In situ hybridization for the detection of cytomegalovirus in blood or bone marrow leucocytes after allogeneic bone marrow transplantation

Summary. We report a study of CMV detection by a non-radioactive in situ hybridization (ISH) technique using a biotinylated CMV-DNA probe. The method was applied to blood and bone marrow cells and its results were compared with those of other currently used techniques. First, we analysed peripheral leucocytes on blood donors, and, after comparison with PCR, we obtained 75% sensitivity and 87% specificity rates. We then studied four BMT recipients during the first 105 d post-transplant. A positive signal was detected as a predominantly nuclear staining. The detection of an ISH-positive signal occurred at least 8 d before CMV isolation from viral culture. The number of positive cells was variable according to the patients'clinical evolution, showing a dramatic increase of labelled cells in viraemic patients and then a decrease until complete disappearance of labelled cells when an efficient anti-viral treatment was initiated. This study suggests that the detection of CMV-DNA by ISH performed on peripheral leucocytes is a valuable tool for the early diagnosis of CMV infection and could lead to the initiation of optimal ganciclovir treatment.     

Maintenance of platelet viability after platelet-labeling with fluorescein isothiocyanate

Possible effects of an ex vivo fluorescent labeling procedure of blood platelets on the platelet viability were examined by different function tests. The degree of the impairment of the platelet function proved to be principally correlated with the dye concentration used during the labeling procedure. However, the physiological properties of the labeled platelets obviously remain unrestricted by using intraplatelet fluorescein isothiocyanate (FITC) concentrations below 0.55 mg/10(11) platelets, corresponding to 8.5 X 10(6) FITC molecules bound to one platelet. A procedure for fluorescent labeling of blood platelets is described which is considered to be preferably suitable to prepare platelets for vital microscopic platelet observations.     

In Situ immunophenotyping of lymphocytes in human bone marrow: an immunohistochemical study

Lymphoid cells in human bone marrow are either assembled focally or occur in a diffuse, loosely scattered infiltrate. While the focal lesions are easily detected, the lymphoid cells of the diffuse infiltrate are hardly recognizable with conventional stains. Quantitative immunohistological analysis of 103 trephine biopsies, including cases with reactive disorders (e.g. myeloid hyperplasia, aplastic anaemia) and neoplastic processes (e.g. myeloproliferative disorders, B-cell non-Hodgkin's lymphomas) and some specimens with normal architecture yielded the following results: (1) Various antibodies recognizing B cells (L26, 4KB5, MB1, Ki-B3), T cells (UCHL1, MT1) and NK cells (Leu-7) are effective in paraffin-embedded bone marrow sections, thus enabling analysis of the in situ distribution of normal lymphocyte subsets and subtyping of lymphomatous infiltrates. (2) The lymphocytes of the diffuse infiltrate constituted about 1-5% of all nucleated cells in normal bone marrow. (3) In the diffuse infiltrate, T lymphocytes were regularly observed in higher numbers than B cells, and Leu-7+ cells were rare or virtually absent, irrespective of the diagnosis. (4) The focally assembled lymphoid cells were mainly B lymphocytes, but many T cells were always intermingled. This was true for both reactive follicles and neoplastic lymphomatous infiltrates, which generally cannot be differentiated on the basis of immunohistological findings alone.     

Allogeneic bone marrow transplantation for leukemia with marrow grafts depleted of lymphocytes by counterflow centrifugation

