Study of thymic size and function in children and adolescents with treatment refractory systemic sclerosis eligible for immunoablative therapy.

Following hematopoietic stem cell transplantation (HSCT), thymic reconstitution of peripheral T lymphocytes is essential to avoid a chronically immunodeficient state and disease recurrence. The purpose of this study was to determine if children and adolescents with treatment refractory SSc, awaiting HSCT, have sufficient thymic function to reconstitute T lymphocyte function after transplantation. Thirteen children with systemic scleroderma were enrolled and assessed by physical exam, chest MRI, measurement of autoantibodies, B and T cell immuno-phenotyping, and quantization of T cell receptor rearrangement excision circles (TREC) as a marker of thymopoiesis. MRI detected thymic tissue in 9/13 children. TREC levels were detectable in all but one child but were significantly reduced (p<0.001) when compared to a control population. SSc patients also had a reduced percentage of naïve (CD45RA+CD31+) CD4+ T lymphocytes, further indicating diminished thymopoiesis. Our data suggest that thymic function in children with SSc might be insufficient for an adequate immunoreconstitution following transplantation in some patients. A thorough evaluation of immune and thymic functions to identify those patients prior to HSCT is recommended.

A summary of boron surface water quality data throughout the European Union.

The distribution of boron in rivers across European Union countries is described. The data have been collected from national and international monitoring programmes. The data show a wide range of concentrations but only in a few instances do concentrations exceed environmental quality standards. Although there is a good body of data for the analysis presented here, it is clear that there is not a full coverage of environments and for some studies there is a clear bias towards sampling of the main rivers or specific rivers where there are environmental concerns. The variations in concentration link directly to pollutant sources although assessment of the details of the spatial variations is clouded by factors such as the location of monitoring points in relation to pollutant discharges, the variation in dilution potential of the various rivers and contrasting hydrological and geological environments. The data are presented to complement more detailed studies on particular river basins as a basis for the further development of regional environmental impact water quality modelling frameworks and within the newly developing field of typological settings.

Detection of leukemic cells in the CD34(+)CD38(-) bone marrow progenitor population in children with acute lymphoblastic leukemia.

Successful autologous hematopoietic stem cell (HSC) transplantation in childhood acute lymphoblastic leukemia (ALL) requires the ability to either selectively kill the leukemia cells or separate normal from leukemic HSC. Based on previous studies showing that more than 95% of childhood B-lineage ALL express CD38, this study evaluated whether normal CD34(+)CD38(-) progenitors from children with B-lineage ALL could be isolated by flow cytometry. CD34(+) cells from bone marrow samples from 10 children with B-lineage ALL were isolated at day 28 of treatment, when clinical remission had been attained. The CD34(+) progenitor cells were flow cytometrically sorted into CD34(+)CD38(+) and CD34(+)CD38(-) populations. The absolute numbers of CD34(+)CD38(-) cells that could be isolated ranged from 401 to 6245. The cells were then analyzed for the presence of clonotypic rearrangements of the T-cell receptor (TCR) Vdelta2-Ddelta3 locus. Only patients whose diagnostic marrow had an informative TCR Vdelta2-Ddelta3 rearrangement were included in this study. Detection thresholds were typically 10(-4) to 10(-5) leukemic cells in normal marrow. In 6 of 10 samples analyzed, the sorted CD34(+)CD38(-) cells had no detectable Vdelta2-Ddelta3 rearrangements. In 4 cases, the clonotypic leukemic Vdelta2-Ddelta3 rearrangement was detected in the CD34(+)CD38(-) population, indicating that the putative normal HSC population also contained leukemic cells. The data indicate that although most childhood ALL cells express CD34 and CD38, leukemic cells are also frequently present in the CD34(+)CD38(-) population. Therefore, strategies to isolate and transplant normal HSC from children with ALL will require a more stringent definition of the normal HSC than the CD34(+)CD38(-) phenotype. (Blood. 2001;97:3925-3930)

Hematopoietic stem cell transplantation for severe combined immune deficiency.

Hematopoietic stem cell transplantation (HSCT) has been the definitive therapy for severe combined immune deficiency (SCID) since the first successful transplant for SCID in 1968. Improvements in the use of HSCT to treat patients with SCID are continuing. For example, during the last 5 years, the first successful in-utero HSCT, and the first success with gene therapy have occurred in patients with SCID. Debate still continues about the role of pretransplantation therapy for SCID patients, and the biology of post-HSCT immune reconstitution is under investigation.

T cell depleted haploidentical bone marrow transplantation for the treatment of children with severe combined immunodeficiency.

