Depletion Of Donor Research Articles

Application of the Activated Sludge Model for Nitrogen to Elevated Nitrogen Conditions

The Activated Sludge Model for Nitrogen (ASMN) was evaluated by conducting simulations under both steady-state and dynamic conditions using a wastewater containing high concentrations of chemical oxygen demand (COD) and nitrogen, and an inhibitor of ammonia-oxidizing bacteria. The adopted wastewater characteristics were based on data from several industrial wastewater treatment facilities. The simulations were performed at a variety of temperatures, solids retention times, dissolved oxygen concentrations, pH values, and salt concentrations. The nitrification operating window was defined, and denitrification performance was characterized. The pH and temperature were found to be the most important variables affecting nitrification performance under upset or startup conditions, with lower pH values allowing better performance at higher temperatures for the high-nitrogen wastewater used in the simulations. Emissions of nitric oxide and nitrous oxide were higher than generally thought to occur and were directly linked to depletion of the electron donor in the anoxic reactor. The findings concerning pH, temperature, and gaseous emissions were all consistent with the known growth characteristics of nitrifying and denitrifying bacteria. Parameter and process variable sensitivity studies were performed, and guidelines for improved biological nitrogen removal were developed.

T Cell Therapy in Allogeneic Stem Cell Transplantation

T cells derived from healthy stem cell donors can mediate curative immune responses after allogeneic stem cell transplantation (SCT) by eliminating (residual) hematopoietic tumor cells life-threatening viral infections. The first evidence for the crucial role of T cells came from the observations that depletion of T cells from a stem cell graft to prevent the development of acute graft versus host disease (GVHD) was associated with an increased incidence of relapse of the malignancy and life threatening viral infections including CMV disease and EBV mediated post-transplant lymphoproliferative disease (PTLD). Apparently, after allogeneic SCT T cells are not only responsible for the detrimental GVHD, but also can mediate a graft versus tumor (GVT) effect and are essential for long term of control of persistent endogenous viral infections. Unmanipulated T cell populations derived from the stem cell donor administered to the patient may result in concurrent development of GVHD, GVT and anti-virus responses. Immune suppression to control GVHD may lead to reduction or elimination of GVT and pathogen specific T cell responses. Obviously, if T cell responses leading to GVHD could be separated from GVL or pathogen-specific reactivity, morbidity and survival after allogeneic SCT can be significantly improved. One approach to manipulate the effect of donor T cells after transplantation has been the depletion of donor T cells from the graft with postponed administration of donor T cells 1. The rationale for this approach came from several mouse and human studies illustrating that the recipient microenvironment plays a significant role in the development of GVHD2. Tissue damage during the conditioning regimen leading to the initiation of a ‘cytokine storm’, the strong lymphodepletion after transplant leading to homeostatic proliferation of donor T cells shortly after transplant, and the presence of patient derived activated dendritic cells (DC) which are required for the induction of allo-immune responses all contribute to these strong allo-response immediately after transplantation. Postponed T cell administration at the time of replacement of the majority of recipient DC by donor DCs, after repair of the tissue damage, and after partial reconstitution of T cells in peripheral blood of patients is associated with lower risk of developing severe acute GVHD. Administration of T cells from 6 months after transplantation can be performed with relatively low risk of inducing severe GVHD, although chronic GVHD remains a significant complication. However, recurrence of the malignancy and the development of severe opportunistic infections frequently take place within the first half year after transplantation. Thus, although postponed administration of T cells can result in less severe GVHD, overall survival did not significantly improve. Delivering stem cell transplants that are free from GVHD while conferring enhanced protection against malignant disease recurrence and viral reactivation is considered by many transplanters to be a key goal (the “Holy Grail”) for improving transplant success. We will discuss several approaches for the selective administration of non-GVHD reactive donor T cells: selective allodepletion, adoptive transfer of virus-specific T cells, allo-reactive anti-tumor T cells recognizing minor histocompatibility antigens (mHag) or MHC molecules, or T cells recognizing tumor-associated self antigens.

