GVHD after unrelated cord blood transplant in children: characteristics, severity, risk factors and influence on outcome.

We reviewed our experience in 79 children who had unrelated cord blood transplant (UCBT) between 1996 and 2007 with a major focus on GVHD, comparing both traditional and National Institute of Health (NIH) criteria. The cumulative incidence (CI) of acute GVHD (aGVHD, by day +100) was 0.42 for grade II-IV and 0.22 for grade III-IV. The CI of all aGVHD (NIH, that is, no time limit) at 1 year was 0.45 for grade II-IV and 0.32 for grade III-IV. Infused CD34 cell dose (>1 × 10(5)/kg), pretransplant bacterial infection and nonmalignant disorders were risk factors for grade II-IV aGVHD on univariate analysis. Infused CD34 cell dose remained significant on multivariate analysis. At 1 year, the CI of chronic GVHD (cGVHD) using the Seattle criteria was 0.27, whereas that for cGVHD (NIH) was 0.08. By NIH criteria, the classic form of cGVHD was uncommon (5%) after UCBT. Instead, the acute (71%) and overlap (24%) GVHD variants predominated. Grade II-IV aGVHD was a significant risk factor for cGVHD by both Seattle and NIH criteria. We conclude that GVHD after day +100 after UCBT typically carries features of aGVHD. Moreover, and in marked contrast to adult unrelated donor hematopoietic stem cell transplantation, the GVHD observed in this series did not adversely affect survival.

Monosomy 7 associated with pediatric acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS): successful management by allogeneic hematopoietic stem cell transplant (HSCT).

Pediatric acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) with monosomy 7 is associated with poor disease-free survival when treated by conventional chemotherapy, immunosuppression or supportive measures. Hematopoietic stem cell transplant (HSCT) may improve outcomes; however, data to support this are limited. To better understand the curative potential of HSCT in these patients, all cases of AML and MDS with monosomy 7 treated by two transplant programs (1992 to present) were reviewed. A total of 16 patients were treated, all by allogeneic HSCT. Primary diagnoses were MDS (N = 5), therapy-related MDS (N = 3), AML (N = 5) and therapy-related AML (N = 3). In all, 11 patients (69%) survive event-free at 2 years with median follow-up of 986 days (range 330-2011 days). Toxicity caused deaths of the five nonsurviving patients, four of whom were transplanted with active leukemia. Allogeneic HSCT is effective therapy for childhood AML and MDS associated with monosomy 7, particularly for patients with AML in complete remission and MDS.

Successful cord blood transplantation for sickle cell anemia from a sibling who is human leukocyte antigen-identical: implications for comprehensive care.

We report the successful transplantation of umbilical cord blood stem cells from a sibling who is human leukocyte antigen-matched to a child with sickle cell anemia. Conditioning was with busulfan, cyclophosphamide, and antithymocyte globulin. Time to neutrophil count >500/microL was 23 days and to platelet count >50,000/microL was 49 days. Full donor engraftment was achieved without graft-versus-host disease. This case demonstrates the potential usefulness of harvesting cord blood from full siblings of patients with sickle cell disease. Routine collection of umbilical cord blood from siblings should be considered for patients with sickle cell disease, and may increase acceptance and use of transplantation by families.

Invasive Aspergillus sinusitis in pediatric bone marrow transplant patients. Evaluation and management.

OBJECTIVES: To evaluate the following: the incidence of invasive Aspergillus sinusitis (AS); the value of surveillance nasal cultures and screening radiologic studies in predicting AS; the clinical criteria used to decide on surgical biopsy in patients suspected of having AS; the surgical and medical management of AS; and the outcome of AS in the peritransplantation period of children who underwent bone marrow transplantation. DESIGN: Retrospective medical chart review. SETTING: Tertiary care children's hospital. PATIENTS: Eighty pediatric patients who underwent bone marrow transplantation for a variety of refractory malignant neoplasms or lymphohematopoietic disorders at the Children's National Medical Center, Washington, DC, from April 1, 1988, to September 30, 1993. INTERVENTION: Diagnostic surgical biopsies, surgical débridement, and treatment with amphotericin B. MAIN OUTCOME MEASURE: Resolution of AS and discharge from the hospital. RESULTS: Seventy-two patients had screening sinus radiographs, 27 of which showed abnormalities. Aspergillus sinusitis developed in three of the patients with abnormal screening radiographs. Fifty-eight patients had screening nasal cultures. One culture was positive for Aspergillus, and histopathologically proved AS developed in this patient. Twelve diagnostic biopsies were done in nine patients. Three biopsy specimens showed histopathologic evidence of AS. The three patients with AS were successfully treated with aggressive surgical and medical therapy and were discharged from the hospital. CONCLUSION: The incidence of AS was 4% (3/80) in the patients who underwent bone marrow transplantation. Screening radiographs, while not a good predictor of AS, have a role in evaluation of patients undergoing bone marrow transplantation to define preexisting sinus disease. Screening nasal cultures do not reliably predict AS. When AS is suspected and diagnostic biopsy is considered, the seven clinical criteria outlined in this article should be used. Survival of immunocompromised patients with AS requires early diagnosis and aggressive surgical and medical therapy.