Severe combined immunodeficiency (SCID) is fatal in early childhood if unrecognized and if not treated. The aim was to determine the efficacy of T cell depleted bone marrow transplantation (TCD BMT) in the treatment of children with SCID. Eleven children diagnosed with SCID received histocompatible related donor bone marrow transplantation--HRD BMT (group I). Thirty seven children diagnosed with SCID who did not have histocompatible donors were treated with TCD haploidentical parental bone marrow transplantation (BMT) (group II). TCD was performed by in vitro soybean lectin agglutination followed by E-rosette depletion. Patients were longitudinally assessed for the presence and function of T and B lymphocytes. In group I all children survived. The mean age of children in this group at the time of HRD BMT was 15.4 months. All surviving patients normalized their specific T cell function. Two out of 11 require treatment with intravenous immunoglobulin i.v. Ig. In group II 17 out of 37 (46%) children survived. At the time of TCD BMT the mean age of survivors was 7.5 months, vs. 11.4 months in patients who died. Death was caused most commonly by opportunistic infections, Epstein-Barr virus induced lymphoproliferative disease (EBV-LPD), and graft versus host disease (GvHD). Seventeen out of 17 surviving patients recovered normal numbers of CD3+ cells and antigen specific T cell function. Five out of 17 never recovered their B cell function and require i.v. Ig injections. Early diagnosis, prevention or treatment of opportunistic infections, and enhancement of immune recovery will be necessary to improve survival in patients with SCID treated with TCD BMT.

Gene therapy for adenosine deaminase deficiency.

The clinical gene therapy trials for adenosine deaminase (ADA) deficiency have defined both the potential benefits and the present limitations of gene therapy with hematopoietic stem cells (HSC). Current clinical results indicate that (a) both umbilical cord blood and neonatal bone marrow HSC can be transduced with murine retroviral-based vectors, (b) the transduced HSC can engraft in nonmyeloablated patients, (c) the frequency of HSC transduction/engraftment is low (1/10,000), (d) an in vivo selective advantage can exist for transduced T lymphoid progeny, and (e) the transduced ADA gene is not expressed in nondividing T lymphocytes. Improving the clinical results of gene therapy for ADA deficiency and other genetic diseases involving HSC will require (a) developing new vectors that express the transduced gene in nondividing cells and (b) increasing the frequency of stable HSC transduction.

Spontaneous apoptosis in lymphocytes from patients with Wiskott-Aldrich syndrome: correlation of accelerated cell death and attenuated bcl-2 expression.

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, eczema, and a progressive deterioration of immune function. WAS is caused by mutations in an intracellular protein, WASP, that is involved in signal transduction and regulation of actin cytoskeleton rearrangement. Because immune dysfunction in WAS may be due to an accelerated destruction of lymphocytes, we examined the susceptibility to apoptosis of resting primary lymphocytes isolated from WAS patients in the absence of exogenous apoptogenic stimulation. We found that unstimulated WAS lymphocytes underwent spontaneous apoptosis at a greater frequency than unstimulated normal lymphocytes. Coincident with increased apoptotic susceptibility, WAS lymphocytes had markedly attenuated Bcl-2 expression, whereas Bax expression did not differ. A negative correlation between the frequency of spontaneous apoptosis and the level of Bcl-2 expression was demonstrated. These data indicate that accelerated lymphocyte destruction by spontaneous induction of apoptosis may be one pathogenic mechanism by which the progressive immunodeficiency in WAS patients develops.

Serum levels of IL-7 in bone marrow transplant recipients: relationship to clinical characteristics and lymphocyte count.

IL-7 is produced by stromal cells and is the major lympho- and thymopoietic cytokine. IL-7 induces proliferation and differentiation of immature thymocytes, and protects thymocytes from apoptosis by induction of bcl-2 expression. The regulation of IL-7 production is poorly characterized, although down-regulation by transforming growth factor-beta (TGF-beta) has been described. We measured the serum levels of IL-7 before and after bone marrow transplant (BMT) in 32 children undergoing BMT for genetic diseases (severe combined immune deficiency (SCID) and thalassemia), aplastic anemia, and acute lymphoblastic and non-lymphoblastic leukemia (ALL and ANLL). Prior to BMT, the highest IL-7 levels were observed in patients with SCID and ALL, i.e. those patients with genetic or acquired lymphopenia. Patients with thalassemia and ANLL had normal levels of IL-7. Over the 8 weeks following BMT, the IL-7 levels of patients with SCID and ALL fell as the absolute lymphocyte count (ALC) increased. No detectable change in IL-7 levels was observed in the patients with thalassemia and ANLL. Levels of IL-7 were highest in the young infants with SCID compared to the age-matched controls. Together, the data demonstrate that serum levels of IL-7 in lymphopenic patients are inversely related to patient age and the absolute lymphocyte count (ALC). The inverse relationship to ALC suggests that there is either direct regulation of stromal production or more likely, binding of secreted IL-7 to lymphocytes expressing IL-7 receptors.

