Oral delivery of Lactobacillus casei Shirota modifies allergen-induced immune responses in allergic rhinitis.

BACKGROUND: Changes in the composition of the gut microbiota have been implicated in the pathogenesis of allergic disorders, suggesting beneficial interactions between the intestinal immune system and specific bacterial strains. Lactobacilli are naturally present within the complex gastrointestinal microbiota of humans and they are currently present in many probiotic supplements. OBJECTIVE: We sought to investigate the role that Lactobacillus casei Shirota (LcS) may play in modulating seasonal allergic rhinitis (SAR). METHODS: The study format was double-blinded, placebo-controlled with 10 SAR sufferers in each group. We have documented and compared changes in immune status arising through the daily ingestion of a milk drink with or without live LcS, over a period of 5 months. Pre-, peak- and post-grass pollen season blood samples were collected for determination of plasma total IgE and grass pollen-specific IgG and IgE levels by an enzyme immunoassay. At the same time, cytokine levels were determined by flow cytometric bead array technology following culture of peripheral blood mononuclear cells for 6 days in the presence or absence of specific grass pollen antigens. RESULTS: Volunteers treated with LcS showed a significant reduction in levels of antigen-induced IL-5, IL-6 and IFN-gamma production compared with volunteers supplemented with placebo. Meanwhile, levels of specific IgG increased and IgE decreased in the probiotic group. CONCLUSION: Changes in antigen-induced production of cytokines were observed in patients treated with probiotics. These data show that probiotic supplementation modulates immune responses in allergic rhinitis and may have the potential to alleviate the severity of symptoms.

Reticulated platelets interfere with flow cytometric reticulocyte counts.

As part of an iron absorption study, we needed to accurately count reticulocytes in the peripheral blood of healthy human volunteers before measuring their enrichment with stable iron isotopes given in an oral dose. Recent studies have suggested the usefulness of reticulocyte counting by flow cytometry, through a combination of differential light scatter and measurement of the stoichiometric binding of thiazole orange (TO) to RNA within the maturing erythrocyte. Using this method we set out to improve the precision of our quantitative analysis by counting more cells, as reticulocytes normally comprise <2% of the red cell population. To ensure exclusion of other cell types, we identified WBCs and platelets with CD16+CD45- allophycocyanin and CD61- phycoerythrin, respectively. After removal of CD16(+) CD45(+) TO(+) WBCs and CD61(+) TO(-) platelets from analysis, the remaining cells were a combination of CD61(-) TO(-) erythrocytes, CD61(-) TO(+) reticulocytes and CD61(+) TO(+) reticulated platelets. Reticulocyte counts were lower after exclusion of CD61(+) TO(+) cells from analysis. They were similarly lower when erythrocyte precursors were positively identified through their glycophorin A expression and TO uptake. We conclude that it is necessary to exclude reticulated platelets from flow cytometric reticulocyte analysis.

Synthetic and naturally occurring COX-2 inhibitors suppress proliferation in a human oesophageal adenocarcinoma cell line (OE33) by inducing apoptosis and cell cycle arrest.

Epidemiological studies suggest that the use of NSAIDs and/or a high intake of fruit and vegetables reduce the risk of oesophageal adenocarcinoma. Since COX-2 is up-regulated in Barrett's oesophageal carcinogenesis, the protective effect of NSAIDs and natural food components might reflect COX-2 inhibition. We explored the effects of quercetin, a natural flavonoid with a potent COX-2 inhibitory activity, and two commercially available selective COX-2 inhibitors (NS-398 and nimesulide) on cell proliferation, apoptosis, PGE2 production and COX-2 mRNA expression in a human oesophageal adenocarcinoma cell line (OE33). Changes in the relative numbers of adherent and floating cells were quantified and apoptotic cells were identified using ethidium bromide and acridine orange staining under fluorescence microscopy. Flow cytometric analysis of adherent and floating cells was used to quantify apoptosis and to examine the effects of the agents on the cell cycle. After 48 h exposure at concentrations of > or =1 microM both COX-2 inhibitors and quercetin suppressed cell proliferation (P < 0.01) and increased the fraction of floating apoptotic cells. At higher concentrations (50 microM) and longer exposure (48 h) the effects of quercetin were significantly greater than those of the selective COX-2 inhibitors (P < 0.01). Cell cycle analyses showed that quercetin blocked cells in S phase, while the selective COX-2 inhibitors blocked cells in G1/S interphase. COX-2 mRNA expression was suppressed by quercetin and the synthetic COX-2 inhibitors in a time- and dose-dependent manner. Quercetin and the synthetic COX-2 inhibitors (10 microM) suppressed PGE2 production by approximately 70% after 24 h exposure (P < 0.001). We conclude that OE33 is a useful model for the study of COX-2 expression and associated phenomena in human adenocarcinoma cells. Synthetic COX-2 inhibitors and the food-borne flavonoid quercetin suppress proliferation, induce apoptosis and cell cycle block in human oesophageal adenocarcinoma cells in vitro, and future studies should assess their effects in vivo.