Encephalopathy with parkinsonian features in children following bone marrow transplantations and high-dose amphotericin B.

Encephalopathy, leukoencephalopathy, and secondary parkinsonism occurred in 3 children with refractory leukemia undergoing allogenic bone marrow transplantation (BMT) who were treated with high-dose amphotericin B for pulmonary aspergillosis or sinus aspergillosis that did not involve the nervous system. Treatment included high-dose cytosine arabinoside, cyclophosphamide, and total body irradiation prior to the BMT. The children developed a progressively worsening encephalopathy and parkinsonian features, characterized by resting tremor, cogwheel rigidity, and masklike facies. Neuroimaging studies showed cerebellar, cerebral, and basal ganglia atrophy, as well as frontal and temporal lobe white matter involvement. Two of the 3 patients recovered, although 1 has residual intellectual impairment. The third succumbed to non-central nervous system Epstein-Barr virus-lymphoproliferative disease and had autopsy-confirmed leukoenephalopathy.

Effect of gamma irradiation of red blood cell units on T-cell inactivation as assessed by limiting dilution analysis: implications for preventing transfusion-associated graft-versus-host disease.

Transfusion-associated graft-versus-host disease can be prevented by treating cellular blood products with gamma irradiation. A wide range of gamma irradiation dose levels has been used in routine practice. We used limiting dilution analysis, which measures clonable T cells, to assess the influence of 500 to 3,000 cGy of gamma irradiation delivered from a 137Cs source on T cells when delivered in situ to ADSOL-preserved red blood cell (RBC) units in blood bags. In a series of experiments using RBC units irradiated within 24 hours after collection, 1,500 cGy inactivated > 4 log10 of T cells; however, viable T cells were detected in all experiments. With 2,000 cGy, a > or = 4.7 log10 decrement in T-cell growth occurred in 7 of 8 experiments. With 2,500 or 3,000 cGy, no T-cell growth (> 5 log10 depletion) was detected. Comparable effects were observed with ADSOL-preserved RBC units in the standard PL 146 plastic container and in the recently developed PL 2209 plastic container. T-cell inactivation, as a function of gamma irradiation dose, was similar when either a 137Cs or a linear accelerator source was used. T cells isolated from ADSOL-preserved RBC units after storage for 7 and 21 days, although reduced in number as compared with a fresh unit stored for 24 hours, were viable, capable of proliferation, and susceptible to inactivation by gamma irradiation. Using a sensitive in vitro assay for T-cell proliferation, we found that a gamma irradiation dose of 2,500 cGy may be required to completely inactivate T cells in RBC units.

Minimal allogeneic donor exposure with the use of dedicated donors and a sterile connecting device in a newborn undergoing bone marrow transplantation.

PURPOSE: Enhanced engraftment and reduced viral complications may be achieved in bone marrow transplantation (BMT) by limiting homologous transfusions. We report on limiting donor exposures before and after BMT in a newborn with severe combined immunodeficiency (SCID) using dedicated whole blood and plateletpheresis donors as well as a sterile connecting device (SCD). PATIENTS AND METHODS: A 1-day-old neonate was admitted for an allogeneic, human leukocyte antigen-disparate, T-cell-depleted BMT performed on day 43 of hospitalization. All transfused red blood cells (RBCs) and platelets were cytomegalovirus negative, and were irradiated and leukodepleted (via a Pall filter). Using the SCD, tubing above the filter was connected to the product bag, and the distal tubing was connected to a transfer pack for collection of the filtered product. Additional transfer packs were connected to the filtered product using the SCD to separate small aliquots as needed. RBC aliquots were irradiated individually before each transfusion. RESULTS: During a total of 134 days of hospitalization, only four donor exposures occurred. Eleven RBC transfusions (mean volume 46.4 +/- 12.6 ml) from three donors and five plateletpheresis transfusions (mean volume 74.2 +/- 7.5 ml) from one donor constituted all the patients' transfusion requirements. Evidence of engraftment was seen on day 18 post-BMT with an absolute neutrophil count sustained at > 500 cells/mm3. The last transfusion was received on day 35 post-BMT. CONCLUSIONS: Current blood transfusion technology enables patients undergoing bone marrow transplantation to have limited donor exposures. This practice should decrease viral complications without effecting bone marrow engraftment.

