Human embryo research in France

The French law on bioethics, voted upon in July 1994, is going to be revised. This is the occasion for France to reconsider its position concerning research on human embryos, which is currently prohibited in France, as it is in Germany, Switzerland and Austria. However, such research is authorised in other European countries such as the UK, Spain, Belgium, Italy and The Netherlands. The establishment of human embryonic stem (ES) cells has reopened the debate in France because of their potential in human therapy. Indeed, ES cells, derived from early embryos (5-6 days old), preserve in vitro a pluripotent character, and they could provide an infinite source of different tissues that could be used in replacement therapy. This consists of ES cells differentiated in vitro into the desired tissues or cell types and grafted into the patient. The use of human ES cells in replacement therapy raises the major problem of graft rejection. One of the proposed solutions would be to carry out a 'therapeutic cloning' and to derive ES cells from the embryos obtained in this way. We do consider that, for the moment, the interest of the cloning study lies mainly in the understanding of the mechanisms responsible for reprogramming the nuclei. This research can be performed first on animal models. France is now thinking to allow human embryo research. We present here the French law proposed on human embryo research. French government is proposing to allow research exclusively on frozen supernumerary embryos, which no longer have any parental or adoption potential. Creation of human embryos for research purposes will still be prohibited. However, allowance of studies on human cloning in order to realise therapeutic cloning is mentioned in the proposal. We think that allowing research in humans on therapeutic cloning is premature and contradicts the prohibition of the creation of human embryos for research.     

Regulation of the CD4 T cell allo-immune response by endothelial cells

Recent studies have revealed the presence of pro-inflammatory and/or regulatory CD4⁺ T cells within allografts promoting either graft rejection or tolerance. Histological evidence has identified the microvascular endothelium as the primary site of allograft damage as it is the first site of encounter with the recipient’s immune system. The initial view of the human endothelial cell inducing an effector Th1 response leading to graft rejection has been extended by recent results which demonstrate the endothelial cell ability to generate other CD4⁺ T cell sub-populations including Th17 and Treg cells. In the transplantation setting, the allo-reactivity of the endothelium with the CD4⁺ T cell populations is likely to depend upon multiple factors including the vascular origin of the endothelial cell, the cytokine environment, the presence or absence of pro-inflammatory stimuli including ligands of Toll like receptors and alloantibodies. This review provides an update on the characteristics of the endothelial cell activation of the CD4⁺ T cell response and an analysis of the factors, which can modify this activity in favor of either graft rejection or tolerance.     

MicroRNA-155 may affect allograft survival by regulating the expression of suppressor of cytokine signaling 1

Immune rejection of organ transplants has life-threatening implications. It is believed that allograft rejection is initiated by the activation of lymphocytes following recognition of donor antigens, leading to generation of effector T lymphocytes, alloantibody production, and graft infiltration by alloreactive cells. There is solid evidence that miRNAs are integral for maintaining immune homeostasis and self-tolerance. A deeper understanding of the regulation of the immune response by miRNAs could define new mechanisms for manipulating graft immunity and preventing rejection. The miRNA miR-155 is of particular interest due to its known roles in regulating the expression of genes relevant to allograft rejection and the induction of immune tolerance. Indeed, miR-155 has been shown to dramatically impact both innate and adaptive immune processes, including inflammation, antigen presentation, T-cell differentiation, cytokine production, and T regulatory cell (Treg) functions. The suppressor of cytokine signaling 1 (SOCS1) is a critical regulator of immune cell function and an evolutionarily conserved target of miR-155 in breast cancer cells. We propose that suppression of miR-155 could enhance SOCS1 expression in immune cells and suppress allograft rejection. Further studies on the specific role of miR-155 in allograft rejection may lead to the identification of new targets for therapeutic intervention.     

Therapeutic cloning by xenotransplanted oocytes, supplemented with species specific reprogramming factors

For therapeutic medication, a lot of work has been done regarding human embryonic stem (ES) cell lines derivation, but immunorejection is a major dilemma of this cell based therapy. Since long time, derivation of patient matched stem cells have been hoped to overcome this problem. Oocytes after nuclear transfer are the most reliable source for patient matched ES cells derivation, for therapeutic use. In humans, utilization of oocytes for stem cell research raises sensitive logistical and ethical questions; primarily surrounding participation of women as oocyte donors. It has been claimed that therapeutic cloning would lead to commercial exploitation of poor women. On the other hand, the therapeutic promise of embryonic stem cell is so huge that there is a strong incentive to find some alternate sources of human oocytes. Here we propose to utilize the cross species oocytes supplemented with human ES cellular extracts to establish patient specific ES cell lines.     