Monocyte and neutrophil adhesion molecule expression during acute hyperglycemia and after antioxidant treatment in type 2 diabetes and control patients.

OBJECTIVE: We hypothesized that acute hyperglycemia (an independent cardiovascular risk factor) increases the expression of proatherogenic leukocyte adhesion molecule in type 2 diabetes and controls and that the expression of these adhesion molecules would be antioxidant sensitive. METHODS AND RESULTS: Twenty-three type 2 diabetes patients and 13 control patients underwent two oral glucose tolerance tests 14 days apart and took placebo or 800 IU daily of oral alpha tocopherol between tests. Monocyte and neutrophil expression of adhesion molecules Mac-1, LFA-1 and 3, ICAM-1, and VLA-4 were measured at 0, 120, and 240 minutes by using laser flow cytometry. Baseline adhesion molecule expression did not differ between groups, but there was a rapid, highly significant increase (P<0.0001) in the intensity of monocyte Mac-1 expression after a glucose load in both groups. Alpha-tocopherol supplementation reduced only Mac-1 expression in the diabetes group (P=0.03). CONCLUSIONS: Acute glycemic excursions of any degree cause highly significant, rapid increases in monocyte Mac-1 expression in type 2 diabetes patients and controls. Mac-1 mediates leukocyte vascular infiltration and is prothrombotic. These data suggest a mechanism for the link between glycemic excursions and increased vascular event rates.

The flow cytometric analysis of telomere length in antigen-specific CD8+ T cells during acute Epstein-Barr virus infection.

Acute infectious mononucleosis (AIM) induced by Epstein-Barr virus (EBV) infection is characterized by extensive expansion of antigen-specific CD8+ T cells. One potential consequence of this considerable proliferative activity is telomere shortening, which predisposes the EBV-specific cells to replicative senescence. To investigate this, a method was developed that enables the simultaneous identification of EBV specificity of the CD8+ T cells, using major histocompatibility complex (MHC) class I/peptide complexes, together with telomere length, which is determined by fluorescence in situ hybridization. Despite the considerable expansion, CD8+ EBV-specific T cells in patients with AIM maintain their telomere length relative to CD8+ T cells in normal individuals and relative to CD4+ T cells within the patients themselves and this is associated with the induction of the enzyme telomerase. In 4 patients who were studied up to 12 months after resolution of AIM, telomere lengths of EBV-specific CD8+ T cells were unchanged in 3 but shortened in one individual, who was studied only 5 months after initial onset of infection. Substantial telomere shortening in EBV-specific CD8+ T cells was observed in 3 patients who were studied between 15 months and 14 years after recovery from AIM. Thus, although telomerase activation may preserve the replicative potential of EBV-specific cells in AIM and after initial stages of disease resolution, the capacity of these cells to up-regulate this enzyme after restimulation by the persisting virus may dictate the extent of telomere maintenance in the memory CD8+ T-cell pool over time.

Fluticasone propionate-induced regulation of the balance within macrophage subpopulations.

In asthma, treatment with inhaled corticosteroids reduces chronic peribronchial inflammation and restores the balance within macrophage subpopulations. This study investigates whether corticosteroids can regulate monocyte differentiation in vitro and thereby influence the balance of functionally distinct macrophages. Graded doses of fluticasone propionate (FP) were added to cultures of normal peripheral blood monocytes in the presence or absence of IL-4. Cells were harvested after 7 days' culture. Double immunofluorescence studies were performed on cytospins of differentiated macrophages using the MoAbs RFD1 and RFD7 to distinguish inductive and suppressive macrophages by their respective phenotypes. Macrophage function was determined by quantifying allostimulation in a mixed leucocyte reaction and by measuring tumour necrosis factor-alpha (TNF-alpha) production. FP reduced the number of mature cells with a D1+ antigen-presenting phenotype and up-regulated the development of cells with the D1/D7+ and D7+ phenotypes. Functionally, this was associated with reduced stimulation of T cell proliferation in a mixed leucocyte reaction (MLR). Fluticasone also reversed the increase in both D1+ expression and TNF-alpha production induced by IL-4. The effect of FP persisted for 24 h after removal of FP from the culture medium. These results suggest that FP treatment of asthmatics may have a direct beneficial effect by normalizing the macrophage subset imbalance that contributes to the chronic peribronchial inflammation present in this condition.