Eighty consecutive patients were transplanted with human leukocyte antigen (HLA)-identical sibling marrow for acute myelogenous leukemia (AML, N = 29), acute lymphoid leukemia (ALL, N = 23), or chronic myelogenous leukemia (CML, N = 28). Donor marrow was depleted of lymphocytes using counterflow centrifugation. Median age of the recipients was 31 years. Pretransplant conditioning consisted of cyclophosphamide and fractionated total body irradiation (TBI) with a low (4.1 +/- 0.3 cGy/min) or high (13.1 +/- 1.6 cGy/min) midline average dose rate. In 43 patients, cytosine-arabinoside or anthracyclines were added to the conditioning regimen. Immunoprophylaxis posttransplant consisted of methotrexate (MTX) alone, cyclosporine A (CsA) in combination with MTX, or CsA alone; two patients received no immunoprophylaxis at all. Graft failure occurred in 4 of 77 evaluable patients (5%). The probability of acute graft-versus-host disease (GVHD) greater than or equal to grade 2 at day 100 after transplantation was 15%. The projected 3-year estimate of extensive chronic GVHD was 12%. Only three patients died of cytomegalovirus-interstitial pneumonitis. The projected 3-year probability of relapse was 30% (95% confidence interval [CI], range 8% to 53%) in transplants for AML in first complete remission (CR1), 35% (95% CI, 1% to 69%) after transplantation for ALL in CR1, and 38% (95% CI, 2% to 74%) after transplantation for CML in first chronic phase (CP1). The projected 3-year probability of leukemia-free survival (LFS) was 56% (95% CI, 35% to 77%) after transplantation for AML-CR1, 42% (95% CI, 16% to 69%) in patients transplanted for ALL-CR1, and 49% (95% CI, 18% to 80%) after transplantation for CML-CP1. After transplantation for AML-CR1, ALL-CR1, or CML-CP1, the median follow-up time for leukemia-free survivors was 31+, 30+, and 21+ months, respectively. Probabilities of relapse, survival, and LFS in AML-CR1 and ALL-CR1 transplants were comparable with those reported in recipients of untreated grafts. In patients transplanted for CML-CP1, probability of relapse was higher and probability of LFS was lower than in recipients of untreated grafts. In transplants for leukemia in CR1 and CP1, preparative regimen and immunoprophylaxis posttransplant were not associated significantly with the probability of acute GVHD greater than or equal to grade 2, extensive chronic GVHD, relapse, survival, or LFS. In bone marrow transplantation for leukemia, counterflow centrifugation is a useful technique for the prevention of GVHD.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chimerism studies using in situ hybridization for the Y chromosome after T cell-depleted bone marrow transplantation

In situ hybridization for the Y chromosome (Y-ISH) was used to monitor engraftment in 10 patients with hematological malignancies who had received T cell-depleted marrow transplants from sex-mismatched donors, seven of whom were only partially HLA-matched. In the three patients who engrafted, as the peripheral counts rose, the percentage of host peripheral blood and marrow mononuclear cells decreased steadily, although host cells (less than 1%) could still be detected as late as day 252. The percentage of host granulocytes fell rapidly to less than 0.2%. Seven patients did not achieve full engraftment by day 28. Those with a low percentage of host cells (less than 1%) improved with observation or treatment with steroids, while those with a high or increasing percentage of host cells did not improve even after treatment with GM-CSF or with repeat marrow infusion without reconditioning. In one patient with graft failure, the residual host cells were predominantly CD8+ CD57+ and CD3+ CD56+, phenotypes consistent with non-MHC-restricted cytotoxic T cells. Lack of full engraftment in recipients of T cell-depleted marrow is not always associated with autologous reconstitution and does not always require retransplantation. Y-ISH may be useful for monitoring patients at high risk for graft failure in order to detect adverse trends in mixed chimerism that will alter therapy early after transplantation.     

Early lymphocyte development in bone marrow and thymus

Haematopoietic stem cells (HSCs), a very rare cell type in the bone marrow, are responsible for the life-long production of all cells of the blood including T and B cells. Until recently, it was thought that the differentiation of HSCs into the various haematopoietic cells was rather hierarchical in that differentiation along a given lineage was associated with a progressive loss of potential to give rise to other blood cell lineages. The recent development of very sensitive and quantitative in vitro assays, together with the identification of new progenitor subpopulations, has challenged this idea. Thus, lymphocyte progenitors can be shown to keep their developmental potential to give rise to myeloid, dendritic and NK cells until just prior to their final commitment stage. Here we review these new findings and concepts.     