Embryonic stem cells: protecting pluripotency from alloreactivity

There can be little doubt that 2006 turned out to be the annus horribilis for therapeutic cloning by somatic nuclear transfer (SNT). As the full extent of the fraud surrounding the generation of patient-specific embryonic stem (ES) cell lines became apparent, hopes began to fade for the advent of cell replacement therapies (CRT), free from the confounding issues of immune rejection. While the dust begins to settle, it is perhaps pertinent to ask whether the promise of SNT is still worth pursuing or whether alternative strategies for immune evasion might help fill the void.     

Embryonic stem cells generate airway epithelial tissue

Embryonic stem (ES) cells are self-renewable and pluripotent cells derived from the inner cell mass of a blastocyst-stage embryo. ES cell pluripotency is being investigated increasingly to obtain specific cell lineages for therapeutic treatments and tissue engineering. Type II alveolar epithelial cells have been derived from murine ES cells, but the capacity of the latter to generate differentiated airway epithelial tissue has never been reported. Herein, we show by RT-PCR and immunocytochemistry that murine ES cells are able to differentiate into nonciliated secretory Clara cells, and that type I collagen induces this commitment. Moreover, when cultured at the air-liquid interface, ES cells give rise to a fully differentiated airway epithelium. By quantitative histologic examination, immunohistochemistry, and scanning electron microscopy, we show that the bioengineered epithelium is composed of basal, ciliated, intermediate, and Clara cells, similar to those of native tracheobronchial airway epithelium. Transmission electron microscopy and Western blotting reveal that the generated epithelium also exhibits the ultrastructural features and secretory functions characteristic of airway epithelial tissue. These results open new perspectives for cell therapy of injured epithelium in airway diseases, such as bronchopulmonary dysplasia, cystic fibrosis, or bronchiolitis obliterans.     

Tumor growth or regression: powered by inflammation

Malignant cells thrive in a highly specialized, stromal environment, which harbors support cells, blood vessels, and diverse leukocyte populations. There is increasing evidence that "by default", intratumoral inflammation fosters angiogenic and vasculogenic processes and simultaneously creates an immunosuppressive micromilieu. This self-amplifying loop of proangiogenic inflammation represents a serious obstacle for adaptive anticancer immune responses. However, angiogenesis is a highly dynamic process, which can be reversed in the "right" inflammatory context; this in turn facilitates immune effector cell entry and tumor rejection. Thus, we propose that a shift from proangiogenic to antiangiogenic inflammation creates a tumor environment permissive for immune destruction. This is a new concept, which integrates antiangiogenic and immune therapeutic treatment modalities.     

血管抑制素和内皮抑制素在大肠杆菌中的融合表达与鉴定

血管抑制素（AS）和内皮抑制素（ES）是两种具有较强活性的内源性血管抑制剂，联合应用具有协同作用。本研究通过大肠杆菌表达AS-ES融合蛋白，观察其对血管生成的抑制作用。首先用RT-PCR方法分别获得AS和ES基因，通过基因拼接获得融合基因，构建了含有该融合基因的原核表达质粒——pET-42（b）／AS-ES。表达菌株经IPTG诱导后目的蛋白以包涵体形式表达，表达量为14％，分子量约65KD。Western blotting检测表明表达产物可分别与AS和ES抗体产生特异的免疫反应。经复性、肝素亲和层析柱纯化的表达产物对鸡胚尿囊膜血管有明显抑制作用。本研究获得了AS、ES在大肠杆菌中的融合表达．目的蛋白具有特异的免疫反应性和血管抑制活性。     

Endothelial and smooth muscle cells express leukocyte adhesion molecules heterogeneously during acute rejection of rabbit cardiac allografts.