Common visions, shared goals: leadership in action.

A senior health care executive describes the strength of the Continuous Improvement philosophy in assisting senior and middle managers in transforming a children's hospital. Emphasizing the power of positive leadership in developing common goals and shared visions, and the relationship of midlevel managers to that process, examples are cited from a patient care delivery redesign project and in child advocacy.

Intracellular cytokine production by human CD4+ and CD8+ T cells from normal and immunodeficient donors using directly conjugated anti-cytokine antibodies and three-colour flow cytometry.

Using three-colour flow cytometry, we have measured intracellular IL-2, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) induced in human CD4+ and CD8+ T cells from normal donors and patients with common variable immunodeficiency (CVID). Since a new range of directly FITC-conjugated anti-cytokine antibodies was used, conditions were optimized for the concentration of antibody, for cell permeabilization and fixation, and for the time of exposure to monensin to retain the cytokines within the cell. Kinetics of intracellular cytokine production were measured for up to 20 h in culture with phorbol myristate acetate (PMA) and ionomycin, or with phytohaemagglutinin (PHA). Kinetic studies of activation with PMA and ionomycin show that a higher proportion of normal CD4+ cells can make IL-2 than the other two cytokines, and that there are more TNF-alpha-positive CD4+ cells than cells with IFN-gamma. For normal CD8+ cells the highest production of cytokine is of IFN-gamma (up to 50% of the cells) especially at longer times (10-20 h) of stimulation. For CD8+ cells, IL-2-positive cells exceed those with TNF-alpha. The other mitogenic stimulus used (PHA) was grossly inferior to PMA and ionomycin in its ability to induce intracellular cytokines. The time of exposure to monensin was also examined. Its continuous presence in the cultures (up to a maximum of 20 h) increased the detection of IL-2-positive cells without apparently reducing the percentage of cytokine-positive CD4+ or CD8+ cells. Finally, using optimal conditions, we compared cytokine production in cells from patients with the disease CVID and showed normal cellular levels of ability to produce IL-2 and TNF-alpha but significantly raised levels of production of IFN-gamma in both CD4+ and CD8+ lymphocytes. This suggests that the pathology of this disease may involve an excessive Th1-type response.

Leukemia-associated changes identified by quantitative flow cytometry. IV. CD34 overexpression in acute myelogenous leukemia M2 with t(8;21).

During the immunodiagnosis of 517 cases of acute myelogenous leukemia (AML) entered into the Medical Research Council (MRC) AML 10 trials, we have observed the CD34 precursor cell antigen more frequently in AML of M2 morphology, especially in the 84% of cases with the t(8;21) chromosomal translocation, than in any other French-American-British classification group. CD34 expression was then quantified (using QIFI and Quantum Simply Cellular beads [Flow Cytometry Standards, Research Triangle Park, NC] and CD34+ standard cells). When CD34 antibody-binding capacity (ABC) of normal bone marrow (BM) precursors and leukemic blasts was compared, it was shown that AML M2 cases with t(8;21) not only had the highest percentages of CD34+ blasts, but in > 80% of CD34+ cases the individual blasts expressed higher than normal levels of CD34 antigen (> 60 x 10(3) ABC per cell). In addition, in 73% of this group CD34 antigen was overexpressed in an asynchronous combination with cytoplasmic myeloperoxidase (MPO). Other signs of asynchrony included high CD34 expression with CD15 and/or CD56, as well as aberrant combinations of CD13 with terminal deoxynucleotidyl transferase (TdT) and CD19. These findings demonstrate that asynchrony is identifiable in virtually every case of AML with t(8;21), although it does not always involve the same antigens. M2 cases with t(8;21), mostly CD34+, had a 100% remission rate and 71% 5-year survival rate; other patients with CD34+ or CD34- AML showed 69% and 84% remission rates and 31% and 36% 5-year survival rates, respectively. Consequently, individual markers such as CD34 should be interpreted in relation to other features such as chromosomal changes. These simple methods, which are well suited to quantify the expression of ligands, are a useful contribution to diagnosis: 60% to 65% of M2 cases with t(8;21) are rapidly identified by CD34 overexpression alone. This aberration, together with the other signs of asynchrony seen at presentation, can be used to search for residual leukemia after therapy.

