Transplantation of hematopoietic stem cells for induction of unresponsiveness to organ allografts

Although it has been recognized since the early days of Owen and Medawar that engraftment of donor stem cells, induced in utero spontaneously or intentionally neonatally, results in life-long unresponsiveness to donor alloantigens. However, successful induction of transplantation tolerance in adult life still represents an unsolved problem. Engraftment of donor stem cells using conventional modalities involves intensive myeloablative or lymphoablative immunosuppression, which is associated with toxicity and mortality and such methods are not suitable for organ allograft recipients. In this chapter, we present an innovative approach for induction of donor-specific unresponsiveness to bone marrow and organ allografts without myeloablative conditioning. Our methods is based on cyclophosphamide-induced, alloantigen-primed lymphocyte depletion. Cyclophosphamide is administered 1 day following infusion of donor hematopoietic cells, thus eliminating predominantly host T lymphocytes reacting against donor cell challenge, and resulting in relative unresponsiveness to donor alloantigens. Subsequently, life-long tolerance to fully mismatched donor skin allografts can be accomplished by a second infusion of stem cells from the same donor, with donor T cells displacing residual alloreactive host cells that may have escaped deletion. Taken together, we believe that induction of true permanent and specific tolerance to organ allografts using donor hematopoietic cells could become a clinical reality in the foreseeable future.     

Analysis of CD154 and CD40 expression in native coronary atherosclerosis and transplant associated coronary artery disease

T cells have roles in the pathogenesis of native coronary atherosclerosis (CA) and transplant-associated coronary artery disease (TCAD). The mechanisms by which T cells interact with other cells in these lesions are not fully known. CD154 is an activation-induced CD4⁺ T cell surface molecule that interacts with CD40⁺ target cells, including macrophages and endothelial cells, and induces the production of pro-inflammatory molecules, including CD54 (ICAM-1) and CD106 (VCAM-1). To investigate whether CD154-CD40 interactions might be involved in the pathogenesis of CA or TCAD we performed immunohistochemical studies of CD154 and CD40 expression on frozen sections of coronary arteries obtained from cardiac allograft recipients with CA (n=10) or TCAD (n=9). Utilizing four different anti-CD154 mAb we found that CD154 expression was restricted to infiltrating lymphocytes in CA and TCAD. CD40 expression was markedly up-regulated on intimal endothelial cells, foam cells, macrophages and smooth muscle cells in both diseases. Dual immunolabeling demonstrated many CD40⁺ cells co-expressed CD54 and CD106. The extent of CD40, CD54 and CD106 expression showed statistical significant correlation with the severity of disease and the amount of intimal lymphocytes. Together these studies demonstrate the presence of activated CD154⁺ and CD40⁺ cells in both CA and TCAD lesions and suggest that CD154-mediated interactions with CD40⁺ macrophages, foam cells, smooth muscle cells and/or endothelial cells may contribute to the pathogenesis of these diseases. Received: 17 December 1999 / Accepted: 20 January 2000     

Intra-bone marrow-bone marrow transplantation facilitates hemopoietic recovery including dendritic cells

In this report, we provide evidence using a serial bone marrow transplantation (BMT) protocol that intra-bone marrow (IBM)-BMT (IBM-BMT) can efficiently reconstitute the hemopoietic system with cells of donor origin, in contrast to conventional intravenous (IV)-BMT (IV-BMT). Furthermore, the hematolymphoid system of secondary recipients that had received bone marrow cells (BMCs) from primary recipients treated with IBM-BMT recovered earlier than that of the secondary recipients of BMCs from primary recipients treated with IV-BMT. This was the case when the Lin-/c-kit+ progenitor cells of the secondary and tertiary recipients were examined. These findings indicate that IBM-BMT can facilitate the development of not only cells of various lineages but also the effective generation and, more importantly, the maintenance of the progenitor cells. Furthermore, we show that IBM-BMT can reconstitute the dendritic cell (DC) subsets (myeloid and lymphoid DCs), which are critical for the initiation of both adaptive and innate immune responses. The frequency of both myeloid and lymphoid DC subsets was approximately equal to that of normal age-matched untreated controls and, after second and third BMT, this ratio was close to that observed in the normal controls. However, the lymphoid DCs were clearly reduced in the secondary and tertiary recipients of BMCs from mice that had received IV-BMT. Therefore, the development of DC subsets is also normally maintained in the IBM-BMT group.     