Interactions of leukocytes with vascular wall cells figure prominently in acute rejection and development of vascular occlusive disease after cardiac transplantation. To investigate the time course and distribution among different types of vessels of expression of endothelial leukocyte adhesion molecules, issues difficult to address in humans, we studied heterotopic transplants of Dutch-Belted rabbit hearts into New Zealand white recipients without immunosuppression (average time to graft failure 8.2 +/- 0.4 days). We found constitutive expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) by coronary arterial endothelium in normal rabbits, whereas myocardial capillaries and the endocardial lining cells showed little or no expression of VCAM-1. VCAM-1 expression increased within 1 day after transplantation on the endothelium of the transplanted aorta and endocardium and on myocardial microvascular endothelial cells. ICAM-1 expression increased remarkably on all endothelia studied from 2 to 8 days after transplantation. Adhesion molecule expression on coronary artery endothelial cells also increased during severe allograft rejection (from a histological score of 1.7 +/- 0.6 pretransplant to 4.8 +/- 0.2 8 days after transplant for VCAM-1 and from 0.9 +/- 0.6 to 4.4 +/- 0.3 for ICAM-1, n = 43 arteries in 5 animals, mean +/- SD). In addition, coronary artery and aortic smooth muscle cells also showed induction of VCAM-1 and ICAM-1 8 days after transplant. We conclude that endothelial activation in a transplanted organ can occur rapidly and varies among microvascular, endocardial, and coronary artery endothelial cells, a point germane to the interpretation of endomyocardial biopsies. Augmented expression of adhesion molecules precedes temporally leukocyte accumulation in vessels. In addition, our finding of activation of coronary artery smooth muscle cells during acute rejection suggests that such episodes may contribute to the development of accelerated coronary arteriosclerosis.     

Mechanisms of tissue injury in hyperacute xenograft rejection.

Vascularized organs transplanted between phylogenetically distant species are subject to hyperacute rejection. The pathologic features of hyperacute rejection appear to be caused by profound dysfunction of small blood vessels in the graft. The author describes a model for the initiation and pathogenesis of hyperacute rejection. The model focuses on the endothelial cell as a target of the recipient immune reaction. Rejection is initiated by the binding to donor endothelial cells of recipient xenoreactive natural antibodies, which, in turn, triggers the complement cascade. These events lead to activation of graft endothelial cells, which promotes the thrombus formation and loss of vascular integrity characteristic of hyperacute rejection.     

肾移植免疫学研究进展

随着免疫抑制剂的不断开发应用，肾移植术后移植肾短期存活率已大大提升，然而其长期存活率仍较低。对于肾移植术后移植肾存活而言，免疫排斥是其中最重要的危险因素。近年来，基于肾移植移植免疫排斥反应，肾移植移植免疫系统逐渐建立，各种免疫细胞在肾移植中的作用及其机制逐渐被揭示，尤其是B细胞及其在肾移植中的作用，逐渐被人们所重视。本文以免疫细胞与移植肾的关系为出发点，详细阐述适应性免疫与天然免疫在肾移植中的作用及其机制，以期梳理肾移植中移植免疫机制，为今后肾移植免疫研究探索新的方向。     

Immunological barriers to embryonic stem cell-derived therapies

Replacement of diseased tissues with healthy cells derived from embryonic stem (ES) cells has a potential to become, in the future, a better alternative to current treatments of a number of conditions characterized by irreversible tissue injury, such as heart and liver failure, diabetes mellitus and neurodegeneration. However, several obstacles have to be overcome before this new therapeutic modality becomes part of a standard clinical practice. First of all, ethical and safety issues have to be resolved, the methodologies must be developed to enable obtaining sufficient amounts of differentiated cells, and the immune rejection of allogeneic cells must be prevented in order to ensure their long-term engraftment and function. Data on immunological properties of human and murine ES cells and their differentiated derivatives are controversial, ranging from those claiming unique immune-privileged properties for ES cells to those which refute these conclusions. This indicates that much more research is required to definitively understand the immunological features and engraftment capacity of ES cell derivatives. We review here the current state of the art in this new and exciting field of ES cell immunology and discuss the implications of these findings for the development of ES cell-based therapies.     