Depletion of Alloreactive Donor T Lymphocytes by CD95-Mediated Activation-Induced Cell Death Retains Antileukemic, Antiviral, and Immunoregulatory T Cell Immunity

In allogeneic hematopoietic stem cell transplantation (AHSCT) graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effect are closely but not invariably linked. Thus, harnessing donor lymphocyte mediated GVL immunity and separating it from GVHD is of particular interest. Based on results obtained in murine models we have explored the CD95-mediated activation-induced cell death (AICD) strategy to selectively deplete alloreactivity in human donor T lymphocytes in vitro. Following stimulation of CD3 + T cells isolated from HLA-A ∗0201-positive donors with HLA or minor histocompatibility antigen mismatched hematopoietic or nonhematopoietic cells in the presence of agonistic anti-CD95 antibody, we achieved efficient and selective allodepletion across major and minor histocompatibility mismatched barriers. Residual alloreactivity was in the range of 10% and 25% using hematopoietic cells and primary keratinocytes as alloantigen-presenting cells, respectively. CD8 + T cells specific for HLA-A ∗0201-associated cytomegalovirus (CMV), Epstein-Barr virus (EBV), and Wilms tumor 1 peptide epitopes were retained at significant numbers within the allodepleted donor lymphocyte subsets. Additionally, CD4 + FoxP3 + regulatory T cells persisted after the allodepletion procedure. Our results show that AICD induced by an agonistic anti-CD95 antibody might be useful to generate allodepleted donor lymphocyte products with preserved beneficial immune functions for patients undergoing AHSCT.

Targeting CD134 Results in Selective Depletion of Alloreactive Human T Cells without Loss of Virus-Specific and Leukemia-Specific Effectors.

Graft versus host disease (GvHD) remains a frequent and severe complication of allogeneic stem cell transplantation (SCT) and is mediated by recognition of recipient alloantigens by donor T cells. Numerous interventions have been employed against GvHD which target the number and function of T cells including pharmacological non-specific immunosuppression, depletion of donor T cells or positive selection of donor CD34+ stem cells. While these strategies reduce the risk of GvHD, the benefits are often offset by prolonged immunodeficiency and increased risk of infection and disease relapse. Thus, research efforts have focused on developing a method to selectively deplete alloreactive T cells without compromising other immune responses. One approach to reducing alloreactivity is to remove donor T cells that are activated after ex-vivo co-culture with recipient antigen-presenting cells (APC). Clinical studies have been performed using anti-CD25-conjugated immunotoxin to deplete alloreactive T cells after incubation with haplo-identical PBMC ex-vivo. Although this approach is promising, post-transplant infections, disease relapse and residual GvHD remain a problem. These observations mandate further experimentation to improve our strategies for allodepletion. We undertook studies to identify an inducible surface activation marker expressed selectively on alloreactive effector T cells following culture with HLA-mismatched allostimulators. Costimulatory molecules play a major role in activation of the effector T cells involved in GvHD. In contrast to CD28, which is constitutively expressed, TNF receptor (TNFR) superfamily members are induced and therefore their expression can also be used as an indicator of T cell activation during GvHD, in experimental animal models and allogeneic human transplantation. Here, using peripheral blood mononuclear cells as responders and HLA-mismatched dendritic cells as allostimulators, we observed that compared to other activation markers the TNFR superfamily member, CD134, was superior because of its completely negative base-line expression and rapid upregulation upon activation. Depletion of CD134+ cells from responder populations dramatically reduced specific alloreactivity as determined by reduction of helper T cell precursor (HTLp) frequencies below the threshold predicting development of clinical GvHD and reduction of cytolytic T cell precursor (CTLp) frequencies by 92% against original allostimulators, while retaining responses to third party alloantigens. However, effector CD8+ T cells expressing perforin and granzyme B were preserved in the CD134 allo-depleted populations. Detailed analysis revealed that, by this approach, functional CMV-specific effectors were retained as determined by their identification with CMV-specific pentamers and assessment of their functional responses by CMV-specific ELISpot. Effectors specific for WT1 leukemia antigen were also retained. In addition, Foxp3+ Treg cells were preserved in the allodepleted populations. These observations suggest that CD134-based selective allodepletion may allow allogeneic SCT without requirement for global T cell depletion or non-specific immunosuppression and without loss of pathogen-specific and leukemia-specific immunity.

Reconstitution of FOXP3+ regulatory T cells (Tregs) after CD25-depleted allotransplantion in elderly patients and association with acute graft-versus-host disease

AbstractSelective depletion (SD) of host-reactive donor T cells from allogeneic stem-cell transplants (SCTs) using an anti-CD25 immunotoxin (IT) is a strategy to prevent acute graft-versus-host disease (aGvHD). There is concern that concurrent removal of regulatory T cells (Tregs) with incomplete removal of alloactivated CD25+ T cells could increase the risk of aGvHD. We therefore measured Tregs in the blood of 16 patients receiving a T-cell–depleted allograft together with anti–CD25-IT–treated SD lymphocytes, in 13 of their HLA-identical donors, and in 10 SD products. Tregs were characterized by intracellular staining for forkhead box protein 3 (FOXP3) and by quantitative reverse-transcription–polymerase chain reaction (qRT-PCR) for FOXP3 gene in CD4+ cells. Patients received a median of 1.0 × 108/kg SD T cells and a stem cell product containing a median of 0.25 × 104/kg residual T cells. Tregs reconstituted promptly after SCT and underwent further expansion. Of the CD4+ T cells in SD products, 1.5% to 4.8% were CD25− Tregs. Acute GvHD (≥ grade II) was restricted to 5 patients whose donors had significantly (P = .019) fewer Tregs compared with those without clinically significant aGvHD. These results suggest that rapid Treg reconstitution can occur following SD allografts, either from CD25− Tregs escaping depletion, or from residual CD25− and CD25+ Tregs contained in the stem-cell product that expand after transplantation and may confer additional protection against GvHD.

