The impact of transplantation on the clinical laboratory. Experience at the University of Nebraska with bone marrow and liver transplantation.

The number of both bone marrow and solid organ transplantation programs has increased significantly during the past several years. The implementation of these programs and their growth have had a significant impact on the clinical laboratories that are involved in the support of these programs. Between 1983 and 1985, two major organ transplantation programs were implemented at the University of Nebraska Medical Center, Omaha. The clinical laboratories at the University of Nebraska Medical Center have seen a 217% increase in procedure volume and a 165% increase in full-time equivalents during an 8-year period from 1983 to 1991 as a result of these programs. The laboratory procedures that are performed on patients who are undergoing transplantation currently generate approximately $9 million in charges per year. In the present article, I discuss the effect of bone marrow and liver transplantation programs on the entire laboratory and individual laboratory sections.     

Role of alloimmunity in clinical transplantation

HLA-specific humoral immunity, as a result of recipient allosensitization, induces hyperacute rejection of allogeneic kidney grafts. Cross-match tests are performed to avoid this complication. However, present techniques do not allow determination of HLA specificity of donor-reactive antibodies in the acute necro-donor situation. New methods are described and discussed in this communication as well as the alloantibody specificities which are of clinical importance. Alloantibodies not only mediate hyperacute rejection, but may also participate in the acute rejection of organ grafts. Clinical associations between early immunological complications, such as acute rejection, in heart, liver and kidney allografted patients and pre-transplantation humoral alloimmunity emphasize the need for proper determination of donor-specific humoral immunity prior to transplantation. Acute rejection episodes are associated with an increased risk of subsequent chronic rejection. Cytomegalovirus (CMV) infection is also an important risk factor for chronic organ graft rejection as well as for chronic graft-versus-host (GvH) disease in bone marrow transplanted patients. CMV infection triggers autoantibody formation against CD13 in transplanted patients which, in turn, has been shown to be associated with the development of chronic GvH disease. Recently, alloactivation of immune reactivity in vitro was found to induce reactivation of latent CMV infection. We present here a hypothesis to explain the series of clinically associated events: acute rejection--CMV infection--chronic rejection.     

Minor histocompatibility antigens: from transplantation problems to therapy of cancer

The idea of transferring healthy marrow for the therapeutic treatment of the various diseases of the blood and of the immune system made a significant contribution to controlling diseases and to advancing modern clinical sciences. The first series of bone marrow transplantations in the 1960s were confronted with severe complications. It became clear that matching for the human leukocyte antigen (HLA) system between donor and recipient significantly improved the clinical results. Nonetheless, an unacceptable percentage of severe complications remained that is mainly attributable to non-HLA histocompatibility systems, i.e., minor histocompatibility antigens. Observations in the 1970s that minor histocompatibility antigens cause serious problems in human bone marrow transplantation laid the basis for their use as curative antigens in stem cell transplantation to date. Thus, the allo-immune T cell activities caused by minor histocompatibililty antigen disparities between HLA-matched donor and recipient can now be applied for the benefit of the transplant patient.     

Post-transplant HHV-6 Diseases

Human herpesvirus 6 (HHV-6) infection is common in the general population. Primary infection occurs mainly in childhood, after which the virus establishes latency in the host and can be reactivated in immunocompromised individuals such as recipients of a solid organ or bone marrow transplant (BMT). HHV-6 infection occurs in 38--60% of BMT recipients and in 14--82% of solid organ recipients, usually 2--4 weeks post-transplantation. Two HHV-6 variants are recognized (HHV-6A and HHV-6B) and transplant patients are infected almost exclusively with variant B. Following BMT, the clinical diseases most commonly associated with HHV-6 infection are bone marrow suppression, interstitial pneumonitis, encephalitis and graft versus host disease. It has also been suggested that HHV-6 infection may be associated with fever and bone marrow suppression following liver transplantation and may contribute to rejection episodes after kidney transplantation. Infection with HHV-6 induces marked immunodepression, and an association both with cytomegalovirus disease and with fungal infection in transplant recipients has been reported. A direct causal relationship between HHV-6 infection and disease following transplantation, however, has yet to be proven conclusively.     