Defective anticarbohydrate antibody responses to naturally occurring bacteria following bone marrow transplantation.

The long-term recipients of allogeneic bone marrow transplantation (BMT) are at an increased risk of death due to bacterial infections. We evaluated the anticarbohydrate antibody responses of BMT recipients to a naturally occurring bacterial carbohydrate, polyribose phosphate (PRP). The recipients of autologous BMT achieved protective anti-PRP levels (>100 ng/mL) by 3 years after transplantation, with a pattern consistent with a recapitulation of the ontogeny of anticarbohydrate antibody responses. None of the six recipients of unrelated BMT who were off immunosuppressive therapy had protective anti-PRP levels, though their response to a protein antigen (tetanus toxoid) was normal. Of 48 recipients of histocompatible BMT, 22 (46%) had protective anti-PRP antibody levels, whereas 13 (27%) recipients who were >3 years post-BMT did not have protective levels. Therefore, all unrelated recipients and a significant proportion of histocompatible recipients without clinical graft-vs.-host disease had persistent and prolonged defects in their capacity to produce antibodies to naturally occurring bacterial carbohydrate antigens. These results suggest that allogeneic BMT recipients should be longitudinally evaluated for their anticarbohydrate antibody responses and that patients with defective antibody responses should receive prophylactic antibiotics or replacement immunoglobulin therapy or both to reduce their risk of late bacterial infections.

Hematopoietic stem cell transplantation for primary lymphoid immunodeficiencies.

Hematopoietic stem cell (HSC) transplantation is curative therapy for many primary immunodeficiencies. All forms of severe combined immune deficiency (SCID) can be cured, but the extent of the immunologic correction is dependent on the pathophysiology of the primary defect. Defects involving lymphocyte differentiation are more easily corrected than defects in lymphocyte function because a selective advantage exists for the progeny of the normal donor HSC when the primary defect affects lymphocyte differentiation. T-cell-depleted (TCD), haploidentical-HSC transplantation can cure many forms of SCID, but not other primary immunodeficiencies like the Wiskott-Aldrich syndrome (WAS). Unrelated bone marrow and umbilical cord blood are alternative sources of HSC for patients who do not have histocompatible donors.

T lymphocytes with a normal ADA gene accumulate after transplantation of transduced autologous umbilical cord blood CD34+ cells in ADA-deficient SCID neonates.

Adenosine deaminase-deficient severe combined immunodeficiency was the first disease investigated for gene therapy because of a postulated production or survival advantage for gene-corrected T lymphocytes, which may overcome inefficient gene transfer. Four years after three newborns with this disease were given infusions of transduced autologous umbilical cord blood CD34+ cells, the frequency of gene-containing T lymphocytes has risen to 1-10%, whereas the frequencies of other hematopoietic and lymphoid cells containing the gene remain at 0.01-0.1%. Cessation of polyethylene glycol-conjugated adenosine deaminase enzyme replacement in one subject led to a decline in immune function, despite the persistence of gene-containing T lymphocytes. Thus, despite the long-term engraftment of transduced stem cells and selective accumulation of gene-containing T lymphocytes, improved gene transfer and expression will be needed to attain a therapeutic effect.

Neonatal thymectomy: does it affect immune function?

OBJECTIVE: The purpose of this study was to determine whether thymectomy in the newborn has a negative effect on immune function. METHODS: Twenty-five neonates (<30 days) who had thymectomy at congenital heart repair were prospectively studied to determine immune function. The percentage of T-cell subtypes including CD3 (all T cells), CD4 (helper T cells), and CD8 (suppressor T cells) was determined. In six patients, further testing of CD4 cells was done to determine whether they were newly formed, recent thymic emigrants (CD4, CD45, and RA+), or older educated lymphocytes (CD4, CD45, and RO+). Response to the mitogen phytohemagglutinin and to tetanus toxoid were determined, as were antibody titers to tetanus. Samples were drawn before the thymectomy, at approximately 3 months after immunization and at 1 year. Ten age-matched control patients were tested. At follow-up, parents were asked about infections. RESULTS: Prethymectomy T-cell subsets were all normal and comparable to controls. At 12 months, the percent of CD3 was significantly less than in the control group (48% +/- 3% versus 64% +/- 2% [mean +/- standard error of the mean]; p < 0.01) as was CD4 (31% +/- 2% versus 46% +/- 2% [mean +/- standard error of the mean]; p = < 0.01). CD8 did not drop. Surprisingly, the percent of CD4 that were recent thymic emigrants did not decrease significantly (50% +/- 8% versus 60% +/- 6% [mean +/- standard error of the mean]; p = not significant). Lymphocyte blastogenesis to phytohemagglutinin and tetanus toxoid and antibody to tetanus were all normal at 12 months. No patient required readmission for infection, and there were the expected number of minor infectious events (median 3; 95% confidence interval 1,4). CONCLUSION: Thymectomy in neonates results in a modest but significant decrease in T-lymphocyte levels, but there is no compromise in immune function.