The Role of Selective Depletion of Alloreactive Donor T Cells in Graft-Versus-Host Disease after Allogeneic Bone Marrow Transplantation.

One of the main goals in allogeneic bone marrow(BM) transplantation is the abrogation of graft-versus-host disease (GVHD) with the preservation of antileukemia and antiviral activity. The Study present a selective T cell depletion strategy based on the physical separation of the alloreactive T cells, which were identified by expression of two activation-induced antigens (CD25 and CD69). T cells from C57BL/6(H-2b) mice were first activated with BALB/c (H-2d) recipient spleen cells in a 2-day mixed-lymphocyte-culture (MLC). Following this activation, this compound is selectively depleted based on expression of two activation-induced antigens CD25 and CD69 using magnetic cell sorting. The depleted cells or the untreated cells were then rechallenged respectively in a secondary MLC, with the same stimulator cells or a third-party (DBAH-2k) or tumor- specific (SP2/0, BALB/c-origin myeloma) cells. Cells proliferation were assayed at the indicated time points(1, 2, 3, 4, 5 days). These treated cells or control-cultured cells (2.0×106) mixed with 5.0×106 BM cells from C57BL/6 were transfused respectively by the trail vain into the lethally irradiated BALB/c to observe the survival time, GVHD incidence and pathological analysis. MLC assays demonstrated that this technique led to a significant decrease in alloreactivity of donor cells(29.02~64.17%), which at the same time preserved reactivity against third party cells(49.61~75.69%)and anti-tumor cells(61.14~68.62%). The mice in the group of control-coclutured were died of acute GVHD within 24days. The 7 recipient mice in the treated group were free of acute GVHD, and 3 mice were died of acute GVHD (aGVHD) within 23 days. MACS-based ex-vivo depletion of alloreactive donor T cells based on expression of two activation-induced antigens (CD25 and CD69) could inhibit anti-host responses, by contrast, anti-SP2/O and anti-third-party responses were preserved. Cotransplantation of these selected depleted cells and BM cells could reduce aGVHD.

The cyanobacterial genome core and the origin of photosynthesis.

Comparative analysis of 15 complete cyanobacterial genome sequences, including "near minimal" genomes of five strains of Prochlorococcus spp., revealed 1,054 protein families [core cyanobacterial clusters of orthologous groups of proteins (core CyOGs)] encoded in at least 14 of them. The majority of the core CyOGs are involved in central cellular functions that are shared with other bacteria; 50 core CyOGs are specific for cyanobacteria, whereas 84 are exclusively shared by cyanobacteria and plants and/or other plastid-carrying eukaryotes, such as diatoms or apicomplexans. The latter group includes 35 families of uncharacterized proteins, which could also be involved in photosynthesis. Only a few components of cyanobacterial photosynthetic machinery are represented in the genomes of the anoxygenic phototrophic bacteria Chlorobium tepidum, Rhodopseudomonas palustris, Chloroflexus aurantiacus, or Heliobacillus mobilis. These observations, coupled with recent geological data on the properties of the ancient phototrophs, suggest that photosynthesis originated in the cyanobacterial lineage under the selective pressures of UV light and depletion of electron donors. We propose that the first phototrophs were anaerobic ancestors of cyanobacteria ("procyanobacteria") that conducted anoxygenic photosynthesis using a photosystem I-like reaction center, somewhat similar to the heterocysts of modern filamentous cyanobacteria. From procyanobacteria, photosynthesis spread to other phyla by way of lateral gene transfer.

Preserving the cornea: corneal storage media

The aim of this article is to review the recent literature regarding corneal storage media since the last review that discussed this topic in substantial detail in this journal. During the last few years, despite the development of new corneal storage media and the addition of new additives to established corneal storage media, Optisol GS (Bausch & Lomb, Irvine, California, USA) has continued to remain the popular choice among storage media used in the United States, and traditional organ culture methods are still used in Europe. Recognizing that persistent epithelial defects after corneal transplantation can be a serious complication, however, attention has started to focus on not only preserving the endothelium, but also the epithelium. In addition, there has been more research towards antimicrobial prophylaxis. With recent evidence suggesting that longer storage times may allow better outcomes in high-risk grafts because of the depletion of donor T cells from the donor cornea into the storage media, storage media will need to be optimized for preserving the endothelium for longer periods of time. In addition, because the epithelium is typically unable to be sustained for longer than 1 week in storage media, research toward preserving the epithelium will also be essential.

