Support; Self; AstaReal. Advisory Panel; Self; Boehringer Ingelheim Pharmaceuticals, Inc.. Consultant; Self; Bristol-Myers Squibb Company. Advisory Panel; Self; Elcelyx Therapeutics, Inc.. Research Support; Self; Eli Lilly and Company, Hitachi, Ltd.. Advisory Panel; Self; AstraZeneca. Consultant; Self; Boehringer Ingelheim Pharmaceuticals, Inc.. Advisory Panel; Self; Intarcia Therapeutics, Inc.. Consultant; Self; Intarcia Therapeutics, Inc., Ionis Pharmaceuticals, Inc., Janssen Pharmaceuticals, Inc.. Advisory Panel; Self; Johnson &amp; Johnson Services, Inc.. Research Support; Self; Lexicon Pharmaceuticals, Inc.. Consultant; Self; Ligand Pharmaceuticals, Inc.. Advisory Panel; Self; Merck &amp; Co., Inc.. Consultant; Self; Merck &amp; Co., Inc.. Research Support; Self; Viacyte, Inc.. Consultant; Self; Sanofi-Aventis. Advisory Panel; Self; Sanofi-Aventis. J. Pettus: Advisory Panel; Self; Sanofi, Novo Nordisk Inc.. Consultant; Self; MannKind Corporation. Advisory Panel; Self; Insulet Corporation. Consultant; Self; Senseonics. J. Wilensky: None. A. Shapiro: Consultant; Self; Viacyte, Inc., Protokinetix. P.A. Senior: Consultant; Self; Abbott, AstraZeneca. Research Support; Self; AstraZeneca, Prometic Life Sciences Inc., Novo Nordisk Inc., Sanofi. Consultant; Self; Eli Lilly and Company. Speaker's Bureau; Self; Eli Lilly and Company, Novo Nordisk Inc.. Consultant; Self; Novo Nordisk Inc., Janssen Pharmaceuticals, Inc.. Speaker's Bureau; Self; Janssen Pharmaceuticals, Inc.. Consultant; Self; Boehringer Ingelheim Pharmaceuticals, Inc.. Speaker's Bureau; Self; AstraZeneca, Merck &amp; Co., Inc., Abbott. Research Support; Self; Viacyte, Inc. B. Roep: Consultant; Self; Viacyte, Inc.. Research Support; Self; Viacyte, Inc. R. Wang: Employee; Self; Viacyte, Inc.. Stock/Shareholder; Self; Sanofi. E.J. Kroon: Employee; Self; Viacyte, Inc. M. Scott: Stock/Shareholder; Self; Viacyte, Inc.. K. D'Amour: None. H.L. Foyt: Employee; Self; Viacyte, Inc.. Stock/Shareholder; Self; Viacyte, Inc..

The development of induced pluripotent stem cell-derived mesenchymal stem/stromal cells from normal human and RDEB epidermal keratinocytes

BackgroundEpidermolysis bullosa (EB) is a group of hereditary disorders caused by mutations in the genes encoding structural molecules of the dermal-epidermal junction (DEJ). Cell-based therapies such as allogeneic mesenchymal stem/stromal cell (MSC) transplantation have recently been explored for severe EB types, such as recessive dystrophic EB (RDEB). However, hurdles exist in current MSC-based therapies, such as limited proliferation from a single cell source and limited cell survival due to potential allogenic rejection. ObjectivesWe aimed to develop MSCs from keratinocyte-derived induced pluripotent stem cells (iPSCs). MethodsKeratinocyte-derived iPSCs (KC-iPSCs) of a healthy human and an RDEB patient were cultured with activin A, 6-bromoindirubin-3′-oxime and bone morphogenetic protein 4 to induce mesodermal lineage formation. These induced cells were subjected to immunohistochemical analysis, flow cytometric analysis and RNA microarray analysis in vitro, and were injected subcutaneously and intravenously to wounded immunodeficient mice to assess their wound-healing efficacy. ResultsAfter their induction, KC-iPSC-induced cells were found to be compatible with MSCs. Furthermore, with the subcutaneous and intravenous injection of the KC-iPSC-induced cells into wounded immunodeficient mice, human type VII collagen was detected at the DEJ of epithelized areas. ConclusionsWe successfully established iPSC-derived MSCs from keratinocytes (KC-iPSC-MSCs) of a normal human and an RDEB patient. KC-iPSC-MSCs may have potential in therapies for RDEB.

