T cell regeneration after allogenic bone marrow transplantation

Various T cell subsets were characterized by double immunofluorescent staining using monoclonal antibodies (MoAb) in blood, bone marrow (BM) and tissues of 29 patients after allogeneic BM transplantation (BMT). In an attempt to prevent graft versus host disease (GvHD), 15 patients received cyclosporin A (Cy A). In the remaining 14 patients the BM was pre-incubated with a MoAb, OKT3. The regeneration of T4⁺ subset was delayed and the level of T8⁺ cells was abnormally high even 1 year after engraftment. This did not have any predictive value for the appearance of complications such as GvHD or severe viral infections. The number of T8⁺ cells was lower in the group of patients who received Cy A than in the OKT3 group (0·7±0·2 vs 1·5±0·3×10⁹/1 at day 90). In contrast to normal individuals, the T4/T8 ratio in both blood and regenerating BM of BMT patients was <1. A sizeable subset of circulating T cells expressed the phenotype T8⁺,T10⁺,HNK-1⁺,DR⁺. Circulating cells of this phenotype were transiently very high (up to 50%) in patients with active GvHD or suffering from severe viral infection. This subpopulation of lymphocytes was not found in the epidermal infiltrate that accompanied GvHD where the predominant phenotype was T8⁺,T1^-,T10^-,HNK-1^-,DR^-. We conclude therefore that after BMT the number and phenotype of circulating T cells reflects the T cell distribution seen in the regenerating BM.     

T lymphocyte regeneration after transplantation of T cell depleted allogeneic bone marrow

The regeneration of T cell subsets was studied with double immunofluorescence marker methods in 37 patients who received HLA matched T lymphocyte depleted bone marrow transplants (BMT) as part of the treatment for their haematological disease. A cocktail of anti-pan-T (CD6: MBG6) and anti-suppressor/cytotoxic-T cell (CD8: RFT8) monoclonal antibodies was used with rabbit serum as a source of cytolytic complement to achieve selective T cell lysis. The T8+ cells reached low normal values around 60 days post-transplant and remained within the normal range throughout the study (greater than 150 days). This observation is in contrast to our previously published results in patients who, after receiving BMT without efficient T cell depletion, had increased numbers of circulation T8+ cells from 60 days post-transplant. In the present study Leu-7+, RFT8- cells reached normal values rapidly but the reconstitution of T4+ lymphocytes was slow: low normal levels were reached only around day 150 following BMT. The degree of graft-versus-host disease (GVHD) seemed to be related to the number of residual T cells infused: two of the three patients who received the highest numbers of T cells developed Grade II III; otherwise GVHD was minimal. Among the clinical parameters studied cytomegalovirus (CMV) immune status moderately influenced reconstitution: at 55-90 days post-transplant T8+ cells were present at the upper normal levels in seven out of 15 patients receiving BMT from CMV seronegative donors, but in none of the 16 individuals receiving BMT from seropositive donors. CMV related complications were relatively uncommon. Thus the most significant factor in preventing 'T8+ cell overshoot' and T cell imbalance during regeneration appears to be the depletion of T (including T8+) lymphocytes from marrow. The differences of T8+ cell reconstitution in this and previous studies may reflect a different regeneration pattern from T cell precursors as opposed to inoculated mature T cells.     

Muscle regeneration and transplantation enhanced by bone marrow cells

This work was undertaken in an attempt to improve the usually poor results of muscle regeneration and grafting in cases of muscle injury. Since bone marrow cells markedly increase myogenic growth in vitro (in Millipore diffusion chambers), we added autogenous and isologous bone marrow cells to minced muscle grafts or to sites of excised muscle in rats. In each case the opposite side was left without added marrow cells, to serve as a control. The results showed that addition of bone marrow produces more extensive muscle regeneration with little fibrosis. The myogenesis-stimulating and antibacterial properties of bone marrow cells warrant further study, aiming towards clinical application of the method.     

Histopathology of bone marrow reconstitution after allogeneic bone marrow transplantation

In order to study haematopoietic reconstitution in allogeneic bone marrow transplantation we investigated bone marrow histology in 61 biopsies of 37 patients, treated with HLA-compatible bone marrow grafts for leukaemia or severe aplastic anaemia. The biopsies were taken from the day of transplantation until 100 d after transplantation. Stromal changes, in particular oedema, fibrosis and granulomas, were found during the whole period of observation. These changes were more prominent in biopsies from leukaemia patients than from patients with aplastic anaemia. The cellularity in the biopsies increased until 28 d after bone marrow transplantation and was stable thereafter. Initially, only clusters of cells belonging to a single cell lineage were seen, suggesting that the first outgrowth of haematopoietic cells is by proliferation of committed precursor cells. Long-lasting abnormalities in localization of haematopoietic cells in the bone marrow space and of the myeloid: erythroid ratio were seen; dyserythropoiesis was common.     