Temporal Transcriptomic Analysis as Desulfovibrio vulgaris Hildenborough Transitions into Stationary Phase during Electron Donor Depletion

Desulfovibrio vulgaris was cultivated in a defined medium, and biomass was sampled for approximately 70 h to characterize the shifts in gene expression as cells transitioned from the exponential to the stationary phase during electron donor depletion. In addition to temporal transcriptomics, total protein, carbohydrate, lactate, acetate, and sulfate levels were measured. The microarray data were examined for statistically significant expression changes, hierarchical cluster analysis, and promoter element prediction and were validated by quantitative PCR. As the cells transitioned from the exponential phase to the stationary phase, a majority of the down-expressed genes were involved in translation and transcription, and this trend continued at the remaining times. There were general increases in relative expression for intracellular trafficking and secretion, ion transport, and coenzyme metabolism as the cells entered the stationary phase. As expected, the DNA replication machinery was down-expressed, and the expression of genes involved in DNA repair increased during the stationary phase. Genes involved in amino acid acquisition, carbohydrate metabolism, energy production, and cell envelope biogenesis did not exhibit uniform transcriptional responses. Interestingly, most phage-related genes were up-expressed at the onset of the stationary phase. This result suggested that nutrient depletion may affect community dynamics and DNA transfer mechanisms of sulfate-reducing bacteria via the phage cycle. The putative feoAB system (in addition to other presumptive iron metabolism genes) was significantly up-expressed, and this suggested the possible importance of Fe2+ acquisition under metal-reducing conditions. The expression of a large subset of carbohydrate-related genes was altered, and the total cellular carbohydrate levels declined during the growth phase transition. Interestingly, the D. vulgaris genome does not contain a putative rpoS gene, a common attribute of the delta-Proteobacteria genomes sequenced to date, and the transcription profiles of other putative rpo genes were not significantly altered. Our results indicated that in addition to expected changes (e.g., energy conversion, protein turnover, translation, transcription, and DNA replication and repair), genes related to phage, stress response, carbohydrate flux, the outer envelope, and iron homeostasis played important roles as D. vulgaris cells experienced electron donor depletion.

Erythrocyte transmembrane electron transfer in haemodialysis patients

Background and aims Patients with chronic renal failure, especially those treated with haemodialysis, have an increased risk of developing atherosclerotic vascular disease probably as a result of enhanced oxidative stress. The human cell membrane possesses electron transfer systems which protect against extracellular pro-oxidant challenge. We evaluated (1) the erythrocyte velocity of ferricyanide reduction (RBC vfcy) in 25 uraemic patients (aged 25–71 years; 14 males), (2) the changes induced by a single haemodialysis session and (3) biomarkers of oxidative stress. Methods and results Before and after a mid-week dialysis session, we measured RBC vfcy, erythrocyte glutathione (RBC GSH), plasma and red cell membrane malondialdehyde (P and RBC MDA), plasma sulphydryl groups (P SH), plasma vitamin C levels and haemolysis percentage. Pre-dialysis RBC GSH (0.68 ± 0.13 vs 0.80 ± 0.13 mg/mL, p < 0.01), P SH (266 ± 74 vs 406 ± 78 μmol/L, p < 0.01) and plasma vitamin C (7.0 ± 5.1 vs 21.5 ± 8.5 mg/L, p < 0.001) were lower than in 25 age-sex-matched healthy controls; P MDA (1.57 ± 0.52 vs 0.54 ± 0.29 nmol/mL, p < 0.001), RBC MDA (0.42 ± 0.13 vs 0.34 ± 0.16 nmol/mL, p < 0.05) and haemolysis (1.2 ± 0.3 vs 0.7 ± 0.3%, p < 0.001) were increased. Baseline RBC vfcy did not differ from normals (13.1 ± 5.2 vs 12.9 ± 3.2 mmol/mL/h). Following dialysis, RBC vfcy (to 8.9 ± 4.5 mmol/mL/h, p < 0.001) decreased, as well as P MDA, RBC MDA and plasma vitamin C (to 2.5 ± 1.4 mg/L, p < 0.001), whereas P SH groups increased (to 413 ± 99 μmol/L, p < 0.001); haemolysis percentage remained high. RBC vfcy values were correlated to RBC GSH and vitamin C levels. Conclusions Uraemic patients showed signs of oxidative stress. Pre-dialysis RBC vfcy is maintained in the normal range on account of a reduced intracellular content of GSH and in spite of low plasma ascorbate. A single haemodialysis treatment reduced biomarkers of protein and lipid oxidation but markedly impaired transmembrane electron transfer, which could be explained by acute depletion of electron donors.