Chronic graft-versus-host disease.

Chronic graft-versus-host disease (GVHD) continues to be the major problem in the long-term survivors of allogeneic hematopoietic stem cell transplants. Because of its similarities to autoimmune disease, the pathogenesis of chronic GVHD has been thought to differ from acute GVHD. Autoreactive T lymphocytes are an important effector mechanism with interferon gamma playing a central role in the increased collagen deposition that is the central histopathologic feature of chronic GVHD. Therapies that prevent the development of acute GVHD have been unsuccessful in the prevention of chronic GVHD. None of the present therapies for established chronic GVHD are successful in the majority of patients.

Gene therapy for newborns.

Application of gene therapy to treat genetic and infectious diseases may have several advantages if performed in newborns. Because of the minimal adverse effect of the underlying disease on cells of the newborn, the relatively small size of infants, and the large amount of future growth, gene therapy may be more successful in newborns than in older children or adults. The presence of umbilical cord blood from newborns provides a unique and susceptible target for the genetic modification of hematopoietic stem cells. In our first trial of gene therapy in newborns, we inserted a normal adenosine deaminase gene into umbilical cord blood cells of three neonates with a congenital immune deficiency. The trial demonstrated the successful transduction and engraftment of stem cells, which continue to contribute to leukocyte production more than 3 years later. A similar approach may be taken to insert genes that inhibit replication of HIV-1 into umbilical cord blood cells of HIV-1-infected neonates. Many other metabolic and infectious disorders could be treated by gene therapy during the neonatal period if prenatal diagnoses are made and the appropriate technical and regulatory requirements have been met.

Immunological reconstitution following bone marrow transplantation.

The recipients of hematopoietic stem cell transplants are characterized by an immunodeficiency of varying severity and duration. Their immunoincompetence is due in part to: 1) a lack of sustained transfer of donor immunity, 2) a recapitulation of lymphoid ontogeny, 3) the effects of graft-versus-host disease and its therapy, and 4) a reduction in thymic function. Recipients can have delays in the production of naive T lymphocytes following transplantation which result in defects in the production of new antigen-specific T lymphocytes and an inability to produce antibodies, especially to carbohydrate antigens.

The effect of thymic function on immunocompetence following bone marrow transplantation.

We investigated the role of thymic function in the immunodeficiency that characterizes bone marrow transplantation (BMT) recipients. The capacity of histocompatible BMT recipients to generate new CD4+ T lymphocytes was determined by FACS analysis with antibodies to the two isoforms of CD45: CD45RA, which is expressed on newly generated CD4 T lymphocytes, and CD45RO, which is expressed on antigen-specific memory CD4 T lymphocytes. Immediately following BMT, all patients had low levels of CD45RA-expressing CD4 T lymphocytes, which increased during the first year and then stabilized. Since thymic function decreases with age in normal individuals, the impact of recipient age on the generation of new CD45RA,CD4 T lymphocytes was determined in BMT recipients with and without chronic graft-vs.-host disease (GVHD). In addition, antigen-specific immune function was determined by assessing in vitro blastogenic response to tetanus toxoid (TT). More than 1 year after transplantation, the results from BMT recipients without chronic GVHD were similar to those of normal individuals; there was an age-dependent decline in the number of CD45RA,CD4 T lymphocytes, and antigen-specific immune function was age-independent. On the other hand, recipients with chronic GVHD had an age-dependent decline in their immune function (r = 0.45 and p = 0.02) that correlated with the number of new CD45RA,CD4 T lymphocytes (p = 0.027) but not the number of memory CD45RO,CD4 T lymphocytes (p = 0.11). Thus recipients with chronic GVHD have decreased antigen-specific immune function that may be due to an acceleration of the normal thymic aging process induced by GVHD and its therapy.