PD-L1-engineered pancreatic islet grafts overcome rejection in allogeneic recipients

Abstract The use of chronic immunosuppression is a major hurdle for clinical islet transplantation as a curative modality for the treatment of type 1 diabetes. T effector cells (Teffs) are a main culprit of allograft rejection, and their pathogenic response is kept in check by CD4+CD25+FoxP3+ T regulatory cells (Tregs). The PD-1/PD-L1 immune checkpoint pathway plays an important role in the Teff/Treg balance with demonstrated clinical efficacy in cancer immunotherapy. This study was designed to assess the immunoregulatory function of PD-L1 in preventing allogeneic islet graft rejection. A chimeric protein (SA-PD-L1) containing the extracellular portion of PD-L1 fused to a modified form of core streptavidin was generated. SA-PD-L1 protein was expressed in insect cells and purified. The protein was functional as it enhanced TGF-beta-induced conversion of Teffs into Tregs and suppressed the proliferation of Teffs in response to alloantigens. Importantly, SA-PD-L1 was transiently displayed on the surface of biotinylated islets, and such engineered islets survived for over 100 days in allogeneic recipients treated with a short course (15 days) of low dose rapamycin. In marked contrast, unmodified islets under the same rapamycin regimen were rejected within 20 days. SA-PD-L1-engineered islets without rapamycin, although showing prolonged survival, were eventually rejected within 40 days. These results provide strong proof-of-efficacy and feasibility for the use of SA-PD-L1 protein as an immunomodulator to promote allogeneic islet graft survival in the absence of continued immunosuppression.

SA-FasL-engineered PEG microgels as a novel means of modulating immune response to allogeneic islet grafts

Abstract Allogenic islet transplantation is an effective therapy for type 1 diabetes in the clinic. Sustained graft survival requires chronic immunosuppression that has adverse effects. Islet graft rejection is initiated and perpetuated by T effector cells (Teffs). Teffs upregulate Fas death receptor on their surface and undergo apoptosis following engagement with FasL. Therefore, FasL has the potential as an immunomodulator to establish immune tolerance to islet grafts by eliminate alloreactive Teffs. This study used PEG microgels engineered with a novel form of FasL, SA-FasL, for the prevention of allogeneic islet graft rejection. Poly(ethylene glycol) microgels were produced with biotin on their surface. Biotinylated microgels were engineered with SA-FasL (1 μg/103 microgels) by taking advantage of the high affinity interaction between biotin and the streptavidin (SA) domain of the molecule. SA-FasL-PEGs induced apoptosis in Fas expressing A20 lymphoid cells in vitro. Co-transplantation of SA-FasL-PEGs with naïve islets led to prolonged survival of grafts as compared with the control group (islets + unmodified PEGs), with 20% surviving for a 200-day observation period. A short (15 days) treatment with low dose rapamycin further improved the immunomodulatory efficacy of SA-FasL-PEGs, with &amp;gt; 90% of grafts surviving for a 200-day observation period. CD4+CD25+FoxP3+ Tregs were required for graft survival, as depletion of this cell population on day 50 post-transplantation resulted in prompt rejection. These findings provide strong proof-of-efficacy and feasibility for the use of SA-FasL-engineered microgels as an off-the-shelf product for the modulation of alloreactive responses with significant therapeutic potential.

Brain Region-Dependent Rejection of Neural Precursor Cell Transplants.