Fludarabine Treatment Is Associated with Depletion of Host CD4+CD25high, FOXP3+, CTLA-4+ Cells and Increased Incidences of Full Donor Chimerism and GVHD in Non-Myeloablative Haploidentical Hematopoietic Cell Transplant Recipients.

Purpose: To evaluate T cell recovery and donor chimerism following haploidentical hematopoietic cell transplantation (HCT) with a non-myeloablative conditioning approach that includes T cell depletion of host and donor and delayed DLI.Methods: Eighteen patients, 3 cohorts of 4 patients each and 1 cohort of 6 evaluable patients/10 transplanted, with chemorefractory hematologic malignancies, received related HLA 1–3 of 6, A, B, or DR antigen mismatched donor HCT after non-myeloablative conditioning with Medi-507 (anti-CD2 humanized mAb; Biotransplant, Inc.), cyclophosphamide, thymic irradiation and peritransplant cyclosporine. The patients in Protocols A received a MEDI-507 test dose of 0.1 mg/kg on Day -2 followed by 0.6 mg/kg on Days −1, 0 and +1 and transplantation of unmanipulated bone marrow. In Protocol B, the timing and dose of Medi-507 was modified. The patients in Protocol C and D received the latter Medi-507 protocol, but were given Isolex ®-selected CD34+ cells from G-CSF mobilized PBSC. Protocol D differs from Protocol C with the addition of fludarabine to more reliably achieve sustained chimerism. Donor leukocyte infusions were administered in an effort to convert mixed to full donor chimerism and to achieve a graft-versus-tumor effect. Chimerism was measured by peripheral blood microsatellite markers or by flow cytometry using HLA-specific mAbs. T cell recovery and phenotype were followed by flow cytometry. Because a high percentage of CD4 T cells post- transplant were CD25high, we performed quantitative RTPCR for Foxp3 and CTLA-4 on sorted PBMC populations.Results: T cell depletion early post-HCT was detected in all patients. There was a marked difference in the percentage of graft acceptance/loss, GVHD prevalence, and T cell phenotype related to each protocol modification. The majority (>90%) of CD4 T cells appearing in the first 100 days post-SCT were CD45RO+/CD45RA- “memory” cells and CD8 T cells were CD45RO+CD45RA-/CD62L-. In addition, a remarkably high percentage (19.5–75%, mean 38.1%) of CD4 T cells expressed high levels of CD25 in recipients of Protocols A, B, and C early post-HCT. CD25 expression decreased as T cell counts increased. By quantitative RTPCR, we found that sorted CD25highCD4 T cells expressed Foxp3 and CTLA-4, consistent with a regulatory phenotype. The addition of fludarabine in Protocol D resulted in an overall reduction in the percentage of peripheral CD4CD25high T cells compared to Protocol C at 4 weeks post-HCT (C 23.43% +/−4.7% versus D 2.1% +/− 0.9%, p<0.00005). The addition of fludarabine improved sustained engraftment from 2/4 in Protocol C to 5/6 in Protocol D. In Protocol C, T cell chimerism conversion occurred following DLI in 2/4 patients with no or grade II skin-limited GVHD. In Protocol D, full or near full donor chimerism was achieved in 5/6 patients, 3 spontaneously and 2 following DLI, and grade I–IV GVHD developed in 5/6 patients.Conclusions: These data suggest that the addition of fludarabine may increase the incidence of sustained and full donor chimerism. Fludarabine efficiently depleted host CD4+CD25high Foxp3+ cells, which may have a regulatory role, preventing spontaneous chimerism conversion and associated GVHD.

Selective depletion of alloreactive donor lymphocytes: a novel method to reduce the severity of graft-versus-host disease in older patients undergoing matched sibling donor stem cell transplantation