Lung diseases after bone marrow transplantation

Summary The case histories of 72 subsequently treated patients — 44 with acute leukemia, 10 with chronic myeloid leukemia, 16 with severe aplastic anemia and 2 with neuroblastoma — were analyzed after bone marrow transplantation (BMT) with respect to pulmonary diseases. Thirty-eight patients suffered from a total of 51 pulmonary complications, which led to death in 20. Of 13 patients, 3 died of bacterial pneumonia, all of them during granulocytopenia; 2 of 6 patients died of fungal pneumonia and 2 out of 3 of a mixed bacterialmycotic infection. Adult respiratory distress syndrome (ARDS) led to death in 2 patients. A granulocyte count under 500/µl correlated significantly (P<0.002) with the fatal outcome of bacterial, fungal and ARDS pneumonia as well as with bronchitis. Viral pneumonia led to death in 8 of 9 patients; in each there was a significant correlation (P<0.05) with graft-versus-host disease (GvHD). Patients with repeated episodes of pulmonary illness had significantly more chronic GvHD (P<0.05); several of these patients displayed a reduction in helper T cells and an increase in suppressor T cells in the peripheral blood. The natural killer (NK) cells were reduced and the percentage of activated NK cell level lay between 6% and 69%. B-cells were absent or deficient. These findings explain in part the absence of specific antibody reactivity. Five of these patients also contracted GvHD-associated obstructive bronchiolitis, which did not respond to therapy. Pulmonary infiltrates of unknown origin (including idiopathic interstitial pneumonia) occurred in 8 of the patients (11.1%), with a fatal outcome in 3 patients. Significant changes (P<0.05) in lung function after BMT appeared in the form of reduced vital capacity (VC) increased residual volume (RV) and an increase in RV expressed as the percentage of total lung capacity. Pulmonary diseases were the most common complication and cause of death in our patients after BMT.Abbreviations AEL acute erythroid leukemia - AHTCG anti-human T-cell globulin - ALL acute lymphoblastic leukemia - AML acute myeloid leukemia - ARDS adult respiratory distress syndrome - AUL acute undifferentiated leukemia - BMT bone marrow transplantation - CR complete remission - CML chronic myeloid leukemia - CMV cytomegalovirus - Ext. extensive - GvHD graft versus host disease - Lim. limited - PR partial remission - Rel. relapse - SAA severe aplastic anemia - VZV varicella zoster virus.Lung function tests - DLCO single-breath CO-diffusion capacity - FEV₁ forced expiratory volume in 1 s - KCO Krogh's constant - MBC maximal breathing capacity - RAW airway resistance - RV residual volume - SRAW specific airway resistance - TGV thoracic gas volume - TLC total lung capacity - VC vital capacity Supported in part by SFB 120, A2, B1, C1, F2     

Muscle regeneration and transplantation enhanced by bone marrow cells.

This work was undertaken in an attempt to improve the usually poor results of muscle regeneration and grafting in cases of muscle injury. Since bone marrow cells markedly increase myogenic growth in vitro (in Millipore diffusion chambers), we added autogenous and isologous bone marrow cells to minced muscle grafts or to sites of excised muscle in rats. In each case the opposite side was left without added marrow cells, to serve as a control. The results showed that addition of bone marrow produces more extensive muscle regeneration with little fibrosis. The myogenesis-stimulating and antibacterial properties of bone marrow cells warrant further study, aiming towards clinical application of the method.     

Regularities of hemopoietic development in bone marrow erythroblastic islands during early ontogenesis

Erythroblastic islands (EIs) of the bone marrow are multicellular associations of erythroid and macrophage cells. EIs serve as morpho-functional units of erythropoiesis. Erythropoiesis was studied in the rat liver, spleen, and bone marrow on days 1, 3, 7, 10, 15, and 30 after birth. Changes in the number of EIs and in their composition were simultaneously examined. After birth, erythropoiesis was shown to decrease in the liver and spleen, but it became more active in the bone marrow. There were islands of nondifferentiated hemopoietic cells on day 1 after birth, mixed hemopoietic islands on day 3, and a drastic increase in bone marrow EIs on day 7. On day 30, the levels of EIs were the same as in adult rats. There was a close relationship between the number of EIs and that of erythroid cells in the bone marrow, as well as the count of red blood cells. The age-specific dynamics of quantitative and qualitative changes in EIs and erythropoiesis is presented.     