In situ localization of T lymphocytes in disseminated coccidioidomycosis

Immunohistochemical techniques using monoclonal antibodies to T lymphocyte subpopulations were used to characterize further the granulomas of disseminated coccidioidomycosis. Skin biopsy specimens from patients with disseminated coccidioidomycosis were studied and compared with tissues from experimentally infected mice. In human skin biopsy specimens and infected mouse tissues, discrete granulomata were seen in which T lymphocytes formed a peripheral mantle surrounding central aggregates of macrophages. This unusual pattern of granuloma formation may represent an ineffective host response because these individuals are unable to clear their infection. Because of the close similarity of immunopathology in both human and mouse infections, the mouse model should serve as a useful tool in elucidating the factors contributing to ineffective host responses in systemic fungal infections.     

In vitro studies on the radiosensitivity of multipotent hemopoietic progenitors in canine bone marrow

The in vitro radiation response to 280-kV x-rays (does rate 72 cGy/min) of multipotent hemopoietic progenitor cells, mixed colony-forming units (CFU-mix), from canine bone marrow was assayed and compared to the radiation response characteristics of early erythroid progenitors, erythroid burst-forming units (BFU-E). To improve the colony-forming efficiency, the effect of various bone marrow cell separation techniques on colony formation of both progenitors was examined. The separation of bone marrow aspirates by discontinuous buoyant gradient centrifugation using the lymphocyte separation medium Lymphoprep with a density of 1.070 g/ml allowed the establishment of reproducible survival curves. The survival curves for both progenitors were strictly exponential, and CFU-mix were found to be more radiosensitive (D0 = 12 +/- 2 cGy) than BFU-E (D0 = 16 +/- 2 cGy).     

Resolution of sarcoidosis after allogeneic bone marrow transplantation with donor lymphocyte infusions

Abnormalities of immune surveillance may contribute to the development of myeloid malignancy as well as immune-mediated diseases. In leukaemia, allogeneic haemopoietic stem cell transplantation (alloHSCT) has been used to induce disease remission, in part by restoring mechanisms of immune regulation. Although, by the same principle, allogeneic stem cell transplantation is an attractive option for the treatment of immunological disorders, it is unclear whether remission after transplantation is due to pre-transplant conditioning, or modulation of auto-reactive lymphocytes by cells in the allograft. We report the case of a patient with chronic myeloid leukaemia (CML) who received an allogeneic bone marrow transplant (alloBMT) from his brother. He subsequently suffered a cytogenetic and molecular relapse of CML. At the same time, sarcoidosis involving the marrow was diagnosed. He was treated with donor lymphocyte infusions (DLI) and attained remission from CML; in addition, no giant cell granulomas were detected in the marrow, indicating resolution of sarcoidosis. This case illustrates the need for further studies on the role of T cell-based therapies in the management of immune-mediated disorders.     

Reconstitution of lymphocyte subpopulations after paediatric bone marrow transplantation

We prospectively studied the reconstitution of lymphocyte subpopulations in a group of 22 children, who survived disease-free at least 6 months after allogeneic BMT for a haematological malignancy. Absolute counts of total lymphocytes, B lymphocytes, T lymphocytes, and CD4+ helper T lymphocytes reached the 5th percentile (p5) of age-matched reference values within 6 months after BMT in 15, 17, 7 and 2 patients, respectively. In particular, CD4+ helper T lymphocyte reconstitution was very slow. Unexpectedly, CMV reactivation had a profound positive influence upon the number of CD4+ helper T lymphocytes in the children. In five patients, absolute B lymphocyte counts above the 95th percentile were reached from 6 months after BMT onwards, mimicking normal ontogeny. Unlike normal ontogeny, the percentages of helper T lymphocytes expressing the 'naive' CD45RA isoform were low and those expressing the 'memory' CD45RO isoform were high in the first 3 months after BMT, as described before. Thereafter, the CD45RA:CD45RO ratio slowly normalised. Also, CD7 expression was absent on up to 90% of T lymphocytes in the first months after BMT, and on a steadily decreasing percentage thereafter, as recently described in adults. However, the absolute counts of CD45RO+/CD4+ and CD7-/CD4+ helper T lymphocytes did not change significantly. So, we found no evidence of peripheral expansion of previously primed donor-derived 'memory' T lymphocytes during the follow-up period which spanned 1-18 months after BMT. The absolute counts of 'naive' CD45RA+ helper T lymphocytes did not show a faster increase after BMT than in adults, despite the presumed presence of a non-involuted thymus in children. Bone Marrow Transplantation (2000) 25, 267-275.     