AbstractWe have selectively depleted host-reactive donor T cells from peripheral blood stem cell (PBS153) transplant allografts ex vivo using an anti-CD25 immunotoxin. We report a clinical trial to decrease graft-versus-host disease (GVHD) in elderly patients receiving selectively depleted PBSC transplants from HLA-identical sibling donors. Sixteen patients (median age, 65 years [range, 51-73 years]), with advanced hematologic malignancies underwent transplantation following reduced-intensity conditioning with fludarabine and either cyclophosphamide (n = 5), melphalan (n = 5), or busulfan (n = 6). Cyclosporine was used as sole GVHD prophylaxis. The allograft contained a median of 4.5 × 106 CD34 cells/kg (range, 3.4-7.3 × 106 CD34 cells/kg) and 1.0 × 108/kg (range, 0.2-1.5 × 108/kg) selectively depleted T cells. Fifteen patients achieved sustained engraftment. The helper T-lymphocyte precursor (HTLp) frequency assay demonstrated successful (mean, 5-fold) depletion of host-reactive donor T cells, with conservation of third-party response in 9 of 11 cases tested. Actuarial rates of acute GVHD were 46% ± 13% for grades II to IV and 12% ± 8% for grades III to IV. These results suggest that allodepletion of donor cells ex vivo is clinically feasible in older patients and may reduce the rate of severe acute GVHD. Further studies with selectively depleted transplants to evaluate graft-versus-leukemia (GVL) and survival are warranted.

Field induced polarisation and relaxation of tungsten oxide thick films

We have investigated the time dependent conductance of a WO 3 film (∼ 700 K) after a voltage step. From the beginning to some 100 ms, the region next to the positive electrode shows a strong increase in resistance. The region next to the negative electrode shows a decrease in resistance. A possible explanation for this first effect is the depletion (respectively accumulation) of mobile donors. After 10 s, a decrease in resistance next to the positive electrode is observed. The region next to the negative electrode shows an increase in resistance. The resistance of the two regions nearly meets its value prior to the perturbation. A possible explanation for this second effect is desorption (respectively adsorption) of charged surface oxygen.

Thrombotic Microangiopathy after Allogeneic Blood and Marrow Transplantation Is Associated with Dose-Intensive Myeloablative Conditioning Regimens, Unrelated Donor, and Methylprednisolone T-Cell Depletion

Allogeneic blood and marrow transplantation (BMT)-associated thrombotic microangiopathy (TM) contributes to transplant-related morbidity and mortality. This report examines the incidence of and risk factors for allogeneic BMT-associated TM in two patient cohorts treated before and after changes in myeloablative conditioning regimen intensity (high vs. standard intensity). Cohort 1 includes 153 consecutive allogeneic BMT patients who underwent transplantation between April 1994 and October 1997 with an allogeneic BMT-associated TM crude incidence of 12%. Cohort 2 includes 75 consecutive allogeneic BMT patients who underwent transplantation from November 1997 to November 2000 with an allogeneic BMT-associated TM crude incidence of 1%. In cohort 1, matched unrelated donor transplant and methylprednisolone (MP) T-cell depletion (TCD) of donor bone marrow were significantly associated with allogeneic BMT-associated TM by univariate analysis; therefore, a logistic model incorporating these effects was constructed to calculate the expected number of allogeneic BMT-associated TM cases in cohort 2. Seven cases would have been expected, but only one was observed (P = 0.003; bayesian predictive test). The multivariate analysis of both cohorts yielded MP-TCD (P<0.001), high-intensity myeloablative conditioning regimens used in cohort 1 (P = 0.02), and matched unrelated donor (P = 0.03) as significant predictors of time to allogeneic BMT-associated TM. Avoidance of high-intensity conditioning regimens may decrease the incidence of allogeneic BMT-associated TM.

Separation of GvH- and GvL-Immune Responses in Allogeneic Stem Cell Transplantation by CD95-Mediated Depletion of Alloreactive T-Cells Using CD178 Based Proapoptotic Chimeric Molecules.