Expression of human minor histocompatibility antigen on cultured kidney cells

Incompatibility of human minor histocompatibility (hmH) antigens can induce rejection of grafts in organ transplantation and graft-versus-host reactions in bone marrow transplantation. In spite of their importance in clinical transplantation, hmH antigens are not well studied. Previous studies have demonstrated the expression of hmH antigens on Tand B cells, hematopoietic progenitor cells and keratinocytes. We have for the first time demonstrated the expression of hmH antigens on cultured kidney cells using HLA-B35-restricted, hmH antigen-specific cytotoxic T lymphocyte (CTL) clones, which were previously established from a patient who rejected two kidneys from HLA-identical sisters. The CTL clones could not kill cultured kidney cells. Since cultured kidney cells expressed very low levels of HLA class I antigens it was thought that their failure to be killed by the CTL clones was due to lack of expression of HLA-B35 antigens. After induction of class I antigens on cultured kidney cells by interferon-y (IFN-γ), the IFN-γ-treated cultured kidney cells were killed by the CTL clones. Furthermore, we isolated hmH antigens as peptides from cultured kidney cells after treatment with IFN-γ. These results indicate that cultured kidney cells express hmH antigens when HLA class I antigen is induced by IFN-γ and hmH antigens on cultured kidney cells are recognized by T cells as peptides presented by HLA-B35 molecules.     

Acquired immunologic tolerance: with particular reference to transplantation

The first unequivocally successful bone marrow cell transplantation in humans was recorded in 1968 by the University of Minnesota team of Robert A. Good (Gatti et al. Lancet 2: 1366–1369, 1968). This achievement was a direct extension of mouse models of acquired immunologic tolerance that were established 15 years earlier. In contrast, organ (i.e. kidney) transplantation was accomplished precociously in humans (in 1959) before demonstrating its feasibility in any experimental model and in the absence of a defensible immunologic rationale. Due to the striking differences between the outcomes with the two kinds of procedure, the mechanisms of organ engraftment were long thought to differ from the leukocyte chimerism-associated ones of bone marrow transplantation. This and other concepts of alloengraftment and acquired tolerance have changed over time. Current concepts and their clinical implications can be understood and discussed best from the perspective provided by the life and times of Bob Good. Presented at the First Robert A Good Society Symposium, St. Petersburg, FL, 2006. Keynote Address: Inaugural meeting of the Robert A. Good Immunology Society, June 9–11, 2006, Tampa, Florida. This work was supported by National Institutes of Health Grant RO1 DK64207, and unrestricted gifts from Mr. Robert Eberly and Frank and Athena Sarris.     

Hematopoietic stem cell transplantation with latently infected donors does not transmit virus to immunocompromised recipients in the murine model of cytomegalovirus infection

Hematopoietic stem cell transplantation (HSCT) bears a risk of reactivating latent cytomegalovirus (CMV) in either the transplanted hematopoietic donor cells or in parenchymal and stromal tissue cells of the immunocompromised recipient, or in both. While reactivated human CMV in recipients of organ transplantations is frequently the virus variant of the donor, this is not usually the case in HSCT recipients. Here we have used experimental sex-mismatched HSCT in the BALB/c mouse model to test if latent murine CMV from CMV-immune donors is transmitted with bone marrow cells to naive immunocompromised recipients.     

The role of the blood bank in transplantation.

Blood transfusion supportive care is essential to successful organ, tissue, and bone marrow transplantation, thus making the blood bank an integral part of these programs. The impact of clinical transplantation on the blood bank can be considered in the following categories: (1) use of blood and blood components for transfusion support; (2) special blood component needs; (3) red blood cell serologic needs; (4) collection, processing, and preservation of bone marrow and peripheral blood stem cells; (5) hemotherapy for bone marrow donors, (6) recruitment of bone marrow donors; and (7) the special procedures that must be available for managing patients undergoing transplantation therapy.     