A mouse model of lymphocyte infusion-induced bone marrow failure

OBJECTIVE: To develop a mouse model for the study of the pathophysiologic mechanism and treatment of human bone marrow (BM) failure. MATERIALS AND METHODS: Unmanipulated B6D2F1 or CByB6F1 hybrid mice were infused with 10-40 x 10(6) lymph node (LN) cells from their C57BL/6 (B6) parent. Pancytopenia was monitored by cell counting, while marrow damage was assessed by histological staining. Destruction of BM hematopoietic progenitor/stem cells was measured by colony formation in vitro and irradiation protection in vivo. Serum interferon-gamma (IFN-gamma) concentration was measured by enzyme-linked immunosorbent assay. BM T cell Vbeta and Fas expressions were analyzed by flow cytometry. Treatment effects of immunosupressive agents cyclosporine, antithymocyte globulin (ATG), anti-IFN-gamma, and anti-tumor necrosis factor-alpha (anti-TNF-alpha) were tested. RESULTS: Infusion of 30-40 x 10(6)B6 LN cells led to rapid development of severe pancytopenia, BM hypoplasia, and death. Affected mice had drastically reduced hematopoietic progenitor and stem cells. BM of affected mice showed lymphocyte infiltration, oligoclonal T cell expansion, and upregulated Fas expression. Serum IFN-gamma concentration increased two- to three-fold. Timed administration of cyclosporine or ATG abrogated pancytopenia. Treatment with anti-IFN-gamma antibody reliably rescued mice, and treatment with anti-TNF-alpha antibody extended animal survival significantly. CONCLUSION: This mouse model indicates that activated lymphocytes and type I cytokines play important roles in marrow destruction in lymphocyte infusion-induced BM failure.     

Cytogenetic studies in bone marrow transplant recipients

Summary Chromosome studies were performed in 24 patients who underwent allogeneic bone marrow transplantation (BMT) for severe aplastic anaemia (8), chronic myeloid leukemia (5 in chronic, 2 in accelerated phase and 1 in lymphoid blast crisis), acute myeloid leukemia (6), acute lymphoblastic leukemia in relapse (1) and Hodgkin's disease (1). Donor-cell type engraftment was demonstrated in 21 patients: in all 17 sex-mismatched transplants and — as demonstrated by reconstitution with Ph-negative cell populations — in 4 CML patients with a sex-matched donor. Recipient-type mitoses were seen in the bone marrow of 5 cases (1 SAA, 3 CML, 1 AML) after transplantation. They were only observed on one occasion in patients with SAA (4 of 25 on day 33) and AML (44 of 50 on day 14). Despite the continued demonstration of some Ph-positive mitoses in 3 patients with CML up to day 28, 323 and 451 after BMT, respectively, all surviving CML patients are still in complete haematological and clinical remission. So far the significance of these cytogenetically abnormal persisting host cells remains unknown. Present address: Roswell Park Memorial Institute, Department of Genetics and Endocrinology, 666 Elm Street, Buffalo, NY 14 222, USA     

Chromosomal studies after bone marrow transplantation for leukaemia in children

Chromosomal studies were performed on 114 blood samples and 117 bone marrow samples, taken systematically over a period of 4 months after bone marrow transplantation (BMT) in 42 children grafted for acute lymphoblastic leukaemia (ALL) (n = 20), acute myeloid leukaemia (AML) (n = 16), non-Hodgkin's lymphoma (NHL) (n = 2) and myelodysplastic syndrome (MDS) (n = 4). In some cases, follow-up investigations were performed. In the first 4 months following BMT, mixed chimerism was frequently observed in blood of AML (25%), ALL (30%), NHL (100%) cases and in bone marrow samples of ALL (35%). The presence and relative number of a patient's own metaphases found shortly after transplantation was not related to leukaemia relapse and probably represents residual non-malignant haematopoietic precursor cells of the host. In only one child grafted for MDS with 45,XY, -7 karyotype was the marker clone still detectable in bone marrow at day +437 post-BMT. This patient shows no recurrence of the MDS and has a sustained haematological recovery at the time of writing, i.e. 4.5 years post-BMT. In 11 other patients, various structural chromosomal abnormalities (not related to the original leukaemia) were found in both peripheral blood and bone marrow. In three different patients structural anomalies were also found in bone marrow and blood samples from donor-derived cells. This indicates that, besides irradiation, there are other as yet unidentified factors (e.g. drugs), which are capable of inducing chromosomal anomalies in the post-BMT period.     