Abdominal complications after bone marrow transplantation in children: sonographic and CT findings.

Bone marrow transplantation is increasingly used in children to treat refractory malignant neoplasms, immunodeficiency syndromes, and hematopoietic and genetic disorders. In preparation for the transplantation, patients receive high doses of chemotherapeutic agents and total-body irradiation to destroy residual malignant cells or dysfunctional marrow and to prevent rejection of the graft. A variety of abdominal and pelvic complications may occur after transplantation because of pancytopenia, the direct toxic effects of the preparative regimen, graft-vs-host disease, or immunosuppression. This essay illustrates the CT and sonographic appearances of these complications.

Extended-cycle elutriation to adjust T-cell content in HLA-disparate bone marrow transplantation.

We report the development of a double-cycle elutriation (DCE) technique separating 3 or greater logs of T cells from a stem-cell-enriched marrow fraction and the results of phase I T-cell depletion studies with HLA-disparate related bone marrow transplantation (BMT) donors in two patient groups. In group 1, 10 patients with refractory hematopoietic malignancies received combination chemotherapy, total body irradiation (TBI), and immunosuppression (pre- and post-BMT), and hematopoietic rescue with a marrow transplant, depleted of T cells by elutriation. Potentially to promote engraftment and a graft-versus-leukemia (GVL) effect, 0.5 to 0.75 x 10(5) T cells/kg were added back. All 10 patients engrafted. Five patients developed acute graft-versus-host disease (GVHD; four grade II, one grade III) and two subsequently developed chronic GVHD. Two patients have relapsed (median follow-up, 206 days; range, 46 to 1,035). Four patients died of BMT-related complications (three of infection, one of veno-occlusive disease [VOD]). Four patient are disease-free survivors (median follow-up, 960 days; range, 670 to 1,035). Group 2 included five infants, four with congenital lymphohematopoietic deficiencies and one with refractory acute lymphocytic leukemia (ALL). In these infants, busulfan and increased cyclophosphamide were substituted for TBI. Only the ALL patient received added T cells. Three patients engrafted: one has stable mixed chimerism, one relapsed with ALL, and one rejected the marrow. One patient had primary autologous recovery, while another failed to engraft. None developed GVHD. We conclude that, in this setting of HLA-disparate BMT with post-BMT antithymocyte globulin (ATG) and corticosteroids, DCE significantly depletes T cells from the marrow and that a defined number of T cells can be added without the occurrence of severe GVHD.

Hematopoietic engraftment and graft failure after bone marrow transplantation.

PURPOSE: This article reviews the complex process of establishing functional long-term hematopoiesis required for successful clinical bone marrow transplantation. The failure to establish sustained hematopoiesis, either primary or secondary graft failure, is defined and multiple etiologic factors involved are discussed. DESIGN: Data from published studies of experimental and clinical BMT, as well as in vitro stem cell biology, were used to elucidate the elements required for establishing functional hematopoiesis. These include pleuripotential hematopoietic stem cells; homing of stem cell to the hematopoietic micro-environment; hematopoietic stroma and secreted cytokines acting synergistically to promote expansion and hematopoietic differentiation of the stem cells; and, in the setting of allogeneic transplantation, immunological tolerance between the host and the graft, without which graft rejection or graft-vs.-host disease may occur. CONCLUSIONS: The factors influencing the establishment of functional hematopoiesis and the risks for graft failure vary with the type of transplant performed. In an autologous transplant, damage to the stem cells or to the stromal microenvironment can contribute to prolonged time to engraftment or primary graft failure. In contrast, the hematopoietic stem cells are normal in both syngeneic and allogeneic transplantation. However, in the latter, because of immunological disparity between the host and recipient, there can be either primary or secondary graft failure due to a host-vs.-graft phenomenon, graft rejection. Classic graft rejection is an immunologic phenomenon mediated by residual host immunocompetent cells, either T cells or NK cells, depending on the allogeneic disparity between host and donor. T cell depletion and increased HLA disparity are risk factors for rejection. Numerous strategies have attempted to decrease the risk of rejection; most have focused primarily on increased immunosuppression of the host either with additional radiation, chemotherapy or in vivo anti-T cell serotherapy. Recent attempts have explored preventing rejection by manipulating donor cell composition of the infused graft.