Bcl-2 protein expression in normal human bone marrow precursors and in acute myelogenous leukemia.

The expression of bcl-2 protein that is involved in preventing apoptosis in hemopoietic and other cells was evaluated by quantitative flow cytometry in various subpopulations in the normal fetal bone marrow (FBM) and in different types of acute myelogenous leukemias (AML). In the FBM the highest bcl-2 levels (mean antibody binding capacity 51 x 10(3) molecules/cell) were found in CD34+ intermediate sized blasts and myeloblasts, while a CD34+ subset of CD10+ lymphoblasts had low bcl-2 content (8-10 x 10(3) molecules/cell) and the CD34-, CD10+ lymphoblasts were, as expected from previous studies, bcl-2- (< 5 x 10(3) molecules/cell). Variable levels of bcl-2 (5.1-222 x 10(3)) were found in 43 tested cases of AML. The bcl-2 levels decrease with granulocytic and monocytic differentiation and, accordingly, cases of AML with M1 and M2 features showed significantly higher mean bcl-2 levels than the leukemias with promyelocytic (M3) or myelo-monocytic (M4/M5) features. Nevertheless, in seven cases of AML the bcl-2 levels were higher than seen in the normal FBM cells and none of these patients remain in remission after 2 years. Furthermore, in several AML cases intraclonal heterogeneity was observed. The undifferentiated smaller blasts with Class-IIdim display had higher bcl-2 content than the more differentiated larger blasts with more granular side scatter and Class-bright expression. In the same subsets of AML blasts the proliferative S-G2-M fractions showed a reciprocal correlation to bcl-2 content. Thus the higher bcl-2 levels may give a survival advantage and confer some degree of drug resistance to the least differentiated blast populations. The multi-parameter analysis described in this paper, including a combined bcl-2 and cytokinetic analysis of phenotypically defined subgroups of AML blasts, may detect early population shifts during relapse and also guide combination drug therapy.

Leukemia-associated changes identified by quantitative flow cytometry: I. CD10 expression.

We have compared CD10 antigen expression in normal fetal bone marrow with that of B-linage acute lymphoblastic leukemia (ALL). Both quantitative indirect immunofluoresence (QIFI) and direct immunofluorescence (IF) tests with Quantum beads were used to convert median fluorescence intensity (MFI) values into numbers of antigen molecules expressed per cell (AgE). Lymphoid precursors in the fetal marrow and liver expressed 3-12.5 x 10(3) CD10 molecules/cell with an upper limit of 5 x 10(4)/cell (MaxAgE). The median CD10 AgE in the different cases of acute B-lineage ALL were variable and ranged from undetectable to very high values (> 1.8 x 10(5). In 24 of the 72 cases (33%) tested with QIFI the median CD10 AgE was above the highest values seen in normal samples (> 5 x 10(4)/cell). An additional 23.6% of cases had higher median values than the normal median CD10 AgE. Next, CD10 antigen was quantitated in 78 cases during the routine multiparameter analysis of B-lineage leukemia using CD10/class II/CD34 3-color IF test or CD10/TdT 2-color IF test. The aberrant overexpression was confirmed in 43.6% of ALL cases. The CD10bright display suggested ALL diagnosis even when few cells were available for study, e.g., in early relapse and in ALL masquerading as aplastic anemia. The levels of CD10 expression were maintained in relapse. In addition, different CD10 levels were associated with the various chromosomal alterations: high CD10 levels (> 3 x 10(4)/cell) with hyperdiploidy, low CD10 levels (1.8-4 x 10(3)/cell) with the t(1;19) and undetectable levels (< 1.2 x 10(3)/cell) with the t(4;11) translocations.(ABSTRACT TRUNCATED AT 250 WORDS)

Leukemia-associated changes identified by quantitative flow cytometry. III. B-cell gating in CD37/kappa/lambda clonality test.