Phenotypic analysis of bone marrow lymphocytes from children with acute thrombocytopenic purpura

Hematogones are benign immature B cells that commonly populate the bone marrow of children. Their presence has been noted to interfere with the flow-cytometric analysis of acute lymphoblastic leukemia (ALL), because their immunophenotype is similar to B-precursor cell lymphoblasts. Immune-mediated thrombocytopenia is a clinical condition characterized by increased platelet destruction due to sensitization of platelets by autoantibodies. The aim of this study was to determine the incidence and clinical impact of bone marrow hematogones in cases of acute immune thrombocytopenic purpura (ITP) among children. This was done by immunophenotyping of bone marrow lymphocytes of ITP cases and controls and follow up of cases. This study was done on 25 cases of ITP, 12 females and 13 males, their age ranged from 2 to 13 years. A control group was included in the study, 15 cases of apparently healthy children with matching age and sex taken from among bone marrow donors. Cases and controls were subjected to bone marrow lymphocyte immunophenotyping with flow-cytometry to verify the presence of hematogones. A statistically significant increase in the percentage of hematogones was demonstrated in their bone marrows. An increased percentage of CD10+ lymphocytes was demonstrated; with a mean of 18+/-15.2%, CD19+ with a mean of 27+/-16.3% and CD34+ with a mean of 3.7+/-3.2%. No correlation was found between the percentage of hematogones and peripheral platelet count or bone marrow lymphocytic count. In conclusion, there is an increase in the bone marrow hematogones in ITP cases in comparison to normal controls. This could be the sequence of an immunological response to the cause which determined the disease, or the regeneration of the stem cell compartment following transient damage.     

Conformational changes in the intestinal brush border sodium-glucose cotransporter labeled with fluorescein isothiocyanate.

Fluorescein isothiocyanate (FITC) was used to label the rabbit intestinal brush border Na+-glucose carrier, identify the carrier protein on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and monitor the effect of ions and substrates on fluorescence quenching. Enriched brush border preparations were employed to study both glucose transport and FITC binding. FITC and a nonfluorescent analog (phenyl isothiocyanate, PITC) both inhibited Na+-dependent D-glucose transport irreversibly. Inhibition was blocked completely by the presence of Na+ and D-glucose during labeling. PITC was used to label nonspecific amino groups in the presence of glucose and Na+, and then the glucose carrier was labeled with FITC in the absence of substrates. Fluorescence of FITC bound to the carrier was quenched specifically with Na+ in a saturable fashion, and this indicates a Na+-dependent conformational change in the carrier. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of FITC-labeled membranes revealed specific labeling of a 71,000-dalton peptide. We conclude that Na+ induces a conformational shift in the 71,000-dalton glucose carrier, and this is quite consistent with the kinetics of Na+-dependent glucose transport in these membranes.     

Increased apoptosis in bone marrow B lymphocytes but not T lymphocytes in myelodysplastic syndrome

The hallmark of myelodysplastic syndrome (MDS) is enhanced apoptosis in myeloid, erythroid, and megakaryocytic cells in the bone marrow leading to ineffective hematopoiesis. Recent studies suggested that immunological and microenvironmental factors play a role in the pathophysiology of this disease. We report a significant increase in apoptosis in bone marrow B lymphocytes in MDS as compared to that found in acute myeloid leukemia and healthy controls. Furthermore, we demonstrate that patients with refractory anemia with excess blasts in transformation (RAEB-T) had apoptosis levels in lymphocytes similar to those seen in other subtypes of MDS. Our findings suggest that the alterations in B lymphocytes in the form of increased apoptosis can be seen in MDS and support the concept that immune modulation plays a role in the pathophysiology of MDS.