Flow cytometric reticulocyte quantification using thiazole orange provides clinically useful reticulocyte maturity index

Flow cytometric reticulocyte quantification with thiazole orange has been reported to be of potential utility in a clinical hematology laboratory. We have instituted this technique into routine clinical testing for 18 months and we describe this experience. Flow cytometric analysis provided not only reproducible, cost-effective reticulocyte quantification, but a quantitative reticulocyte maturity index proportional to the amount of RNA in the reticulocytes. The reticulocyte maturity index measurement represents an independent parameter of erythropoiesis, which provided clinically valuable information regarding bone marrow engraftment in patients following autologous bone marrow transplantation. The findings of this study demonstrate the clinical utility of thiazole orange reticulocyte analysis and indicate the diagnostic importance of the reticulocyte maturity index measurement in the evaluation of erythropoietic activity.     

Evaluation of autonomic dysfunction by novel methods

The autonomic nervous system innervates every organ in the body. Since autonomic disturbances affect patient survival, an understanding and recognition of these disturbances are important. We adopted several new methods to evaluate autonomic function accurately. 123I-metaiodobenzylguanidine scintigraphy can assess the cardiac autonomic function even in the presence of cardiac arrhythmia. Laser-Doppler flowmetry, ultrasonographic study in the vessels and near-infrared spectrophotoscopy techniques serve as useful markers for screening the dysfunction of vasomotor neurons and blood circulation. Electrogastrography and the circadian rhythms of protein C secretion could be markers of the visceromotor nerves in the abdomen. Electrogastrography is a particularly useful tool for reflecting on functional changes in gastrointestinal motility. The evaluation of anemia could be a marker of autonomic dysfunction in the kidney and bone marrow in patients with familial amyloidotic polyneuropathy, pandysautonomia, and multiple system atrophy. Normocytic and normochromic anemia correlated with the severity of autonomic dysfunction were shown in these patients. We also evaluated the dysfunction of the neuroendocrine system and sudomotor neuron using our new autonomic function tests. The glucose-tolerance test could become one of the most useful clinical tools for detecting autonomic dysfunction in the endocrine system. Microhydrography and thermography could be useful tools for diagnosing the lesion site of dyshidrosis. Moreover, it is clinically important to check the systemic circulation and autonomic function in patients treated with sildenafil citrate and organ transplantation to save their lives. Our new autonomic function tests, such as laser-Doppler flowmetry and 123I-metaiodobenzylguanidine scintigraphy, are crucial tools in supplying the best symptomatic treatment for such patients.     

自体骨髓单个核细胞移植治疗糖尿病下肢周围神经病变

背景：有研究表明移植骨髓单个核细胞治疗糖尿病下肢神经病变动物模型,通过在组织内能促进血管再生和增加血管生成因子及神经营养因子能改善临床症状。                             目的：观察自体骨髓单个核细胞移植治疗糖尿病下肢周围神经病变的临床效果。 方法：30例糖尿病下肢闭塞症患者60条下肢,按治疗方式的不同分为2组：自体骨髓单个核细胞移植的治疗组和对侧下肢非自体骨髓单个核细胞移植的对照组,各30条下肢。 结果与结论：移植4周后,治疗组总有效率高于对照组(P < 0.05)。两组治疗后神经病变自主症状问卷神经病变主觉症状问卷评分均较治疗前明显降低,治疗组评分降低更明显(P < 0.01),治疗组胫神经和腓总神经感觉和运动神经传导速度均较对照组快(P < 0.01),患者未出现并发症和不良反应。说明自体骨髓单个核细胞移植治疗糖尿病下肢周围神经病变的临床效果较好。     

Infection in the bone marrow transplant recipient and role of the microbiology laboratory in clinical transplantation.