The concept of CNS as an immune-privileged site has been challenged by the occurrence of immune surveillance and allogeneic graft rejection in the brain. Here we examined whether the immune response to allogeneic neural grafts is determined by the site of implantation in the CNS. Dramatic regional differences were observed between immune responses to allogeneic neural precursor/stem cell (NPC) grafts in the striatum vs. the hippocampus. Striatal grafts were heavily infiltrated with IBA-1+ microglia/macrophages and CD3+ T cells and completely rejected. In contrast, hippocampal grafts exhibited milder IBA-1+ cell infiltration, were not penetrated efficiently by CD3+ cells, and survived efficiently for at least 2 months. To evaluate whether the hippocampal protective effect is universal, astrocytes were then transplanted. Allogeneic astrocyte grafts elicited a vigorous rejection process from the hippocampus. CD200, a major immune-inhibitory signal, plays an important role in protecting grafts from rejection. Indeed, CD200 knock out NPC grafts were rejected more efficiently than wild type NPCs from the striatum. However, lack of CD200 expression did not elicit NPC graft rejection from the hippocampus. In conclusion, the hippocampus has partial immune-privilege properties that are restricted to NPCs and are CD200-independent. The unique hippocampal milieu may be protective for allogeneic NPC grafts, through host-graft interactions enabling sustained immune-regulatory properties of transplanted NPCs. These findings have implications for providing adequate immunosuppression in clinical translation of cell therapy.

ADAR1 attenuates allogeneic graft rejection by suppressing miR-21 biogenesis in macrophages and promoting M2 polarization.

ADAR1 (adenosine deaminase acting on double-stranded RNA 1) is an RNA-editing enzyme that mediates adenosine-to-inosine RNA editing events, an important post-transcriptional modification mechanism that can alter the coding properties of mRNA or regulate microRNA biogenesis. ADAR1 also regulates the innate immune response. Here, we have demonstrated that ADAR1 expression increased in LPS-stimulated macrophages. Silencing ADAR1 by using small interfering RNA in macrophages resulted in the pronounced polarization of macrophages to M1, whereas ADAR1 overexpression promoted M2 polarization, which indicated that ADAR1 can inhibit macrophage hyperpolarization and prevent immune hyperactivity. The RNA-RNP immunoprecipitation binding assay demonstrated a direct interaction between ADAR1 and miR-21 precursor. Significant up-regulation in IL-10 and down-regulation in miR-21 were observed in ADAR1-overexpressing macrophages. We evaluated miR-21 target mRNAs and macrophage polarization signaling pathways and found that forkhead box protein O1 (Foxo1) was up-regulated in cells that overexpressed ADAR1. In a mouse allogeneic skin transplantation model, grafts in the ADAR1-overexpressed group survived longer and suffered less immune cell infiltration. In ADAR1-overexpressed recipients, splenic macrophages were significantly polarized to M2, and levels of sera IL-10 were markedly higher than those in the control group. In summary, ADAR1 modulates macrophage M2 polarization via the ADAR1-miR-21-Foxo1-IL-10 axis, thereby suppressing allogeneic graft rejection.-Li, J., Xie, J., Liu, S., Li, X., Zhang, D., Wang, X., Jiang, J., Hu, W., Zhang, Y., Jin, B., Zhuang, R., Yin, W. ADAR1 attenuates allogeneic graft rejection by suppressing miR-21 biogenesis in macrophages and promoting M2 polarization.

Extracellular vesicles and their immunomodulatory functions in pregnancy.

Extracellular vesicles (EVs) are membrane-bound vesicles released into the extracellular space by almost all types of cells. EVs can cross the physiological barriers, and a variety of biological fluids are enriched in them. EVs are a heterogeneous population of vesicles, including exosomes, microvesicles, and apoptotic bodies. The different subpopulations of vesicles can be differentiated by size and origin, in which exosomes (~100nm and from endocytic origin) are the most studied so far. EVs have essential roles in cell-to-cell communication and are critical modulators of immune response under normal and pathological conditions. Pregnancy is a unique situation of immune-modulation in which the maternal immune system protects the fetus from allogenic rejection and maintains the immunosurveillance. The placenta is a vital organ that performs a multitude of functions to support the pregnancy. The EVs derived from the human placenta have crucial roles in regulating the maternal immune response for successful pregnancy outcome. Placenta-derived vesicles perform a myriad of functions like suppression of immune reaction to the developing fetus and establishment and maintenance of a systemic inflammatory response to combat infectious intruders. A fine-tuning of these mechanisms is quintessential for successful completion of pregnancy and healthy outcome for mother and fetus. Dysregulation in the mechanisms mentioned above can lead to several pregnancy disorders. In this review, we summarize the current literature regarding the critical roles played by the EVs in immunomodulation during pregnancy with particular attention to the placenta-derived exosomes.