Depletion of donor T cells (TCD) from the graft effectively ameliorates graft-vs-host (GvH) disease (GvHD) in allogeneic stem cell transplantation (SCT). TCD, however, is associated with impaired engraftment, immunosuppression, and abrogation of the beneficial graft-vs-leukemia (GvL) effect. Thus, based on previous results (Hartwig et al., Blood 2002) to eliminate alloreactive specificities but preserve oligoclonal antiviral- and GvL- immunity we explored proapoptotic chimeric molecules composed of the extracellular domains of human CD178 (FasL) and CD28 or RANK to selectively deplete GvH-reactive T cells by AICD in vitro and in vivo. As these fusion proteins are expressed as soluble Flag-tagged trimers and reconstitute bioactivity only upon binding to well defined cell-surface molecules (Samel et al., J. Biol. Chem., 2003), they can be specifically targeted onto e.g. recipient derived DC or donor T cells via the molecules CD80/86 or RANKL/TRANCE, respectively.Using an MHC-mismatched murine BMT model proliferative responses of purified CD90+ alloreactive T cells from C57BL/6 (H-2b) mice were strongly reduced following stimulation with DC from F1 (C57BL/6xBalb/c; H-2bxd) mice in vitro in the presence of CD178-CD28 or CD178-RANK to levels comparably obtained with agonistic anti-CD95 mAb (Jo2). Depletion was specific to alloantigen since H-2d restricted Hemagglutinin (HA)-peptide specific T cells titrated into the system persisted among retained T cells.In addition, adoptive transfer experiments of H-2b derived CD90+ T cells depleted of alloreactivity in vitro prior to injection into irradiated F1 recipients confirmed the in vitro results as these mice did not develop clinical signs of GvHD as compared to controls.Moreover, in current studies the CD178-fusion proteins are applied in vivo in our BMT model by injecting 2.5 to 5.0 x 106 syngeneic (H-2bxd) DC preloaded with CD178-CD28 into lethally irradiated F1 mice reconstituted with 1 x 107 TCD H-2b donor marrow cells. Alternatively, soluble CD178-fusion proteins are applied i.v. following BMT. Preliminary results obtained in first experiments suggest that host DC expressing cell surface bound CD178 fusion proteins confer protection to acute GvH-reactions in recipients challenged with purified CD90+ T cells of H-2b origin as these mice show prolonged survival compared to controls. Furthermore, studies on evaluating GvL-reactivity in this model using donor T cells from HA-primed Balb/c mice and A20 cells expressing HA as a defined tumor surrogate antigen are in progress, and their results will be discussed.Taken together, these results suggest that CD178-based proapoptotic fusion proteins devoid of systemic toxicity due to cell-surface antigen-restricted activation might provide a promising tool to separate GvH- and GvL-responses in vivo without inducing severe side effects on CD95-mediated apoptosis in sensitive tissues.

Selective Depletion of Alloreacting CD25+ Cells from Stem Cell Allografts Can Reduce Acute Graft-Versus-Host Disease Following Matched Related Donor Transplantation.

Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality after allogeneic stem cell transplant (SCT), especially in older patients. We previously showed that host-reactive donor T cells are selectively depleted (SD) from an allograft ex vivo, following a short co-culture of donor cells with irradiated T cell stimulators from the recipient and subsequent treatment with an anti-CD25 immunotoxin. We report a pilot study to test the hypothesis that GVHD could be decreased in a cohort of elderly patients receiving SD allografts from HLA-identical sibling donors. Sixteen patients, median age 65 years (range 51–73), with advanced hematologic malignancies were transplanted following reduced-intensity conditioning with fludarabine and either cyclophosphamide (n=5), melphalan (n=5), or busulfan (n=6). Cyclosporine was used as the only additional GVHD prophylaxis. SD allografts contained a median CD34 dose of 4.5x106/kg (range 3.5–7.3) and an SD CD3 dose of 1.0x108/kg (range 0.2–1.5). Fifteen patients achieved sustained engraftment. The helper T lymphocyte precursor (HTLp) frequency assay demonstrated depletion of host-reactive donor T cells in 9/11 cases tested from a mean of 1/182,089 to 1/822,354 (mean 5.5-fold depletion), while third party responses were conserved. Kaplan-Meier estimates of probability of grade II-IV and grade III-IV acute GVHD were lower than those seen in a historical control group of patients receiving cyclosporine alone for GVHD prophylaxis (35±13% vs. 57±10%, p=0.34) and (7±6% vs. 38±6%, p=0.05), respectively. Of note, the two patients who developed visceral (gut ± liver) GVHD showed ineffective allodepletion by HTLp (figure). Chronic GVHD occurred in five of 14 evaluable patients. At a median follow-up of 212 days (range 60 – 690), seven of sixteen patients remain alive and in remission. Relapse deaths occurred in four patients (refractory AML [2], therapy-related MDS [1], and CMML [1]). Non-relapse mortality in this high-risk cohort of patients included graft failure [1], GVHD [2], infection [1], and myocardial infarction [1]. In summary, CD25-directed allodepletion of stem cell allografts can reduce clinically relevant acute GVHD following matched related donor transplantation. [Display omitted]

Alemtuzumab (Campath IH) in Conditioning Therapy with Cyclophosphamide and Total Body Irradiation in Matched and Mismatched Unrelated Donor Transplantation of Children with Acute Lymphoblastic Leukaemia: A Report from 3 U.K. Centres.

