The role of cell migration and microchimerism in the induction of tolerance after solid organ transplantation

A new hypothesis has been proposed which states that microchimerism is the basis for the clinical tolerance seen in long-term survivors of solid organ transplants. Efforts to enhance microchimerism include simultaneous infusion of bone marrow of donor origin and transplantation of a solid organ. Studies are in progress to verify the phenomenon of microchimerism and its role in clinical tolerance.     

DNA polymorphism, allogenic bone marrow transplantation and peripheral cell chimerism

BACKGROUND: Bone marrow transplantation or transplantation of peripheral stem cells is an effective treatment of a number of diseases. Its increasing success and expanding use in associated with the development of molecular diagnostic methods which enable to follow up the graft from its engraftment in a recipient and then during the whole posttransplantation period at the level extremely small numbers of cells. METHODS AND RESULTS: In peripheral blood of patients, genotypes of the following loci were examined by polymerase chain reaction (PCR): APOB, COL2A1, D17S20, D1S80, HVR/1G, SRY and AMXY. Technique of restriction analysis was used for loci DXYS20 and DXYS75. 1. The first signs of donor bone marrow activity were observed in 50% of patients already at the beginning of the second week after transplantation, while in the second half of patients increasing number of donor cells in peripheral blood was noticed in the second and third week. 2. Engraftment with full and permanent substitution of own bone marrow without presence of recipients cells in peripheral blood--complete chimerism--was achieved only in a part of patients (cca 50%). 3. Peripheral blood of other patients did not contain only donor cells but also recipients cells--mixed chimerism. With regard to its onset, the authors have divided mixed chimerism into early and late, taking into account that some patients can develop both types. In patients under study, early chimerism was found more frequently, which apparently resulted from a shorter period of observation of lately transplanted patients. 4. In cases of oncohaematologic patients, which allowed to study specifically the presence of a pathologic clone, the follow-up of chimerism enabled to distinguish between relapse of the original disease and "biologic" recovery--resurrection of original disease-free haematopoiesis. 5. Regression of mixed chimerism was supposed to be the result of treatment focused at the original disease (CML), in some patients, however, it was a spontaneous process. CONCLUSIONS: Follow-up of cellular chimerism in transplanted patients by means of molecular genetic methods provides substantial information about patient's shape which can be utilized it is necessary to decide on treatment procedures. For this reason it is desirable that examination of chimerism by molecular methods should form integral part of care of these patients.     

The role of macrophages in antigen presentation and T cell tolerance

Bone marrow derived cells (dendritic cells, macrophages and B cells) are involved in antigen presentation and T cell tolerance. However, the precise functions of each cell type remain unclear. To determine the role of macrophages we produced transgenic mice expressing I-E molecules only on macrophages, by introducing the hybrid gene containing the colony stimulating factor-1 (CSF-1) receptor promoter region and the structural gene encoding E alpha d into C57BL/6 mice. In these mice I-E restricted antigen presentation and T cell priming were impaired. With respect to T cell tolerance, I-E reactive T cells were anergized but not clonally deleted. These results clearly demonstrate that macrophages by themselves are defective in efficient I-E restricted antigen presentation, so that T cells exposed to antigens expressed on macrophages are led to anergy.     

A critical role for transforming growth factor-beta in donor transfusion-induced allograft tolerance

To evaluate the relationship between carotenoid concentrations in serum and breast tissue, we measured serum carotenoid concentrations and endogenous carotenoid levels in breast adipose tissue of women with benign breast tumor (n = 46) or breast cancer (n = 44). Before extraction, serum was digested with lipase and cholesterol esterase, and breast adipose tissue was saponified. Serum and tissue carotenoids were extracted with ether/hexane and measured by using HPLC with a C30 column. Serum retinoic acid was extracted with chloroform/methanol and measured using HPLC with a C18 column. There were no significant differences in serum carotenoids [lutein, zeaxanthin, cryptoxanthin (both alpha- and beta-), alpha-carotene, all-trans beta-carotene, 13-cis beta-carotene and lycopene], retinoids (retinol, all-trans and 13-cis retinoic acids), and alpha- and gamma- tocopherol concentrations between benign breast tumor patients and breast cancer patients. A substantial amount of 9-cis beta-carotene was present in adipose tissue and was the only carotenoid that had a significantly lower level in benign breast tumor patients than in breast cancer patients. Correlations between carotenoid concentrations in serum and in breast adipose tissue were determined by combining the data of the two groups. Concentrations of the major serum carotenoids except cryptoxanthin showed significant correlations with breast adipose tissue carotenoid levels. When the concentrations of serum carotenoids were adjusted for serum triglycerides or LDL, correlations between serum carotenoid concentrations and breast adipose tissue carotenoid levels markedly increased, including that of cryptoxanthin (P <0. 001). The strong correlation between serum carotenoid concentrations and endogenous breast adipose tissue carotenoid levels indicate that dietary intake influences adipose tissue carotenoid levels as well as serum concentrations, and that adipose tissue is a dynamic reservoir of fat-soluble nutrients.     

