Brian M. Freed

Unrelated Donor Cord Blood Transplantation for Children with Severe Sickle Cell Disease: Results of One Cohort from the Phase II Study from the Blood and Marrow Transplant Clinical Trials Network (BMT CTN)

The Sickle Cell Unrelated Donor Transplant Trial (SCURT trial) of the Blood and Marrow Transplant Clinical Trials Network (BMT CTN) is a phase II study of the toxicity and efficacy of unrelated donor hematopoietic cell transplantation in children with severe sickle cell disease (SCD) using a reduced-intensity conditioning regimen. Here we report the results for the cord blood cohort of this trial. Eight children with severe SCD underwent unrelated donor cord blood transplantation (CBT) following alemtuzumab, fludarabine, and melphalan. Cyclosporine or tacrolimus and mycophenolate mofetil were administered for graft-versus-host disease (GVHD) prophylaxis. Donor/recipient HLA match status was 6 of 6 (n = 1) or 5 of 6 (n = 7), based on low/intermediate-resolution molecular typing at HLA -A, -B, and high-resolution typing at -DRB1. Median recipient age was 13.7 years (range: 7.4-16.2 years), and median weight was 35.0 kg (range: 25.2-90.2 kg). The median pre-cryopreservation total nucleated cell dose was 6.4 × 10(7) /kg (range: 3.1-7.6), and the median postthaw infused CD34 cell dose was 1.5 × 10(5) /kg (range: 0.2-2.3). All patients achieved neutrophil recovery (absolute neutrophil count >500/mm(3)) by day 33 (median: 22 days). Three patients who engrafted had 100% donor cells by day 100, which was sustained, and 5 patients had autologous hematopoietic recovery. Six of 8 patients had a platelet recovery to >50,000/mm(3) by day 100. Two patients developed grade II acute GVHD. Of these, 1 developed extensive chronic GVHD and died of respiratory failure 14 months posttransplantation. With a median follow-up of 1.8 years (range: 1-2.6), 7 patients are alive with a 1-year survival of 100%, and 3 of 8 are alive without graft failure or disease recurrence. Based upon the high incidence of graft rejection after unrelated donor CBT, enrollment onto the cord blood arm of the SCURT trial was suspended. However, because this reduced-intensity regimen has demonstrated a favorable safety profile, this trial remains open to enrollment for unrelated marrow donor transplants. Novel approaches aimed at improving engraftment will be needed before unrelated CBT can be widely adopted for transplanting patients with severe SCD.

Acrolein inhibits cytokine gene expression by alkylating cysteine and arginine residues in the NF-κB1 DNA-binding domain

Cigarette smoke is a potent inhibitor of pulmonary T cell responses, resulting in decreased immune surveillance and an increased incidence of respiratory tract infections. The α,β-unsaturated aldehydes in cigarette smoke (acrolein and crotonaldehyde) inhibited production of interleukin-2 (IL-2), IL-10, granulocyte-macrophage colony-stimulating factor, interfer-on-γ, and tumor necrosis factor-α by human T cells but did not inhibit production of IL-8. The saturated aldehydes (acetaldehyde, propionaldehyde, and butyraldehyde) in cigarette smoke were inactive. Acrolein inhibited induction of NF-κB DNA binding activity after mitogenic stimulation of T cells but had no effect on induction of NFAT or AP-1. Acrolein inhibited NF-κB1 (p50) binding to the IL-2 promoter in a chromatin immunoprecipitation assay by >99%. Using purified recombinant p50 in an electrophoretic mobility shift assay, we demonstrated that acrolein was 2000-fold more potent than crotonaldehyde in blocking DNA binding to an NF-κB consensus sequence. Matrix-assisted laser desorption/ionization time-of-flight and tandem mass spectrometry demonstrated that acrolein alkylated two amino acids (Cys-61 and Arg-307) in the DNA binding domain. Crotonaldehyde reacted with Cys-61, but not Arg-307, whereas the saturated aldehydes in cigarette smoke did not react with p50. These experiments demonstrate that aldehydes in cigarette smoke can regulate gene expression by direct modification of a transcription factor.

In vitro immunosuppressive properties of cyclosporine metabolites

The in vitro biological activity of cyclosporine (CsA) and four of its metabolites (M1, M8, M17, and M21) was determined. M1, M17, and M21 are primary metabolites, while M8 is a secondary metabolite derived from either M1 or M17. The order of inhibitory activity in production assays was phytohemagglutinin (PHA), con-canavalin A (ConA), mixed lymphocyte culture (MLC), and interleukin-2 (IL-2) CsA > M17 > M1 > M21 “ M8. In the PHA assay, CsA was significantly more inhibitory than M17, but in Con A and MLC assays, the inhibitory activity of M17 approached that of CsA. More importantly, M17 and Ml inhibited the production of IL-2 in the MLC to the same extent as CsA. M21 was significantly less inhibitory than either M17 or M1, and M8 appeared to be largely devoid of biological activity. These experiments demonstrate that single hydroxylations of amino acids 1 (M17) and 9 (M1) do not significantly affect the ability of the molecule to block IL-2 production, but hydroxylation of both amino acids renders the molecule virtually inactive. In addition, the presence of the N-methyl group on amino acid 4 appears to be very important, since removal of this group (M21) greatly diminishes the immunosuppressive activity.