Efficacy of T cell depletion of surgically resected cadaveric marrows by monoclonal antibodies: considerations in HLA-mismatched marrow transplantation.

The potential therapeutic benefits of allogeneic bone marrow transplantation (BMT) remain generally unavailable to patients who have diseases amenable to treatment by allogeneic BMT, but who lack an identifiable HLA-matched marrow donor. If graft versus host disease and graft rejection can be controlled, then the possibility of expanding allogeneic BMT to minimally HLA-matched or fully mismatched combinations exists and a "universal" donor marrow bank might be established. Towards this end, we have evaluated surgical harvest of cadaveric marrow, T cell depletion of such marrow (for prevention of graft-versus-host disease), influence of surgical harvest on final T cell content, and final cell yield. Marrow harvest was coordinated with the donation of other tissues or organs of cadaveric origin. In a series of twenty-two surgical vertebral body harvests, the initial marrow yield per vertebral body was 4.5 x 10(9) with four to six vertebral bodies per harvest. T cell depletion was evaluated by a limiting dilution assay. Since a combination of multiple monoclonal antibodies with specificity for T cell surface molecules provided greater reproducibility in the depletion of T cells from marrow than the utilization of any single antibody, a pool of monoclonal antibodies and complement were used to treat the marrow. The final cell yield per vertebral body was 2.1 x 10(9) for an average total yield of 9.3 x 10(9) cells per harvest. T cell content for each marrow following T cell depletion was less than 0.001%. These marrow have been cryopreserved as a bank of characterized donor marrow for use in HLA minimally matched or unmatched marrow transplantation.

Target cell lysis by cytotoxic T lymphocytes redirected by antibody-coated polystyrene beads.

Cytotoxic T lymphocytes were found to mediate rapid lysis of target cells not normally recognized in the presence of small polystyrene beads coated with a combination of anti-T3 and antitarget cell antibodies. Lysis was not seen with beads bearing one of these antibodies alone, nor with a mixture of two types of beads each coated with a single antibody. The effector cells mediating this lysis include long term allospecific human CTL, and both human and mouse CTL clones recognizing mouse class I MHC Kb Ag. TNP-modified mouse tumor cells, a human lymphoblastoid line, and human red cells were found to be good targets for this cytotoxicity. Polystyrene beads with diameters of 3 to 15 mu caused target lysis, with a dose-response curve which typically went through a maximum and declined at high bead numbers. Maximal bead-redirected lysis by CTL was less efficient than that mediated by soluble antibody heteroconjugates of the same two antibodies. Bead-redirected target lysis was calcium dependent. These results are interpreted as a form of bystander lysis induced by the beads, since the target cell membrane is not directly crosslinked to the region of CTL activation. These observations thus favor a mechanism of lysis involving the polarized secretion of a locally acting lytic agent by CTL.

Inhibition of cytotoxic T cell lysis by anti-CD8 monoclonal antibodies: studies with CD3-targeted cytolysis of nominal antigen-negative targets.

The function of the CD8 molecule in lympholysis mediated by cytotoxic T cells was investigated by examining possible contributions of ligands on the target cell to the inhibition of lysis observed with CD8-specific mAb. In order to evaluate a variety of target cells, including those not expressing the nominal Ag (NA) for which the CTL was specific, lysis was effected by cross-linking the CTL and the target cells with anti-CD3 mAb. Such CD3 redirected cytotoxicity was demonstrated to be inhibited by anti-CD8 mAb when low anti-CD3 mAb concentrations were used. The possibility that inhibition by anti-CD8 mAb resulted for competition for the FcR between the anti-CD3 mAb and anti-CD8 mAb was eliminated by targeting TNP-modified cells with an antibody heteroconjugate prepared from Fab fragments of anti-CD3 and anti-DNP antibodies. Inhibition of the lysis of target cells not expressing NA including those deficient in class I expression, demonstrated that neither NA nor class I expression was required for anti-CD8 mAb inhibition. Whether the anti-CD8 mAb inhibition required CD8 Ag interaction with any ligand on the target cell was further investigated by measuring exocytosis of enzyme granule from CTL activated with CD3-coated poly-styrene beads. CD8-specific mAb inhibited such CTL activation in this target cell-free system. A CD8(+), MHC class II-specific CTL clone, was used to show differential inhibition by anti-CD8 mAb, depending on the target cell, therefore providing evidence that anti-CD8 mAb binding does not generate an absolute off signal. These data are consistent with the hypothesis that anti-CD8 mAb affect the lytic process independent of the recognition of a ligand on the target cell by CD8.

An anti-human-T-cell monoclonal antibody with specificity for a novel determinant.