Immunomodulatory and antimicrobial efficacy of intravenous immunoglobulin in bone marrow transplantation

BACKGROUND. Graft-versus-host disease (GVHD) and infection are major complications of allogeneic bone marrow transplantation. Since intravenous immunoglobulin has shown benefit in several immunodeficiency and autoimmune disorders, we studied its antimicrobial and immunomodulatory role after marrow transplantation. METHODS. In a randomized trial of 382 patients, transplant recipients given immunoglobulin (500 mg per kilogram of body weight weekly to day 90, then monthly to day 360 after transplantation) were compared with controls not given immunoglobulin. By chance, the immunoglobulin group included more patients with advanced-stage neoplasms; otherwise, the study groups were balanced for prognostic factors. RESULTS. Control patients seronegative for cytomegalovirus who received seronegative blood products remained seronegative, but seronegative patients who received immunoglobulin and screened blood had a passive transfer of cytomegalovirus antibody (median titer, 1:64). Among the 61 seronegative patients who could be evaluated, none contracted interstitial pneumonia; among the 308 seropositive patients evaluated, 22 percent of control patients and 13 percent of immunoglobulin recipients had this complication (P = 0.021). Control patients had an increased risk of gram-negative septicemia (relative risk = 2.65, P = 0.0039) and local infection (relative risk = 1.36, P = 0.029) and received 51 more units of platelets than did immunoglobulin recipients. Neither survival nor the risk of relapse was altered by immunoglobulin. However, among patients greater than or equal to 20 years old, there was a reduction in the incidence of acute GVHD (51 percent in controls vs. 34 percent in immunoglobulin recipients; P = 0.0051) and a decrease in deaths due to transplant-related causes after transplantation of HLA-identical marrow (46 percent vs. 30 percent; P = 0.023). CONCLUSIONS. Passive immunotherapy with intravenous immunoglobulin decreases the risk of acute GVHD, associated interstitial pneumonia, and infections after bone marrow transplantation.     

Interaction between carbon composites and bone after intrabone implantation.

Candidate carbon fiber reinforced carbon (CFRC) porous implant materials were evaluated for tissue compatibility in a rat model. Six different CFRCs of constant pore size (about 30 microm) were fabricated that had 9, 12, and 17% porosity with 2-nm3 matrix crystallites and 6, 12, and 20% at 25 nm3. They were implanted as femoral transverse diaphyseal pins that were 1.5 mm in diameter. At 5 and 45 weeks, implant and bone histologic specimens were evaluated histologically and by a scanning electron microscope and an electron microprobe. Also, regional lymph nodes and spleens were examined. By 45 weeks, direct implant-bone contact was observed over most of the interface in most specimens. At the implant surface, there was partial replacement of CFRC with host tissue. However, the microprobe showed that the implant-bone interface was chemically abrupt with no cross-diffusion of ionic species. Besides the surface effects, there was partial filling of the implant pores with tissue, including bone organized de novo deep within. This was observed histologically and confirmed by microprobe. Lymph nodes and spleens were histologically normal, and no carbon particles were found. None of the results were influenced by porosity or matrix crystallite size over the ranges studied. In summary, these porous CFRCs are partially degraded when in contact with bone and appear substantially tissue compatible. They may be useful as scaffolds for regrowth of bone.     

Postirradiation thymocyte regeneration after bone marrow transplantation. III Intrathymic differentiation and development of thymocyte subpopulations

Fluorescence distributions of thymocytes stained for Thy-1 as well as size measurements were used to discriminate between thymocyte subpopulations during regeneration of the thymus after irradiation and bone marrow transplantation. Subpopulations with “low” and “high” Thy-1 density of donor- and recipient-derived progeny were quantitated. They were continuously present in the thymus and developed simultaneously but at different rates of growth. A similar developmental pattern was observed for donor- and host-derived “high” Thy-1⁺ cells, whereas “low” Thy-1⁺ cells of donor and recipient origin showed markedly different growth patterns. This indicated that development of the two subpopulations took place independently. During early regeneration donor-derived “low” and “high” Thy-1⁺ cells contain a high proportion of large cells, indicating the presence of cycling cells in both subpopulations.     