Immunosuppressive metabolites of cyclosporine in the blood of renal allograft recipients.

Cyclosporine levels by radioimmunoassay (RIA) and high-performance liquid chromatography (HPLC) were monitored in serial blood samples (n = 177) from 11 renal allograft recipients. HPLC analysis revealed three primary metabolites of CsA (M17, M1, and M21) in peak and trough blood samples; M17 was the preponderant metabolite. In 4 patients on whom serial metabolite assays were performed, M17 was found in the blood at 86-2004 ng/ml; M1 and M21 were found at up to 100 ng/ml. The immunosuppressive properties of purified metabolites M1, M17, M21, and M8 (which was not detected in the blood) were compared with CsA. M17--and, to a lesser extent, M1 and M21--were found to inhibit the in vitro response of human mononuclear cells in the mixed leukocyte culture and in mitogen (phytohemagglutinin [PHA], concanavalin A [Con A], and pokeweed mitogen [PWM]) assays at 1000 ng/ml. M8 exhibited no in vitro inhibitory activity. M17 was further tested at 10-1000 ng/ml in PHA and mixed lymphocyte culture (MLC) assays. M17 had considerably less inhibitory activity (12-43%) than CsA (18-70%) in the PHA assay. However, in MLC experiments M17 blocked the proliferative response by 39-72% at 100-800 ng/ml, which approached the degree of inhibition exhibited by CsA (63-87%). In 34 of 37 (92%) patient blood samples, the level of metabolite M17 was found to exceed the parent drug level and could not be measured accurately by RIA. The observed in vitro immunosuppressive activity of metabolites (particularly M17) and their presence in the blood of renal allograft recipients suggest a possible role for these metabolites in the immunopharmacology of CsA.

Studies of Lymphocyte Reconstitution in a Humanized Mouse Model Reveal a Requirement of T Cells for Human B Cell Maturation

The hematopoietic humanized mouse (hu-mouse) model is a powerful resource to study and manipulate the human immune system. However, a major and recurrent issue with this model has been the poor maturation of B cells that fail to progress beyond the transitional B cell stage. Of interest, a similar problem has been reported in transplant patients who receive cord blood stem cells. In this study, we characterize the development of human B and T cells in the lymph nodes (LNs) and spleen of BALB/c-Rag2nullIl2rγnull hu-mice. We find a dominant population of immature B cells in the blood and spleen early, followed by a population of human T cells, coincident with the detection of LNs. Notably, in older mice we observe a major population of mature B cells in LNs and in the spleens of mice with higher T cell frequencies. Moreover, we demonstrate that T cells are necessary for B cell maturation, as introduction of autologous human T cells expedites the appearance of mature B cells, whereas in vivo depletion of T cells retards B cell maturation. The presence of the mature B cell population correlates with enhanced IgG and Ag-specific responses to both T cell–dependent and T cell–independent challenges, indicating their functionality. These findings enhance our understanding of human B cell development, provide increased details of the reconstitution dynamics of hu-mice, and validate the use of this animal model to study mechanisms and treatments for the similar delay of functional B cells associated with cord blood transplantations.

Cigarette Smoke Increases Susceptibility to Tuberculosis—Evidence From In Vivo and In Vitro Models

BACKGROUND Cigarette smoke (CS) exposure is an epidemiological risk factor for tuberculosis, although the biological basis has not been elucidated. METHODS We exposed C57BL/6 mice to CS for 14 weeks and examined their ability to control an aerosol infection of Mycobacterium tuberculosis Erdman. RESULTS CS-exposed mice had more M. tuberculosis isolated from the lungs and spleens after 14 and 30 d, compared with control mice. The CS-exposed mice had worse lung lesions and less lung and splenic macrophages and dendritic cells (DCs) producing interleukin12 and tumor necrosis factor α (TNF-α). There were significantly more interleukin 10-producing macrophages and DCs in the spleens of infected CS-exposed mice than in non-CS-exposed controls. CS-exposed mice also showed a diminished influx of interferon γ-producing and TNF-α-producing CD4(+) and CD8(+) effector and memory T cells into the lungs and spleens. There was a trend toward an increased number of viable intracellular M. tuberculosis in macrophages isolated from humans who smoke compared with nonsmokers. THP-1 human macrophages and primary human alveolar macrophages exposed to CS extract, nicotine, or acrolein showed an increased burden of intracellular M. tuberculosis. CONCLUSION CS suppresses the protective immune response to M. tuberculosis in mice, human THP-1 cells, and primary human alveolar macrophages.