The future of transplantation: with particular reference to chimerism and xenotransplantation

The innervation of the thymus was studied in SCID mice: There was a relatively more dense innervation pattern in SCID mice as compared to normal BALB/c mice (from which SCID mice are derived), including nerve fibres immunoreactive for protein gene product 9.5 (PGP 9.5), tyrosine hydroxylase (TH), neuropeptide tyrosine (NPY) and vasoactive intestinal peptide (VIP), although there was no reactivity to substance P (SP) or leucine enkephalin (ENK). Only a few acetylcholinesterase (AChE)-positive nerve fibres were observed in the SCID thymus. Ten weeks after the transfer of bone marrow from normal BALB/c mice into SCID mice no immunoreactivity to the above markers was found, nor was there any AChE reaction, although histologically the thymus appeared normal and dot-blot assays demonstrated the presence of immunoglobulin indicating a return to normal bone marrow function in SCID mice. Both innervation and morphology were restored 6 months after bone marrow transfer. In conclusion, the thymus of SCID mice lacking thymocytes has visible neurotransmitter levels in the nerves, but after thymocyte repopulation by bone marrow transplantation the transmitters are generally not demonstrable. This indicates that the innervation may be more important for the establishment of the microenvironment rather than the maintenance of thymocyte differentiation.     

The donor stage of orthotopic liver transplantation

First clinical experience of the harvesting (donor) stage of orthotopic transplantation of the liver in Russia is represented. During 5-year period in National Research Center of Surgery RAMS 16 orthotopic transplantations of the liver were carried out. Surgical modes of harvesting of the donor liver are detailed ("standard" and "fast"), the regimes and methods of preservation of the liver were determined. The influence of various parameters on function of the graft in postoperative period was established. Adequate selection of donors, correct and proper choice of donor-recipient pair, limited time of preservation allow to decrease the number of graft function failure and to eliminate primarily nonfunctioning grafts.     

Conventional cytogenetics and FISH evaluation of chimerism after sex-mismatched bone marrow transplantation (BMT) and donor leukocyte infusion (DLI)

BACKGROUND AND OBJECTIVES: Sensitive and quantitative cytogenetic methods to better assess the biological significance of post-BMT chimerism have been recently developed. In this study, we compared the results of chimerism analysis and evolution employing conventional cytogenetics and fluorescence in situ hybridization (FISH) in 16 patients after sex-mismatched BMT, and in 5 patients after donor lymphocyte infusion (DLI) to treat post-BMT relapse. DESIGN AND METHODS: FISH studies were performed using separate digoxigenin labeled centromeric DNA probes for the X (pDMX1) and Y (DYZ1/DYZ3) chromosomes. To this purpose, different types of samples were used: bone marrow (BM) and peripheral blood (PB) slides processed for conventional cytogenetics, and routine BM and PB smears. RESULTS: Results of chimerism studies performed on different types of samples showed no significant differences. No significant differences in the ability to identify the sex of each cell with both pDMX1 and DYZ1/DYZ3 probes were found and the results obtained from independent experiments showed a high linear correlation. Chimerism analysis by FISH showed initial mixed chimerism after BMT in 10 patients. Seven of these patients were also studied by conventional cytogenetics and 2 of these showed mixed chimerism. Seven of the former 10 patients evolved to complete donor chimera. 6 patients showed cytogenetic or hematologic bone marrow relapse, 3 of which were preceded by mixed chimaerism as revealed by FISH studies. FISH studies permitted an easy and accurate monitorization of the response to DLI in 5 relapsed patients, showing an increase in the proportion of donor cells in 4 patients as they reached a new complete remission. INTERPRETATION AND CONCLUSIONS: Both FISH and conventional cytogenetics are quantitative methods to assess chimerism. However, FISH is more sensitive, accurate and can even be applied on routine BM and PB smears. Furthermore, its combination with immunophenotyping approaches to quantify chimerism on cell subpopulations, will help to clarify post-BMT chimerism significance.     