Dermal Microvascular Injury in the Human Peripheral Blood Lymphocyte Reconstituted-Severe Combined Immunodeficient (HuPBL-SCID) Mouse/Skin Allograft Model Is T Cell Mediated and Inhibited by a Combination of Cyclosporine and Rapamycin

We have analyzed the mechanism of human endothelial injury in a human peripheral blood lymphocyte-severe combined immunodeficient (huPBL-SCID) mouse/human skin graft model of allograft injury and examined the effect of immunosuppressive drugs on this process. In this model, split-thickness human skin containing the superficial dermal microvessels was grafted onto immunodeficient C.B-17 SCID or SCID/beige mice and allowed to heal. Human peripheral blood mononuclear cells (PBMCs) allogeneic to the skin, when subsequently introduced by intraperitoneal injection, caused destruction of the human dermal microvasculature by day 16, evident as endothelial cell sloughing and thrombosis. In the same specimens, mouse microvessels that invaded the human skin graft were uninjured. Human microvascular cell injury was accompanied by a mononuclear cell infiltrate consisting of approximately equal numbers of human CD4+ and CD8+ T cells, some of which contained perforin-positive granules. We found no evidence of human natural killer cells and noted occasional human, but not mouse, macrophages at a frequency indistinguishable from that resident in skin on animals not receiving human PBMCs. These human T cell infiltrates did not extend into adjacent mouse skin. Human immunoglobulin G antibody was detected in the blood and was diffusely present throughout mouse and human tissues in SCID mice receiving PBMCs. Mouse C3 was detected on human dermal vessels in both unreconstituted control animals and those that received PBMCs. Blood and tissues from mice injected with PBMCs depleted of B cells showed no human immunoglobulin, but circulating CD3+ cells were detected by flow cytometry at levels comparable with those of animals receiving whole PBMCs. Significantly, skin graft infiltration by human T cells and human dermal microvascular injury were equivalent in the B cell-depleted and whole-PBMC-reconstituted mice. Mice inoculated with PBMCs depleted of CD8+ T cells developed microvascular injury and infiltrates containing perforin-expressing CD4+ T cells. These data suggested a cytolytic T cell-dependent mechanism of microvessel injury. We then tested the ability of T cell immunosuppressants, cyclosporine and rapamycin, to attenuate vessel damage. Neither cyclosporine nor rapamycin alone effectively reduced either mononuclear cell infiltration or vascular injury. However, a combination of the two agents reduced both parameters. We conclude that the huPBL-SCID/skin allograft model may be used both to study cytolytic T cell-mediated rejection and to test the effect of immunosuppressive drug strategies in vivo in a small-animal model of human immune responses.     

Widespread immunological functions of mast cells: fact or fiction?

Immunological functions of mast cells are currently considered to be much broader than the original role of mast cells in IgE-driven allergic disease. The spectrum of proposed mast cell functions includes areas as diverse as the regulation of innate and adaptive immune responses, protective immunity against viral, microbial, and parasitic pathogens, autoimmunity, tolerance to graft rejection, promotion of or protection from cancer, wound healing, angiogenesis, cardiovascular diseases, diabetes, obesity, and others. The vast majority of in?vivo mast cell data have been based on mast cell-deficient Kit mutant mice. However, work in new mouse mutants with unperturbed Kit function, which have a surprisingly normal immune system, has failed to corroborate some key immunological aspects, formerly attributed to mast cells. Here, we consider the implications of these recent developments for the state of the field as well as for future work, aiming at deciphering the physiological functions of mast cells.     

Prospects for microtechnology and nanotechnology in bioengineering of replacement microvessels

CONTEXT: Due to its anticipated curative potential, therapeutic angiogenesis recently became a major preoccupation for the biomedical research community. Most of the related work reported to date employs either biochemical or genetic tools. OBJECTIVE: To identify opportunities for application of the current developments in microtechnology and nanotechnology to the field of therapeutic angiogenesis. DATA SOURCES: Survey of recent English-language literature on microvascular tissue engineering in the context of therapeutic angiogenesis. We include our results regarding the role played by microtopographical cues in the progression of angiogenesis, such as those produced during processing of the extracellular matrix by chronic inflammatory cells. CONCLUSION: While notable accomplishments have been identified in the field of tissue engineering of larger vessels, reports on purposeful assembly of microvascular structures with the ability to be transferred in vivo by implantation are still scarce. Under these circumstances, we suggest the development of a new class of implantable biomedical microdevices, that is, "angiogenesis assist devices" (or "angiochips"), and we indicate some of their conceivable applications.     