Desensitization protocols improving access and outcome in transplantation

Sensitization to antigens of the HLA and ABO system has been the biggest barrier to access in renal transplantation and, increasingly, in transplantation of other organs. Additionally, antibody to donor antigens has been shown to result in injury to the graft ranging from catastrophic, irreversible hyperacute rejection to the slower, more insidious, chronic form of rejection. The problem of access has been recognized globally and has been the incentive for measures to overcome the disadvantage experienced by the sensitized patient. However, early attempts to reduce sensitization achieved only transient success. Newer immunosuppressive agents that affect B-cell function or viability have permitted the development of treatment protocols to eliminate and, potentially, downregulate donor-specific antibodies. The use of these protocols has achieved successful transplants that were HLA and/or ABO incompatible prior to treatment and, as such, has provided some patients with their only opportunity for transplantation.     

自体腹膜管道替代血管在冠状动脉搭桥手术中的实验应用

目的 采用实验犬的腹膜片制作腹膜管道替代冠状动脉搭桥手术中的传统移植血管,希望为冠状动脉搭桥提供一种理想的血管替代物.方法 随机选用杂种成年犬10头,雌雄不限,麻醉后气管插管行机控呼吸.所有犬经旁正中切口达腹直肌鞘后层取腹膜片,制成腹膜管道在非体外循环下行主动脉与右冠状动脉主干搭桥.采用电磁血流量计测量搭桥前后腹膜管道的血流通畅度.术毕记录实验犬的早期存活比例,饲养至实验结束处死动物,光镜下观察HE染色腹膜管道的病理变化.结果 实验犬的早期死亡比例为10%(1/10),腹膜管道的血流通畅,冠状动脉搭桥手术前后实验犬右冠状动脉的血流量分别为(126±13)、(117±14)ml/min,差异无统计学意义(P>0.05).腹膜管道的组织学形态改变良好,光镜下,未见腹膜管道瘤样扩张,无明显纤维疤痕形成,腹膜管道弹力纤维结构完好,无断裂,腹膜管道与血管吻合口附近有血管内皮细胞衍生.结论 在非体外循环下采用腹膜管道替代传统的移植血管行冠状动脉搭桥创伤小,实验犬存活比例高,冠状动脉血供效果令人满意,有希望成为理想的血管移植替代物.     

慢性心功能不全时经主动脉移植干细胞的可行性研究

心肌梗死或/和慢性心功能不全时,心脏干细胞移植是一项颇有应用前景的新的治疗技术。但是,对于非缺血性心脏病所致的慢性心功能不全,目前所采用的移植路径有一定的局限性。本试验拟研究慢性心功能不全时,封堵主动脉窦上部经主动脉根部移植干细胞是否可行。阿霉素所致的慢性心功能不全兔模型,采用双球囊封堵主动脉窦上部经主动脉根部自体移植骨髓单核细胞,4周后评价心功能。在封堵主动脉窦上部经主动脉根部移植干细胞时,无1例因移植手术死亡。双球囊充盈时造影显示,主动脉窦显影良好,大部分情况下,左、右冠状动脉可显影。骨髓单核细胞移植4周后,左室射血分数改善明显(P=0 .0 34)。所以,慢性心功能不全时,封堵主动脉窦上部经主动脉根部移植干细胞安全可行。     

Regeneration in grafts of normal and denervated rat muscles

Summary The soleus or extensor digitorum longus muscles of young rats were freely grafted into the bed of the corresponding contralateral muscle. The grafts were of normal muscle or muscles which had been denervated for 14 days. Grafts of normal muscle were characterized by little or no contractile activity for the first 2–4 days after transplantation. In contrast, denervated grafts contracted weakly, but consistently, throughout this early period. The patterns of contraction were complex. In early transplants, the contractions were due entirely to surviving muscle fibers in the graft, and the contractile characteristics were those of denervated muscle fibers. After the first week, contractions of newly regenerating muscle fibers within the grafts were superimposed upon and later took over those from the fibers that survived the original transplantation. The contraction time approached those of the normal soleus or extensor muscles during the second month after grafting, and the grafts contracted like fast or slow muscles.     