Real-time automated PCR for early diagnosis and monitoring of cytomegalovirus infection after bone marrow transplantation

The purpose of this study was to assess the usefulness of real-time automated PCR as a quantitative, highly reproducible, and sensitive method to detect cytomegalovirus (CMV) DNA in blood specimens. Intra- and interassay precision rates were 0.89% (small number of copies [L]), 1.43% (middle number of copies [M]), and 1.12% (high number of copies [H]), and 4.46% (L), 1.51% (M), and 2.28% (H), respectively. The linearity of this assay was obtained between 10 and 10(7) copies/well, with a minimum detection limit of 20 copies/well. Specimens from 55 of 70 healthy subjects were found to be positive for CMV antibody, but CMV DNA was not detected in any of them. In the qualitative assessment of each specimen, the results of the CMV antigenemia assay and those of the real-time PCR assay agreed in 80% (plasma specimens), 79% (all nucleated cells), and 86% (blood) of the cases examined. For eight patients diagnosed as having CMV infection or disease, no sample was positive in the antigenemia assay earlier than in the real-time PCR assay. Furthermore, the results of this assay could be obtained within 8 h. We concluded that the real-time PCR assay is useful for rapid diagnosis of CMV infection and monitoring of clinical courses.     

Prevention of graft-versus-host disease by intrabone marrow injection of donor T cells: involvement of bone marrow stromal cells

We have developed a new and effective method for bone marrow transplantation (BMT): bone marrow cells (BMCs) are injected directly into the bone marrow (BM) cavity of recipient mice. The intrabone marrow injection of BMCs (IBM-BMT) greatly facilitates the engraftment of donor-derived cells, and IBM-BMT can attenuate graft-versus-host reaction (GVHR), in contrast to conventional intravenous BMT (i.v.-BMT). Here, we examine the mechanisms underlying the inhibitory effects of IBM-BMT on GVHR using animal models where GVHR is elicited. Recipient mice (C57BL/6) were irradiated and splenic T cells (as donor lymphocyte infusion: DLI) from major histocompatibility complex-disparate donors (BALB/c) were injected directly into the BM cavity (IBM-DLI) or injected intravenously (i.v.-DLI) along with IBM-BMT. The BM stromal cells (BMSCs) from these recipients were collected and related cytokines were examined. The recipient mice that had been treated with IBM-BMT + i.v.-DLI showed severe graft-versus-host disease (GVHD), in contrast to those treated with IBM-BMT + IBM-DLI. The suppressive activity of BMSCs in this GVHD model was determined. The cultured BMSCs from the recipients treated with IBM-BMT + IBM-DLI suppressed the proliferation of responder T cells remarkably when compared with those from the recipients of IBM-BMT + i.v.-DLI in mixed leucocyte reaction. Furthermore, the level of transforming growth factor-beta and hepatocyte growth factor in cultured BMSCs from IBM-BMT + IBM-DLI increased significantly when compared with those from the recipients of IBM-BMT + i.v.-DLI. Thus, the prevention of GVHD observed in the recipients of IBM-BMT + IBM-DLI was attributable to the increased production of immunosuppressive cytokines from BMSCs after interaction with host reactive T cells (in DLI).     

Fatal dissemination of cytomegalovirus after bone marrow transplantation

The clinical course and necropsy findings are described of an 11-year-old child found to have active cytomegalovirus infection at the time of bone marrow transplantation for acute lymphoblastic leukaemia. Attention is drawn to the presence of primitive mononuclear cells of uncertain origin in the regenerating bone marrow.     

Kinetics of liver repopulation after bone marrow transplantation

Recent work has convincingly demonstrated that adult bone marrow contains cells capable of differentiating into liver epithelial cells in vivo. However, the frequency and time course with which fully functional hepatocytes emerge after bone marrow transplantation remained controversial. Here, we used the fumarylacetoacetate hydrolase knockout mouse to determine the kinetics of hepatocyte replacement after complete hematopoietic reconstitution. Single donor-derived hepatocytes were first detected 7 weeks after lethal irradiation and bone marrow transplantation. Liver disease was not required for this transdifferentiation. In the presence of selective pressure the single cells evolved into hepatocyte nodules by 11 weeks after transplantation and resulted in >30% overall liver repopulation by 22 weeks. The frequency with which hepatocytes were produced was between 10(-4) and 10(-6), resulting in only 50 to 500 repopulation events per liver. Hepatic engraftment was not observed without previous hematopoietic reconstitution even in the presence of liver injury. In addition, significant liver repopulation was completely dependent on hepatocyte growth selection. We conclude that hepatocyte replacement by bone marrow cells is a slow and rare event. Significant improvements in the efficiency of this process will be needed before clinical success can be expected.