Delayed allogeneic skin graft rejection in CD26-deficient mice.

Organ transplantation is an effective therapeutic tool for treating many terminal diseases. However, one of the biggest challenges of transplantation is determining how to achieve the long-term survival of the allogeneic or xenogeneic transplant by, for example, preventing transplant rejection. In the current study, CD26 gene-knockout mice were used to investigate the potential role of CD26/dipeptidyl peptidase-4 (DPPIV) in allogeneic skin graft rejection by tail-skin transplantation. Compared with wild-type (CD26+/+) counterparts, CD26–/– mice showed reduced necrosis of grafts and delayed graft rejection after skin transplantation. Concentrations of serum IgG, including its subclasses IgG1 and IgG2a, were significantly reduced in CD26–/– mice during graft rejection. Moreover, after allogeneic skin transplantation, the secretion levels of the cytokines IFN-γ, IL-2, IL-6, IL-4, and IL-13 were significantly reduced, whereas the level of the cytokine IL-10 was increased in the serum of CD26–/– mice compared with that in the serum of CD26+/+ mice. Additionally, the concentration of IL-17 in serum and the percentage of cells secreting IL-17 in mouse peripheral blood lymphocytes (MPBLs) were both significantly lower, while the percentage of regulatory T cells (Tregs) was significantly higher in MPBLs of CD26–/– mice than in those of CD26+/+ mice. Furthermore, a lower percentage of CD8+ T cells in MPBLs and fewer infiltrated macrophages and T cells in graft tissues of CD26–/– mice were detected during graft rejection. These results indicate that CD26 is involved in allogeneic skin graft rejection and provides another hint that CD26 deficiency leads to less rejection due to lower activation and proliferation of host immune cells.

CD150high Bone Marrow Tregs Maintain Hematopoietic Stem Cell Quiescence and Immune Privilege via Adenosine.

A crucial player in immune regulation, FoxP3+ regulatory Tcells (Tregs) are drawing attention for their heterogeneity and noncanonical functions. Here, we describe a Treg subpopulation that controls hematopoietic stem cell (HSC) quiescence and engraftment. These Tregs highly expressed an HSC marker, CD150, and localized within the HSC niche in the bone marrow (BM). Specific reduction of BM Tregs achieved by conditional deletion of CXCR4 in Tregs increased HSC numbers in the BM. Adenosine generated via the CD39 cell surface ectoenzyme on niche Tregs protected HSCs from oxidative stress and maintained HSC quiescence. In transplantation settings, niche Tregs prevented allogeneic (allo-) HSC rejection through adenosine and facilitated allo-HSC engraftment. Furthermore, transfer of niche Tregs promoted allo-HSC engraftment to a much greater extent than transfer of other Tregs. These results identify a unique niche-associated Treg subset and adenosine as regulators of HSC quiescence, abundance, and engraftment, further highlighting their therapeutic utility.

Janus-Faced Myeloid-Derived Suppressor Cell Exosomes for the Good and the Bad in Cancer and Autoimmune Disease

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells originally described to hamper immune responses in chronic infections. Meanwhile, they are known to be a major obstacle in cancer immunotherapy. On the other hand, MDSC can interfere with allogeneic transplant rejection and may dampen autoreactive T cell activity. Whether MDSC-Exosomes (Exo) can cope with the dangerous and potentially therapeutic activities of MDSC is not yet fully explored. After introducing MDSC and Exo, it will be discussed, whether a blockade of MDSC-Exo could foster the efficacy of immunotherapy in cancer and mitigate tumor progression supporting activities of MDSC. It also will be outlined, whether application of native or tailored MDSC-Exo might prohibit autoimmune disease progression. These considerations are based on the steadily increasing knowledge on Exo composition, their capacity to distribute throughout the organism combined with selectivity of targeting, and the ease to tailor Exo and includes open questions that answers will facilitate optimizing protocols for a MDSC-Exo blockade in cancer as well as for strengthening their therapeutic efficacy in autoimmune disease.