Non-cholinergic afferents determine the distribution of the cholinergic septohippocampal projection: a study of the AChE staining pattern in the rat fascia dentata and hippocampus after lesions,X-irradiation,and intracerebral grafting

Summary The acetylcholinesterase (AChE) activity of the rat hippocampus and fascia dentata depends on an intact septohippocampal connection, and histochemical staining for AChE is commonly used to monitor the distribution of the cholinergic septohippocampal projection. It is also characteristic that the laminae of low or moderate to dense AChE staining in the hippocampus and fascia dentata coincide with the terminal fields of the major non-cholinergic, afferent pathways. While studying lesion-induced collateral sprouting and aberrant axonal growth of these pathways we observed that the AChE staining pattern changed in accordance with the reorganized distribution of the non-cholinergic pathways, and this occurred even without direct interfering with the septohippocampal projection itself. Widening and narrowing of the medial perforant path and mossy fiber terminal zones thus resulted in corresponding changes in the bands of AChE staining normally associated with these zones. Expansion of the commissural-associational hippocampodentate projections and the lateral perforant path was in a similar way paralleled by a widening of the AChE-poor zones which normally overlap with the termination of these projections. Observations of the same kind were made in intracerebral transplants of fascia dentata innervated by various host afferents, and in rats subjected to neonatal X-irradiation, where the mossy fiber projection is reduced and aberrant perforant pathways project into CA3 due to a reduced formation of granule cells. The observed sets of changes with linkage between the different noncholinergic projections and the activity of AChE in their respective terminal fields were accordingly reproduced under several different experimental conditions. It could not be explained alone by interaction between the septal afferents and their target cells. We therefore conclude that the density and laminar distribution of the AChE activities within the hippocampus and fascia dentata are determined at least in part by the major afferent, noncholinergic nerve connections. We suggest that the effect occurs through direct axonal interaction or through changes in the receptiveness of the common dentate and hippocampal target cells.     

An inhibitor of cytotoxic functions produced by CD8+CD57+ T lymphocytes from patients suffering from AIDS and immunosuppressed bone marrow recipients

An inhibitor of the cytotoxic functions (ICF) mediated by human immunodeficiency virus (HIV)- or HLA-specific cytotoxic T lymphocytes, natural killer and lymphokine-activated killer (LAK) cells is secreted by CD8⁺CD57^? T lymphocytes, a subset expanded during infection with HIV and after bone marrow transplantation. We previously showed an apparent molecular mass of 20–30 kDa for this soluble glycosylated concanavalin A-binding inhibitor which is distinct from known cytokines. Here, we report a characterization of the mechanism of action of this CD8⁺CD57⁺ ICF. We show that the ICF-induced inhibition of LAK cell cytolytic activity is transient, with a spontaneous recovery of cytolytic potential after 18 h. When testing interactions of ICF with a large set of cytokines we found that the ICF-mediated inhibition of cytotoxic functions is antagonized by two cytokines: recombinant interleukin (rIL)-4 and recombinant interferon (rIFN)-γ. Finally, we show that ICF acts at the level of cytolytic effector cells, where it induces a significant increase of cyclic AMP (cAMP) level. In contrast, no modification of either cell surface antigen expression or of target/effector cell conjugate formation could be evidenced. Addition of rIL-4 and rIFN-γ reverses such an increase of cAMP levels and in parallel restores the cytolytic activity. Altogether, these data demonstrate that the glycoprotein ICF produced by CD8⁺CD57⁺ cells (1) inhibits cell-mediated cytotoxicity by sensitizing cytolytic effector cells to the cAMP pathway, and (2) is part of a cytokine network controlling cell-mediated cytotoxic functions.     

Transplantation of Pacinian corpuscles of the rat into the brain

Summary In adult inbred rats of the AVN strain, branches of the crural interosseous nerve were dissected out from donors and transplanted into the brain of recipients, together with a cluster of Pacinian corpuscles, (either into a suction cavity or the cerebral cortex) into a slit 1–2 mm deep. The grafts were fixed and processed for electron microscopy 10 days to 6 months after the operation, and their ultrastructure was examined. Sporadic axons of small diameter grew into the nerve branches of some of the grafts from 11 days onward, and became myelinated during the 2nd month after the operation, but none of the transplanted Pacinian corpuscles became reinnervated. The corpuscles, however, survived denervation and grafting. Most of them retained a well-preserved inner core and an intact capsule, consisting of a normal complement of 29.2±1.0 (mean ±SE) capsular layers (n=8), as did the corpuscles previously examined after denervation in situ. Some of the corpuscles underwent degenerative changes, presumably due to a delayed or restricted revascularization. In this group of corpuscles, the inner core underwent disintegration and was gradually replaced by collagen fibrils, whereas the capsule remained preserved but the number of its layers eventually reduced by 40%. It is assumed that the lack of reinnervation of the grafted Pacinian corpuscles was due to the paucity of regenerating axons, and their failure to form correct projections along those Schwann cell columns connected with the corpuscles.