Bristol has previously published extensively on the use of Campath antibodies during the unrelated donor cell transplantation of children with relapsed or otherwise high risk Acute Lymphoblastic Leukaemia (A.L.L.). These former reports (Br J Haematol 1996; 94(3):574–8; Blood 1999; 94(7) 2236–46) concerned the use of Campath IG (a rat monoclonal antibody directed at CD52) given pre-transplant to the patients by intravenous infusion during conditioning therapy that also included cyclophosphamide (120 mgs/kg) and fractionated total body irradiation (1440 Gy). During this treatment the unrelated donor marrow was T cell depleted in vitro using Campath IM (also monoclonal and of rat origin). The results of such treatment demonstrated results comparable with HLA-matched sibling BMT, with low levels of acute graft versus host disease. In this communication transplant centres in 3 UK hospitals (Bristol, Great Ormond Street, London and Manchester) report their combined experience of using Campath IH (Alemtuzumab, a humanised monoclonal antibody - also directed at CD52) in the conditioning therapy of children with relapsed or otherwise high risk A.L.L.. The conditioning was otherwise again with Cyclophosphamide and fractionated total body irradiation. In this protocol a T cell replete graft was given and there was no in vitro T cell depletion of the unrelated donor marrow.We report 34 successive patients where follow up for at least 12 months was available. The median age was 6.5 years and 28 patients received fully matched grafts (matched at HLA-A, -B, -C, -DRB1 and DQB1). The remaining patients received a graft that was mismatched at one of these class I loci. All patients engrafted and the median time to neutrophil count sustained above 0.5x10e9 / l was 20 days (range 12–58 days). Only tow patients experienced grade III acute GvHD and no patient experienced grade IV acute GvHD. 22 patients survive at a median follow up of 23.8 months including 4 of the patients who received a mismatched graft. There were only 3 deaths in the first 100 days following transplant (2 due to disseminated, invasive adenovirus and one to aspergillus). Beyond 100 days 3 further patients have died of adenovirus, 3 have relapsed, 2 have died of chronic GvHD and one died of a pulmonary haemorrhage of uncertain aetiology.We therefore conclude the combination of Aletuzumab with cyclophosphamide and TBI is an effective and safe conditioning therapy for children with relapsed A.L.L. All patients engraft and there are low rates of acute and chronic GvHD.

Therapeutic immunization protects dopaminergic neurons in a mouse model of Parkinson's disease.

Degeneration of the nigrostriatal dopaminergic pathway, the hallmark of Parkinson's disease, can be recapitulated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. Herein, we demonstrate that adoptive transfer of copolymer-1 immune cells to MPTP recipient mice leads to T cell accumulation within the substantia nigra pars compacta, suppression of microglial activation, and increased local expression of astrocyte-associated glial cell line-derived neurotrophic factor. This immunization strategy resulted in significant protection of nigrostriatal neurons against MPTP-induced neurodegeneration that was abrogated by depletion of donor T cells. Such vaccine treatment strategies may provide benefit for Parkinson's disease.

Reduction of uranium(VI) under sulfate-reducing conditions in the presence of Fe(III)-(hydr)oxides

Hexavalent uranium [U(VI)] dissolved in a modified lactate-C medium was treated under anoxic conditions with a mixture of an Fe(III)-(hydr)oxide mineral (hematite, goethite, or ferrihydrite) and quartz. The mass of Fe(III)-(hydr)oxide mineral was varied to give equivalent Fe(III)-mineral surface areas. After equilibration, the U(VI)-mineral suspensions were inoculated with sulfate-reducing bacteria, Desulfovibrio desulfuricans G20. Inoculation of the suspensions containing sulfate-limited medium yielded significant G20 growth, along with concomitant reduction of sulfate and U(VI) from solution. With lactate-limited medium, however, some of the uranium that had been removed from solution was resolubilized in the hematite treatments and, to a lesser extent, in the goethite treatments, once the lactate was depleted. No resolubilization was observed in the lactate-limited ferrihydrite treatment even after a prolonged incubation of 4 months. Uranium resolubilization was attributed to reoxidation of the uraninite by Fe(III) present in the (hydr)oxide phases. Analysis by U L 3 -edge XANES spectroscopy of mineral specimens sampled at the end of the experiments yielded spectra similar to that of uraninite, but having distinct features, notably a much more intense and slightly broader white line consistent with precipitation of nanometer-sized particles. The XANES spectra thus provided strong evidence for SRB-promoted removal of U(VI) from solution by reductive precipitation of uraninite. Consequently, our results suggest that SRB mediate reduction of soluble U(VI) to an insoluble U(IV) oxide, so long as a suitable electron donor is available. Depletion of the electron donor may result in partial reoxidation of the U(IV) to soluble U(VI) species when the surfaces of crystalline Fe(III)-(hydr)oxides are incompletely reduced.

Intimal Exuberance: Veins in Jeopardy

Address reprint requests to Anupam Agarwal, M.D., Associate Professor of Medicine, Biochemistry and Molecular Biology, Box 100224, University of Florida, 1600 SW Archer Road, Gainesville, FL 32610. E-mail: .ude.lfu.cdren.psren@lawraga