Electro-spun Membranes as Scaffolds for Human Corneal Endothelial Cells

ABSTRACTBackground: Corneal endothelial dysfunction remains the most frequent indication for corneal transplantation, limited by donor material shortage, poor long-term graft survival, or allogeneic graft rejection. Therefore, tissue-engineered endothelial grafts (TEEG) represent a promising alternative to human donor tissue. In this study, we generated electro-spun scaffolds and tested these for their suitability for human corneal endothelial cell (hCEC) cultivation.Methods: The polymers poly(methyl-methacrylate) (PMMA), poly(lactic-co-glycolic acid) (PLGA), and polycaprolactone (PCL) were spun with equal parameters. HCEC-12 was cultured on the scaffolds for 3 to 7 days. Scaffolds were evaluated by light microscopy, porometry, light transmission, scanning electron microscopy (SEM), live/dead staining and cell viability assay.Results: Electro-spun fibers from PMMA (2.99 ± 0.24 µm) showed significantly higher diameters than PCL (2.29 ± 0.11 µm; p = 0.003) and PLGA (1.84 ± 0.21 µm; p < 0.001), while fibers from PCL also showed larger diameters than those from PLGA (p = 0.002). PMMA scaffolds (26.77 ± 17.48 µm) had significantly larger interstitial spaces than those from PCL (13.30 ± 5.47 µm; p = 0.04) and PLGA (10.42 ± 6.15 µm; p = 0.002), while PCL and PLGA did not differ significantly (p = 0.26). SEM analysis revealed that only PLGA fibers preserved a normal HCEC-12 morphology. PLGA and PCL did not differ in cell number, death, or viability after 7 days of HCEC-12 cultivation. PMMA showed significantly higher cytotoxicity (p < 0.001; PLGA: 1626.2 ± 183.8 RLU; PMMA: 841.9 ± 92.7 RLU; PCL: 1580.2 ± 171.02 RLU).Conclusions: The biodegradable PLGA and PCL electro-spun scaffolds resulted in equal biocompatibility, while PMMA showed cytotoxicity. Only PLGA preserved hCEC morphology and consequently seems to be a promising candidate for TEEG construction.

Formulation and in vivo pharmacokinetic evaluation of ethyl cellulose-coated sustained release multiple-unit system of tacrolimus

A novel once-a-day sustained-release (SR) system of tacrolimus (FK506), a poorly water-soluble immunosuppressive agent, was designed employing ethyl cellulose (EC) polymer as release retardant. Drug (5 mg) was layered onto sugar spheres (518.3 mg) with hypromellose (5 mg), to transform the drug from a crystalline to an amorphous form. Subsequently, the drug-layered pellets were recoated with EC polymer (0.5-1.5 mg) using a fluid bed granulator. Drug release from the reservoir-type pellets was markedly impeded by the outer EC-based coating layer (EC 1 mg), displaying about 60% of drug release after 8 h, regardless of the acidity of the media. In an in vivo pharmacokinetic study in fasted Cynomolgus monkeys, the drug level in blood was gradually increased over 4.7 h and high drug concentration was maintained until 24 h, with an elimination half-life of 16.6 h. There were no statistical differences between the novel SR pellets and the recently marketed SR capsule (Advagraf®, Astellas Pharma, Japan) in terms of maximum blood concentration, area under the curve, and half-life values, in both fasted and fed states. Therefore, the novel EC-coated pellets are expected to be bioequivalent to the commercial SR capsule, providing a once-daily dosing regimen in patients with allogenic rejection.