Normal and malignant B-lymphoid cells were studied for CD37 antigen expression with three-color immunofluorescence (IF) in combination with kappa/lambda light chain staining, and by quantitative immunofluorescence utilizing the QIFI test. Peripheral B cells brightly expressed CD37 antigen (median 80-114 x 10(3) molecules/cell). Moderate to high levels (> 20 x 10(3)/cell) of CD37 expression were detected in 364 in 366 cases of peripheral B-cell disorders including all cases of B-ALL, B-cell lymphomas and B-CLL as well as eight of ten cases of PLL. By contrast, slg- B-cell precursors and other cell types in normal bone marrow (BM) were CD37-/CD37dull (< 10 x 10(3) molecules/cell). The negativity for CD37 or only CD37dull expression was confirmed in 180 of 182 cases of precursor B-ALL and 196 cases of non-B malignancies. Among the CD37 cluster, the RFB7 antibody of IgM class showed the weakest binding to non-B cells. In 64 normal samples of blood and BM the CD37+ gated cells showed normal kappa/lambda ratios as expected, while in 100 cases of B malignancy striking changes such as kappa/lambda monoclonality (79%) and aberrant slg- or sigdull expression (21%) were seen among the gated CD37+ B cells. The CD37/kappa/lambda test identified as few as 0.5% kappa+ or lambda- monoclonal B cells admixed to normal BM: circulating B-lymphoma cells were seen in nine patients with morphologically normal blood count. The discrimination of the Kolmogorov-Smirnov (KS) test for kappa/lambda excess was also improved by CD37+ B gating. Thus CD37+ B-cell gating and kappa/lambda analysis is a simple and sensitive routine test, e.g. when combined with autogating on a Cytoron-Absolute cytometer, for identifying malignant B cells in minimally involved BM and blood.

Human CD4+CD45R0+ and CD4+CD45RA+ T cells synergize in response to alloantigens.

Alloantigens, unlike recall antigens, activate both CD45RA+ (naive) and CD45R0+ (memory) CD4+ cells to the same extent. These T cell subsets may therefore interact with each other in response to alloantigens on transplanted grafts. We have investigated if the ability of activated CD4+CD45RA+ and CD4+CD45R0+ T cells to produce and respond to interleukin 2 (IL2) and IL4 may be involved in this interaction. After activation, both subsets up-regulate their IL2 receptor (IL2R) and IL4R expression, yet IL4 substantially enhanced the proliferation of the CD4+CD45RA+ but not of the CD4+CD45R0+ T cell subset, while IL2 increased the proliferation of CD4+CD45R0+ but not of the CD4+CD45RA+ T cells. Significantly, the CD4+CD45RA+ T cells synthesized two- to threefold more mRNA for IL2 than the CD4+CD45R0+ subset, while the CD4+CD45R0+ T cells synthesized mRNA for IL4 and interferon-gamma exclusively. The addition of IL2 to alloactivated CD4+CD45R0+ T cells further up-regulated their production of all three lymphokine mRNA; in contrast, IL4 induced an increase in mRNA for IL2 in only the alloactivated CD4+CD45RA+ subset. The reciprocity in the ability of both these CD4+ T cells to synthesize and respond to IL2 and IL4 may provide a rationale for the regulation of lymphokine interactions in vivo. Furthermore, the synergy between these subsets in response to alloantigens, which was directly quantitated by co-culturing CD4+CD45RA+ and CD4+CD45R0+ cells together prior to activation, may potentiate the alloreactivity against transplanted grafts in vivo.

Inhibition of alloresponsive naive and memory T cells by CD7 and CD25 antibodies and by cyclosporine.

Human naive and memory T cells can be isolated from each other by their CD45RA and CD45RO expression, respectively. This enables the assessment of their differential sensitivities to immunosuppressive agents for the first time. We have investigated the ability of cyclosporine or CD7 and CD25 antibodies to selectively block alloantigen stimulated naive and memory T cells in vitro. CD7 antibodies blocked the proliferation of naive (P less than 0.025) but not memory T cells in a primary MLR. CD25 antibody inhibited both naive and memory subsets but a significantly greater effect was found on the memory T cells (P less than 0.005). The constitutive CD7 and CD25 antigen expression on resting naive or memory T cells was related to the inhibitory activities of these antibodies on both subsets. Accordingly, naive T cells expressed more CD7 antigen than memory cells while memory T cells displayed low levels of CD25 antigen that was absent from naive populations before activation. Cyclosporine, like CD25 antibody, inhibited both subsets in a primary MLR but had a greater effect on memory cells (P less than 0.02). Memory T cells, therefore, are more dependent than naive cells on IL-2 for proliferation. There was great individual variation in the ability of CsA to block the MLR. The simultaneous addition of CD25 or CD7 antibody together with CsA, however, enhanced the MLR inhibition as the effects of all three were additive. This suggested interference by these agents at different points during T cell activation. Thus, in CsA sensitive individuals, one-tenth of the optimal CsA concentration together with CD25 antibody maintained maximum immunosuppression in vitro. These results demonstrate the possibility of using CD7 and CD25 antibodies for selective inhibition of naive or memory T cells and also the possibility of augmenting the inhibition of and reducing the CsA concentration required for clinically effective immunosuppression.