Effect of HLA-Matching Recipients to Donor Noninherited Maternal Antigens on Outcomes after Mismatched Umbilical Cord Blood Transplantation for Hematologic Malignancy

Transplantation-related mortality (TRM) is high after HLA-mismatched umbilical cord blood (UCB) transplantation (UCBT). In utero, exposure to noninherited maternal antigen (NIMA) is recognized by the fetus, which induces T regulator cells to that haplotype. It is plausible that UCBTs in which recipients are matched to donor NIMAs may alleviate some of the excess mortality associated with this treatment. To explore this concept, we used marginal matched-pair Cox regression analysis to compare outcomes in 48 NIMA-matched UCBTs (ie, the NIMA of the donor UCB unit matched to the patient) and in 116 non-NIMA-matched UCBTs. All patients had a hematologic malignancy and received a single UCB unit. Cases and controls were matched on age, disease, disease status, transplantation-conditioning regimen, HLA match, and infused cell dose. TRM was lower after NIMA-matched UCBTs compared with NIMA-mismatched UCBTs (relative risk, 0.48; P = .05; 18% versus 32% at 5 years posttransplantation). Consequently, overall survival was higher after NIMA-matched UCBT. The 5-year probability of overall survival was 55% after NIMA-matched UCBTs versus 38% after NIMA-mismatched UCBTs (P = .04). When faced with the choice of multiple HLA-mismatched UCB units containing adequate cell doses, selecting an NIMA-matched UCB unit may improve survival after mismatched UCBT.

Beryllium Presentation to CD4+ T Cells Is Dependent on a Single Amino Acid Residue of the MHC Class II β-Chain

Chronic beryllium disease (CBD) is characterized by a CD4+ T cell alveolitis and granulomatous inflammation in the lung. Genetic susceptibility to this disease has been linked with HLA-DP alleles, particularly those possessing a glutamic acid at position 69 (Glu69) of the β-chain. However, 15% of CBD patients do not possess a Glu69-containing HLA-DP allele, suggesting that other MHC class II alleles may be involved in disease susceptibility. In CBD patients without a Glu69-containing HLA-DP allele, an increased frequency of HLA-DR13 alleles has been described, and these alleles possess a glutamic acid at position 71 of the β-chain (which corresponds to position 69 of HLA-DP). Thus, we hypothesized that beryllium presentation to CD4+ T cells was dependent on a glutamic acid residue at the identical position of both HLA-DP and -DR. The results show that HLA-DP Glu69- and HLA-DR Glu71-expressing molecules are capable of inducing beryllium-specific proliferation and IFN-γ expression by lung CD4+ T cells. Using fibroblasts expressing mutated HLA-DP2 and -DR13 molecules, beryllium recognition was dependent on the glutamic acid at position 69 of HLA-DP and 71 of HLA-DR, suggesting a critical role for this amino acid in beryllium presentation to Ag-specific CD4+ T cells. Thus, these results demonstrate that a single amino acid residue of the MHC class II β-chain dictates beryllium presentation and potentially, disease susceptibility.

Prevalence of donor-specific anti-HLA antibodies during episodes of renal allograft rejection.

Background. Recent studies suggest that the appearance of anti-HLA antibodies in the early posttransplant period is associated with an increased incidence of acute and chronic rejection months later. However, very little is known about the prevalence of anti-HLA antibodies at the time that the rejection episodes are diagnosed. The purpose of this study was to analyze retrospectively 420 sera from 263 renal allograft recipients who were readmitted to the hospital for any reason between 1989 and 1998 in order to determine if a correlation existed between the presence of donor-specific anti-HLA antibodies and graft rejection. Methods. Sera were assayed for IgG HLA class I and II antibodies by ELISA. The ELISA results were analyzed using contingency tables with Fisher’s exact test and compared with mismatched antigens in the donor. Results. Antibodies to donor HLA class I molecules in the posttransplant sera were extremely rare, occurring in only 6 of the 420 sera (1.4%) analyzed. Antibodies to donor class II antigens were slightly more common, occurring in 25 of the 420 sera (6%). In 21 of these 25 cases (84%), the presence of donor-specific HLA class II antibodies was associated with episodes of either acute (n=14) or chronic rejection (n=7). Five patients had antibodies to both class I and class II donor antigens, and all five of them lost their grafts to rejection. Conclusion. Although the presence of donor-specific HLA antibodies presented a significant risk for acute or chronic rejection, 77% of all acute and chronic rejections occurred in patients without detectable HLA antibodies.

Exposure to Cigarette Tar Inhibits Ribonucleotide Reductase and Blocks Lymphocyte Proliferation

Cigarette smoking causes profound suppression of pulmonary T cell responses, which has been associated with increased susceptibility to respiratory tract infections and decreased tumor surveillance. Exposure of human T cells to cigarette tar or its major phenolic components, hydroquinone and catechol, causes an immediate cessation of DNA synthesis without cytotoxicity. However, little is known of the mechanisms by which this phenomenon occurs. In this report we demonstrate that hydroquinone and catechol inhibit lymphocyte proliferation by quenching the essential tyrosyl radical in the M2 subunit of ribonucleotide reductase.