Nuclear UCHL5 Expression Associates with Increased Patient Survival in Pancreatic Ductal Adenocarcinoma

While reef-building corals portray highly complex and specific allorecognition responses, still, no available synthesis on historecognition at the molecular level exists for this group of organisms. Here, we present the first subtractive library of expressed sequence tags (ESTs) from allogeneic challenged coral (Stylophora pistillata) colonies revealing the differential expression of a wide range of immune-related genes. 1760 unique ESTs were clustered and assembled into 230 contigs and 1530 singlets with 28% that showed homology (E-value ≤ 0.005) to known database sequences, of which 16% (n = 80) homologues were identified as immune-relevant genes, encoding for stress proteins, pattern recognition receptors and complement proteins, proteases, cell adhesion proteins, cytokine related proteins, programmed cell death and proteasome-associated proteins. Transcripts that were subjected to quantitative RT-PCR, further supported the library data. In situ hybridization analyses elucidated specific and enhanced expressions of von Willebrand factor-like transcript during S. pistillata allogeneic rejection. Availability of such genome-wide expression tools may lead to significant advances in the research of coral historecognition and comparative immunology.

The Immunogenicity and Immune Tolerance of Pluripotent Stem Cell Derivatives.

Human embryonic stem cells (hESCs) can undergo unlimited self-renewal and differentiate into all cell types in human body, and therefore hold great potential for cell therapy of currently incurable diseases including neural degenerative diseases, heart failure, and macular degeneration. This potential is further underscored by the promising safety and efficacy data from the ongoing clinical trials of hESC-based therapy of macular degeneration. However, one main challenge for the clinical application of hESC-based therapy is the allogeneic immune rejection of hESC-derived cells by the recipient. The breakthrough of the technology to generate autologous-induced pluripotent stem cells (iPSCs) by nuclear reprogramming of patient’s somatic cells raised the possibility that autologous iPSC-derived cells can be transplanted into the patients without the concern of immune rejection. However, accumulating data indicate that certain iPSC-derived cells can be immunogenic. In addition, the genomic instability associated with iPSCs raises additional safety concern to use iPSC-derived cells in human cell therapy. In this review, we will discuss the mechanism underlying the immunogenicity of the pluripotent stem cells and recent progress in developing immune tolerance strategies of human pluripotent stem cell (hPSC)-derived allografts. The successful development of safe and effective immune tolerance strategy will greatly facilitate the clinical development of hPSC-based cell therapy.

Gut Permeability and Glucose Absorption Are Affected at Early Stages of Graft Rejection in a Small Bowel Transplant Rat Model

Intestinal transplantation (IT) faces many challenges, among them, the necessity to early detect rejection processes. Rodent models of IT are used to provide evidence for intervention strategies as well as improve knowledge of small bowel transplant biology. The aim of our study was to determine the kinetics of small bowel rejection using a Heterotopic IT (HIT) model, with emphasis in the characterization of the process using different approaches, including the use of simple functional assays of absorptive and barrier function evaluation that could be eventually translated to the clinic. Allogeneic HIT was performed following standard procedure and rejection was monitored by clinical scoring and histopathology analysis. In order to evaluate graft absorptive function, a solution containing glucose (2 gr/Kg) and ovalbumin (OVA) (150 ug/dose) diluted in 1.5 ml of normal saline was administered by proximal ostomy 5 and 10 to 12 days after HIT. Blood samples were obtained for glucose and OVA determination at different times post-treatment. In allogeneic HIT recipients rejection kinetics was coincident to previously described, with mild rejection beginning by day 5 post-IT, becoming severe by day 10 post-IT (Figure 1). Interestingly, either at 5 or 10 to 12 post-IT days, glucose levels did not change from the basal value (101 +/− 21 mg/dL) in this group. In most of evaluated cases 5 days after HIT and in all cases evaluated at 10–12 post-IT days in allogeneic groups, OVA was detected in the recipient´s serum (24 +/−11 ug/mL, p<0.001 vs. isogenic control) 90 min post treatment. On the other hand, glucose curves were normal with a rise from basal to 240 +/−32 mg/dL at 60 min post-treatment in non-IT controls as well as in isogeneic HIT recipients (p<0.001 vs allogenic group). Furthermore, OVA was undetectable in any serum sample from control groups.FigureThis result indicate that even in early stages of ACR, there is an impairment of barrier function that is evidenced by passage of non-degraded proteins from intestinal lumen to blood. Furthermore, there is impairment on glucose absorptive capacity, even from the initial stages of rejection diagnosed by clinical and histological evaluations. The evaluation of these graft functional features could provide additional tools for rejection diagnosis in the clinics. Normal microscopic (A) and macroscopic (B) gaft appearance 10 days after syngeneic HIT. Microsocpic (C and E) and macroscopic appearance of the transplanted intestine compatible with ACR in allogeneic group.