JM, a Thy-ALL cell line produces both inhibitory and stimulatory lymphokines for bone marrow progenitor cells.

Normal and malignant T cells as well as T-cell hybridomas have frequently been reported to produce factors which stimulate the growth of committed hemopoietic progenitors. One previous report described a lymphokine produced by a T-cell clone which inhibited hemopoietic progenitor cell proliferation. We now describe the simultaneous production of two activities by a Thy-ALL cell line (JM), a sub-line of Jurkat. Two sets of culture conditions were used: the Fauser & Messner and Iscove's assays. We have been able to separate both inhibitory and stimulatory factors for the growth of multipotent and committed bone marrow progenitors (CFU-GEMM, BFU-E, CFU-E and CFU-GM). The stimulatory factor has an apparent mol. wt of less than 30,000 and the inhibitor an apparent mol. wt of 65-80,000. The growth promoting activity for BFU-E and CFU-GEMM could replace that of phytohemagglutinin stimulated leucocyte conditioned medium (PHA-LCM). We do not know if the production of both activities is due to the malignant phenotype or if there is a normal counterpart to JM that could produce both inhibitory and stimulatory factors.

Standardization of T-cell depletion in HLA matched bone marrow transplantation.

The IBM 2991 Blood Cell Processor has been used to isolate a mononuclear cell (MNC) fraction from the marrow of 31 allogeneic donors. The MNC fraction was then incubated with a combination of two murine monoclonal antibodies MBG6 (CD6) and RFT8 (CD8) followed by two rounds of treatment with rabbit complement resulting in a marrow inoculum significantly reduced in the number of T-lymphocytes. We report here new specifications for the use of Ficoll-Metrizoate, the method used to calculate T-lymphocyte depletion and the details of our attempts to improve T-depletion. Following marrow transplantation with this T-depleted fraction, 29 patients are evaluable for engraftment, one patient failed to engraft and one died too early for evaluation. Twenty-two had no acute graft-versus-host disease (aGvHD), at a minimum of 60 d, six had grade I acute GvHD and one grade III. No correlation was found between the absolute number of MNC infused and time to engraftment, nor any relationship between the number of residual viable T-lymphocytes in the infused marrow and the incidence of GvHD, but the patient with the most severe aGvHD also had the highest number of T-lymphocytes infused.

Graft rejection following HLA matched T-lymphocyte depleted bone marrow transplantation.

Bone marrow graft rejection following HLA-matched bone marrow transplantation (BMT) for leukaemia has been a rare problem. However, with the introduction of T-lymphocyte depleted BMT, graft rejection is recognized as a new complication. At the Royal Free Hospital (RFH) in London T-depletion is achieved using two monoclonal antibodies with complement mediated lysis. The methodology was extended to other centres and in total 56 patients have received T-depleted, HLA matched BMT. Twelve of 56 patients have had graft rejection. At the RFH three of 41 (7%) patients have had rejection whereas at collaborating centres nine of 15 (60%) patients have had rejection. We have investigated these rejections in order to identify factor(s) responsible. Rejection was not restricted by patient or donor characteristics, nor disease status. Patient management, chemotherapy conditioning, efficiency of T-depletion, graft versus host disease (GvHD), and infection post BMT, were not consistently implicated. The major difference between the RFH and all other centres was in the radiotherapy (RT) conditioning: The RFH prescribed a single fraction of 7.5 Gy total body irradiation (TBI) whilst collaborating centres gave 10 or 12 Gy fractionated TBI. We conclude that the different incidence of rejection (7% v. 60%) relates primarily to the RT conditioning although the mechanisms(s) of rejection remain unknown. We conclude that where T-depleted BMT is used, compensation by more intensive RT conditioning is required in order to avert graft rejection.