Over the past quarter century, tremendous technological advances have been made in bone marrow and solid organ transplantation. Despite these advances, an enduring problem for the transplant recipient is infection. As immunosuppressive regimens have become more systematic, it is apparent that different pathogens affect the transplant recipient at different time points in the posttransplantation course, since they are influenced by multiple intrinsic and extrinsic factors. An understanding of this evolving risk for infection is essential to the management of the patient following transplantation and is a key to the early diagnosis and treatment of infection. Likewise, diagnosis of infection is dependent upon the quality of laboratory support, and services provided by the clinical microbiology laboratory play an important role in all phases of clinical transplantation. These include the prescreening of donors and recipients for evidence of active or latent infection, the timely and accurate microbiologic evaluation of the transplant patient with suspected infection, and the surveillance of asymptomatic allograft recipients for infection. Expert services in bacteriology, mycology, parasitology, virology, and serology are needed and communication between the laboratory and the transplantation team is paramount for providing clinically relevant, cost-effective diagnostic testing.     

Reactive versus neoplastic bone marrow: problems and pitfalls

Examination of the bone marrow poses several unique challenges to the pathologist: it is a semisolid organ without helpful gross correlation, it exists in a dynamic state with the peripheral blood and other organs of the lymphohemopoietic system, and the diagnosis of diseases affecting bone marrow often depends heavily on ancillary special studies. The bone marrow examination ideally encompasses review of the bone marrow biopsy histology (with or without additional nondecalcified clot preparation material), bone marrow aspirate smear cytology, and the peripheral blood smear; optimal procurement and processing of these samples is critical in ensuring that a maximal level of diagnostic information can be extracted. The pathologist must be aware of the clinical context of the bone marrow and the results of ancillary tests, whether these are ordered by the pathologist or the clinician. A combination of excellent diagnostic samples, appropriate ancillary tests, and knowledge of the clinical context provides the best background to distinguish between the common reactive and neoplastic processes that involve the bone marrow and to avoid diagnostic pitfalls in making these distinctions.     

Path to clinical transplantation tolerance and prevention of graft-versus-host disease

Although organ and bone marrow transplantations are life-saving procedures for patients with terminal diseases, the requirement for the lifelong use of immunosuppressive drugs to prevent organ graft rejection and the development of graft versus host disease (GVHD) remain important problems. Experimental approaches to solve these problems, first in preclinical models and then in clinical studies, developed at Stanford during the past 40 years are summarized in this article. The approaches use fractionated radiation of the lymphoid tissues, a procedure initially developed to treat Hodgkin’s disease, to alter the immune system such that tolerance to organ transplants can be achieved and GVHD can be prevented after the establishment of chimerism. In both instances, the desired goal was achieved when the balance of immune cells was changed to favor regulatory innate and adaptive immune cells that suppress the conventional immune cells that ordinarily promote inflammation and tissue injury.     

骨髓间充质干细胞移植治疗糖尿病足过程中血管内皮生长因子的表达

背景：目前已有研究证明骨髓间充质干细胞移植治疗糖尿病足能够获得良好的效果。 目的：评价骨髓间充质干细胞移植治疗糖尿病足溃疡的效果以及血管内皮生长因子的表达情况。 方法：应用文献检索的方法获取骨髓间充质干细胞移植治疗糖尿病足及血管内皮生长因子表达的相关研究文献,对符合研究标准的文献进行深入的数据分析,文章选取骨髓间充质干细胞移植治疗糖尿病足的实验研究和临床应用进行分析,实验研究中,建立大鼠糖尿病足溃疡模型,给予骨髓间充质干细胞移植治疗,观察创面溃疡的愈合情况,并分析血管内皮生长因子的表达情况。临床应用研究中,对应用骨髓间充质干细胞移植治疗糖尿病足的患者进行观察随访,观察创面溃疡的愈合情况以及不良反应发生情况。 结果与结论：实验研究显示骨髓间充质干细胞移植治疗糖尿病足创面溃疡的愈合快于常规治疗,但是与正常足溃疡愈合相比仍较慢,且移植治疗后血管内皮生长因子的表达升高,但是低于正常足溃疡对照的水平。临床应用研究显示糖尿病足溃疡患者经骨髓间充质干细胞移植治疗后,创面溃疡基本均可完全愈合,且无心、脑血管疾病、肝肾功能损伤以及出凝血时间改变等不良反应发生。     