CD8+ effector memory T cells induce acute rejection of allogeneic heart retransplants in mice possibly through activating expression of inflammatory cytokines

BackgroundTo investigate the effects of CD8+ memory T (Tm) cells and CD8+ effector memory T (Tem) cells on the results of allogeneic heart retransplantations performed in mice. MethodsA skin transplantation model was used to generate sensitized splenic CD8+ Tem cells for infusion into BALB/c mice. One week after infusion, the BALB/c mice underwent allogeneic heart transplantation in the abdominal cavity. Cyclosporin A was administered via intraperitoneal injection starting one day prior to transplantation to arrest immunological rejection of the transplanted heart. The effects of sensitized CD8+ Tem cells on allogeneic heart graft rejection were examined by monitoring survival of the transplanted hearts, the infiltration of effector memory CD8+ T cells into myocardium, and expressions of inflammatory cytokines in blood serum. ResultsAdoptive transfer of sensitized CD8+ Tem cells prior to transplantation induced an acute rejection response which decreased the survival of transplanted hearts. The rejection response was accompanied by an infiltration of CD8+ Tem cells into the transplanted myocardial tissue. Additionally, infusion of sensitized CD8+ Tem cells induced markedly increased expressions of IL-2 and IFN-γ, and decreased expression of TGF-β in the transplanted hearts, as well as higher levels of IFN-γ and CXCL-9 in blood serum. ConclusionsThe infusion of sensitized CD8+ Tem cells induced an acute graft rejection response and decreased the survival of grafted hearts by regulating the expressions of inflammatory cytokines including CXCL-9, IL-2, and INF-γ. Cyclosporin A had no therapeutic effect on the graft rejection response induced by sensitized CD8+ Tem cells.

Comparison of the glycosphingolipids of human-induced pluripotent stem cells and human embryonic stem cells.

High expectations are held for human-induced pluripotent stem cells (hiPSC) since they are established from autologous tissues thus overcoming the risk of allogeneic immune rejection when used in regenerative medicine. However, little is known regarding the cell-surface carbohydrate antigen profile of hiPSC compared with human embryonic stem cells (hESC). Here, glycosphingolipids were isolated from an adipocyte-derived hiPSC line, and hiPSC and hESC glycosphingolipids were compared by concurrent characterization by binding assays with carbohydrate-recognizing ligands and mass spectrometry. A high similarity between the nonacid glycosphingolipids of hiPSC and hESC was found. The nonacid glycosphingolipids P1 pentaosylceramide, x2 pentaosylceramide and H type 1 heptaosylceramide, not previously described in human pluripotent stem cells (hPSC), were characterized in both hiPSC and hESC. The composition of acid glycosphingolipids differed, with increased levels of GM3 ganglioside, and reduced levels of GD1a/GD1b in hiPSC when compared with hESC. In addition, the hESC glycosphingolipids sulf-globopentaosylceramide and sialyl-globotetraosylceramide were lacking in hiPSC. Neural stem cells differentiating from hiPSC had a reduced expression of sialyl-lactotetra, whereas expression of the GD1a ganglioside was significantly increased. Thus, while sialyl-lactotetra is a marker of undifferentiated hPSC, GD1a is a novel marker of neural differentiation.