The thymic way to transplantation tolerance

Within the past three decades, extensive research has been carried out with the aim to prevent graft rejection by minimizing the side effects related to the use of immunosuppressants. The major goal in transplantation research remains the development of strategies that would allow one to achieve a state of donor-specific unresponsiveness in order to promote a condition of true tolerance without the need of immunosuppressants. Recent evidence has been provided that this is a pursuing goal, at least in experimental animals. The thymus plays the major role in the development of self-tolerance, and initial work in the late 1960s indicated that the thymus also plays a critical role in the induction of acquired tolerance to exogenous antigens. Recently, the interest in acquired thymic tolerance has been renewed by the observation that, in the rat, the thymus is an immunologically privileged site in which isolated pancreatic islets can be engrafted and survive indefinitely. Moreover, intrathymic injection of the islets induced donor-specific unresponsiveness, which allowed survival of a second donor-strain islet cell allograft transplanted into an extrathymic site. These findings on cellular allografts have been extended to vascularized organ allografts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-term persistence of hemopoietic chimerism following sex-mismatched bone marrow transplantation

Wegener's granulomatosis is a distinct clinicopathologic entity characterized by granulomatous vasculitis of the upper and lower respiratory tract and glomerulonephritis. This disease can present as a clinical picture which resembles sepsis and adult respiratory distress syndrome (ARDS). Wegener's disease requires immunosuppression which can have detrimental consequences when used in sepsis. The following case report illustrates the diagnostic difficulties encountered by intensive care physicians treating severe pulmonary failure and multiple organ dysfunction in Wegener's granulomatosis appearing as ARDS with sepsis. CASE REPORT: A 19-year-old female patient had developed acute respiratory and renal failure after a prolonged period (many months) of antibiotic resistant otitis, sinusitis and mastoiditis. The patient had required intubation at another hospital and there was a history of tension pneumothorax and cardiopulmonary resuscitation during mechanical ventilation. Emergency extracorporeal membrane oxygenation (ECMO) for acute hypercapnic and hypoxic respiratory failure was instituted and the patient was transported to our institution while on ECMO. The patient was treated empirically for suspected pulmonary and systemic infection and received hydrocortisone (0.18 mg/kg/h) as part of a protocol-driven treatment of septic shock in addition to antibiotic and antimycotic regime. The use of ECMO was required for 10 and mechanical ventilation for another 50 days after admission. After successful extubation, central nervous system dysfunction became evident with a somnolent and generally unresponsive patient. When the hydrocortisone dose was gradually tapered, the clinical status of the patient further deteriorated, pulmonary gas exchange worsened and she developed renal failure with proteinura and hematuria. A renal biopsy was performed demonstrating vasculitis and focal segmental glomerulonephritis, a systemic granulomatous vasculitis was suspected; the serum was tested for anti-proteinase 3 antibodies (PR3-ANCA) and turned out to be positive (17.5 U/ml; normal range < 7 U/ml). The morphologic findings from renal biopsy, the positive test for antiproteinase 3 antibodies and the pulmonary-renal involvement with evidence of multisystem disease established the diagnosis of Wegener's granulomatosis. Immunosuppressive therapy with cyclophosphamide and prednisolone was instituted resulting in rapid improvement with recovery of pulmonary, renal and central nervous system function within two weeks. The use of ECMO in this patient served as a life-saving immediate measure usefull to "buy time" until a definite diagnosis could be established. ARDS represents an uniform pulmonary reaction to a large number of different noxious stimuli and disease entities. This case demonstrates that intensive care physicians caring for critically ill patients with ARDS should include even rare causes of pulmonary injury into their differential diagnosis.