Cytogenetics in patients with chronic myelogenous leukemia treated with bone marrow transplantation

Cytogenetic data are reported from 16 patients with Philadelphia chromosome (Ph) positive chronic myelogenous leukemia (CML) treated with bone marrow transplantation (BMT). The usefulness of cytogenetic investigations for the assessment of marrow engraftment is stressed. The significance of persistence or reappearance of Ph after BMT, possibly due to a defective leukemic clone eradication by the conditioning regimen, is also discussed. Generally, Ph-positive cells are damaged and disappear within the first year of BMT. Sometimes, however, the cells may repair the damage and proliferate again, resulting in disease relapse. Rarely, clinical and hematologic relapse does not follow Ph-positive clone expansion although leukemic cells represent more than 50% of marrow metaphases examined. Finally, the effect of interferon on Ph-positive clones after BMT and random chromosome changes, that appear transiently after BMT and are of uncertain significance, are discussed.     

Bone marrow tissue engineering

The creation of mixed hematopoietic chimerism has become an important clinical strategy for tolerance induction for cellular and organ transplantation, and for the treatment of numerous hematopoietic diseases. Clinical success has been limited however, by host immune response and by competition from host hematopoiesis. Recent data suggests that limited donor stem cell engraftment after minimally myeloablative hematopoietic stem cell (HSC) transplantation may in part be due to MHC associated microenvironmental mismatch resulting in a competitive disadvantage for donor HSC. A strategy to overcome this barrier to stable mixed hematopoietic chimerism would involve concurrent transplantation of a donor bone marrow microenvironment. To test this possibility, we set out to develop a method to tissue engineer a bone marrow microenvironment. One to two murine femurs were mechanically crushed to a fine suspension and were combined in vitro with various delivery vehicles. These constructs were transplanted into syngeneic animals in locations that are known to support transplantation of other tissues. Although bone formation was observed with several conditions, bone marrow formation was noted only within the small bowel mesentery when type I collagen was used as the delivery vehicle. No bone marrow formed when the vehicle was changed to polyglycolic acid or type IV collagen. We have demonstrated that the small bowel mesentery can support bone marrow formation under specific in vivo conditions. Future work will focus on strategies for transplantation of an engineered donor bone marrow environment to facilitate creation of allogeneic mixed hematopoietic chimerism.     

Mesenchymal stromal cells for tolerance induction in organ transplantation

The primary challenge in organ transplantation continues to be the need to suppress the host immune system long-term to ensure prolonged allograft survival. Long-term non-specific immunosuppression can, however, result in life-threatening complications. Thus, efforts have been pursued to explore novel strategies that would allow minimization of maintenance immunosuppression, eventually leading to transplant tolerance. In this scenario, bone marrow-derived mesenchymal stromal cells (MSC), given their unique immunomodulatory properties to skew the balance between regulatory and memory T cells, have emerged as potential candidates for cell-based therapy to promote immune tolerance. Here, we review our initial clinical experience with bone marrow-derived MSC in living-donor kidney transplant recipients and provide an overview of the available results of other clinical programs with MSC in kidney and liver transplantation, highlighting hurdles and success of this innovative cell-based therapy.     

Bone marrow lacks a transplantable progenitor for smooth muscle type alpha-actin-expressing cells

While some studies have suggested that hematopoietic stem cells might give rise to other tissue types, others indicate that transdifferentiation would have to be an extremely rare event. We have now exploited smooth muscle type alpha-actin (alphaSMA) promoter-driven green fluorescent protein (GFP) transgenic mice (alphaSMA-GFP mice) for bone marrow transplantation to evaluate their potential to generate donor-type tissues in irradiation chimeras. There was a highly restricted pattern of GFP expression in the transgenic mice, marking bone marrow stromal cells and mesangial cells in the kidney. However, these characteristics were not transferable to wild-type animals given transgenic marrow cells even though hematopoietic cells were largely replaced. Our findings support earlier studies suggesting that the bone marrow microenvironment is difficult to transplant and indicate that hematopoietic stem cells are unlikely to give rise to alphaSMA-expressing progeny.