The role of B cells in solid organ transplantation

The role of antibodies in chronic injury to organ transplants has been suggested for many years, but recently emphasized by new data. We have observed that when immunosuppressive potency decreases either by intentional weaning of maintenance agents or due to homeostatic repopulation after immune cell depletion, the threshold of B cell activation may be lowered. In human transplant recipients the result may be donor-specific antibody, C4d+ injury, and chronic rejection. This scenario has precise parallels in a rhesus monkey renal allograft model in which T cells are depleted with CD3 immunotoxin, or in a CD52-T cell transgenic mouse model using alemtuzumab to deplete T cells. Such animal models may be useful for the testing of therapeutic strategies to prevent DSA. We agree with others who suggest that weaning of immunosuppression may place transplant recipients at risk of chronic antibody-mediated rejection, and that strategies to prevent this scenario are needed if we are to improve long-term graft and patient outcomes in transplantation. We believe that animal models will play a crucial role in defining the pathophysiology of antibody-mediated rejection and in developing effective therapies to prevent graft injury. Two such animal models are described herein.     

Dendritic cell homeostasis and trafficking in transplantation

Hematopoietic cell transplantation and solid organ transplantation are definitive therapies for several otherwise fatal conditions. Post-transplant immune reactions are the major cause of morbidity after transplantation and limit the extended use of these critical therapies. Post-transplant immune complications include graft rejection by the host and injury to the host mediated by the graft. Dendritic cells (DCs), a population of professional antigen-presenting cells, are thought to be crucial in triggering primary immune responses against both the graft and the host. Here, we review studies on DC homeostasis and trafficking after transplantation, and examine the role of the host and graft DC in post-transplant immune responses. We also discuss the therapeutic implications of these studies.     

Cellular reprogramming for the creation of patient-specific embryonic stem cells

The success of somatic cell nuclear transfer in mammals has opened the possibility to dedifferentiate cells from a patient into embryonic stem cells and in doing so, potentially generate all different cells and tissues of the human body. These cells could be later transplanted to the same patient without immune rejection. Whereas this principle has been demonstrated in laboratory animals, it is yet to be shown to work in primates. Herein we discuss the probability of somatic cell nuclear transfer becoming a real therapeutic alternative as well as the potential emerging dedifferentiation approaches that may eventually replace it.     

Killer artificial antigen-presenting cells deplete alloantigen-specific T cells in a murine model of alloskin transplantation

FasL-expressing killer antigen-presenting cells (KAPCs) have the ability to delete antigen-specific T cells and, therefore, could potentially be used for the treatment of allograft rejection and autoimmunity; however, their cellular nature markedly limits their clinical use. Novel bead-based killer artificial antigen-presenting cells (KaAPCs), which are generated by coupling major histocompatibility complex (MHC) class I antigens together with the apoptosis-inducing anti-Fas monoclonal antibody (mAb) onto magnetic beads, have recently attracted more attention. KaAPCs have a number of advantages over KAPCs and are able to deplete specific T cells in cocultures. However, it remains unknown whether bead-based KaAPCs can also induce apoptosis of alloreactive or autoreactive T cells and, consequently, generate hyporesponsiveness in vivo. In this study, H-2K(b)/peptide monomers and anti-Fas mAb have been covalently coupled to latex beads and administered intravenously into BALB/c mice (H-2K(d)) that had previously been grafted with skin squares from C57BL/6 mice (H-2K(b)). Alloskin graft survival was prolonged for 6 days. A 60% decrease of H-2K(b) antigen-alloreactive T cells was demonstrated by several measures 2 days after each injection of KaAPCs, but intact immune function, including antitumor activity, was maintained. These data provide the first in vivo evidence that bead-based KaAPCs can selectively deplete antigen-specific T cells without the loss of overall immune responsiveness and, therefore, highlight the therapeutic potential of this novel strategy for the treatment of allograft rejection and autoimmune disorders.     

诱导性多能干细胞体外定向诱导分化的研究进展

胚胎干细胞体外定向诱导分化的研究已成为发育生物学和细胞移植治疗学的研究热点,但伦理及免疫排斥问题一直阻碍胚胎干细胞（ES）的研究和临床应用。近两年,科学家成功地从多种成体细胞获得了诱导性多能干细胞（iPS细胞）,iPS细胞具有和胚胎干细胞相似的特性,由于其可来自自体成体细胞,因而可避开伦理及免疫排斥问题,是胚胎干细胞良好的替代材料,具有广阔的应用前景。iPS只有诱导分化为成熟的功能细胞才能用于疾病的治疗。主要介绍iPS细胞向成熟细胞分化的研究现状及目前存在的一些问题。