A new antihuman T cell monoclonal antibody, MAb 22, recognizes an antigen that is present on all mature T cells, but detected on only a subset of thymocytes. Dual color flow microfluorimetry (FMF) demonstrates that MAb 22 staining has concordant distribution with pan-T MAb specific for CD3 and CD5 and includes the CD4 and CD8 subsets; other FMF studies confirm T cell specificity of MAb 22 expression by cells of hematopoietic origin. The antigen recognized by MAb 22 is expressed on only a subset of thymocytes and by dual FMF is expressed at a mature thymocyte stage. Immunoprecipitation by MAb 22 demonstrates a series of molecules with a predominant 45 KD protein and associated 12 and 95 KD proteins under reducing conditions, while a 92 KD protein predominates under nonreducing conditions. Comparisons of expression on a variety of T cell lines, including a mutant line defective in the expression of the T cell receptor, by FMF analysis further distinguishes the determinant recognized by MAb 22 from those detected by MAb of defined T cell specificity.

Alternative donor sources in HLA-mismatched marrow transplantation: T cell depletion of surgically resected cadaveric marrow.

Allogeneic bone marrow transplantation has been largely confined in its application to patients with an HLA-matched marrow donor, thereby limiting potential therapeutic benefits for patients with diseases amenable to treatment by marrow transplantation who lack effective alternative therapies and an identified matched marrow donor. T cells in the donor marrow generate graft-versus-host disease which is a major consideration in attempts to widen the application of allogeneic marrow transplantation to the minimally HLA-matched or mismatched setting. The method of marrow harvest influences both the number of such T cells in the donor marrow inoculum and the functional capacity of T cells in murine marrow. We have therefore evaluated surgically resected cadaveric marrow for T cell content and for T cell depletion. Surgically resected marrow is more easily depleted of T cells than aspirated marrow and clinically useful quantities of marrow have been obtained and cryopreserved in a bank of characterized donor marrow for future use in HLA minimally matched or unmatched marrow transplantation.

Measurement of cytotoxicity by target cell release and retention of the fluorescent dye bis-carboxyethyl-carboxyfluorescein (BCECF).

In order to utilize a newly available scanning microfluorimeter for lymphocyte-mediated cytotoxicity assays, a number of commercially available fluorescent dyes were compared for their suitability as target cell markers. One of them, bis-carboxyethyl-carboxyfluorescein (BCEFCF), was useful for assays with about 10(4) target cells and showed substantially less spontaneous leakage than other fluorescein derivatives, while still leaking more rapidly than 51Cr. For short cytotoxicity incubations (less than 2 h) with cytotoxic T lymphocytes (CTL), the corrected percentage BCECF release into the supernatant parallels that of 51Cr. For 4 h assays cytotoxicity could be quantitated by measuring the BCECF retained by target cells. Using human CTL and natural killer (NK) cells as effectors, with a variety of lymphoid cells and fibroblasts as targets in 4 h assays, the BCECF retention technique was found to give cytotoxicity values comparable to the 51Cr release assay. Cytotoxicity assays measuring BCECF fluorescence in microtiter wells with the scanning microfluorimeter offer advantages of safety, economy, and processing time compared with the 51Cr release assay.

Anti-T3 antibody both activates and inhibits the cytotoxic activity of human T cell clones.

The present report outlines results using several approaches to investigate the effects of antibodies against cell surface molecules on human cytotoxic T lymphocyte (CTL) recognition. First, when interactions between CTL and targets are perturbed by antibody immobilized on the surface of the incubation vessel, anti-T3 antibody was unique in its ability to inhibit at least 10 times more efficiently than when present in solution. Second, anti-T3 antibody at sufficiently high concentrations inhibits lectin-dependent cellular cytotoxicity as well as allospecific cytotoxicity. Third, murine hybridomas expressing anti-T3 antibody induce their own lysis by human CTL clones; but hybridomas bearing antibodies of other specificities are not lysed in a similar manner. We have exploited this anti-T3 triggered lysis of the OKT3 hybridoma as a model system in which to study T cell recognition. Such lysis is susceptible to inhibition not only by anti-T3 but also by anti-LFA-1 antibody; however, conjugate formation between the anti-T3 hybridoma and CTL is not easily inhibited by anti-LFA-1. These data, which are discussed in the context of results from other laboratories, are consistent with a model in which CTL triggering can be induced by interaction of the antigen specific receptor (Ti) with its physiologic ligand, antigen plus MHC, or interaction of the T3 complex with a surrogate ligand, anti-T3/anti-Ti. However, they raise questions regarding the role which LFA-1 plays in CTL recognition.