Suppression of black-hole growth by strong outflows at redshifts 5.8-6.6.

Bright quasars, powered by accretion onto billion-solar-mass black holes, already existed at the epoch of reionization, when the Universe was 0.5-1 billion years old1. How these black holes formed in such a short time is the subject of debate, particularly as they lie above the correlation between black-hole mass and galaxy dynamical mass2,3 in the local Universe. What slowed down black-hole growth, leading towards the symbiotic growth observed in the local Universe, and when this process started, has hitherto not been known, although black-hole feedback is a likely driver4. Here we report optical and near-infrared observations of a sample of quasars at redshifts 5.8 ≲ z ≲ 6.6. About half of the quasar spectra reveal broad, blueshifted absorption line troughs, tracing black-hole-driven winds with extreme outflow velocities, up to 17% of the speed of light. The fraction of quasars with such outflow winds at z ≳ 5.8 is ≈2.4 times higher than at z ≈ 2-4. We infer that outflows at z ≳ 5.8 inject large amounts of energy into the interstellar medium and suppress nuclear gas accretion, slowing down black-hole growth. The outflow phase may then mark the beginning of substantial black-hole feedback. The red optical colours of outflow quasars at z ≳ 5.8 indeed suggest that these systems are dusty and may be caught during an initial quenching phase of obscured accretion5.

Probing negatively charged and neutral excitons in MoS2/hBN and hBN/MoS2/hBN van der Waals heterostructures.

High-quality van der Waals heterostructures assembled from hBN-encapsulated monolayer transition metal dichalcogenides enable observations of subtle optical and spin-valley properties whose identification was beyond the reach of structures exfoliated directly on standard SiO2/Si substrates. Here, we describe different van der Waals heterostructures based on uncapped single-layer MoS2 stacked onto hBN layers of different thicknesses and hBN-encapsulated monolayers. Depending on the doping level, they reveal the fine structure of excitonic complexes, i.e. neutral and charged excitons. In the emission spectra of a particular MoS2/hBN heterostructure without an hBN cap we resolve two trion peaks, T1 and T2, energetically split by about 10 meV, resembling the pair of singlet and triplet trion peaks (T S and T T ) in tungsten-based materials. The existence of these trion features suggests that monolayer MoS2 has a dark excitonic ground state, despite having a 'bright' single-particle arrangement of spin-polarized conduction bands. In addition, we show that the effective excitonic g-factor significantly depends on the electron concentration and reaches the lowest value of -2.47 for hBN-encapsulated structures, which reveals a nearly neutral doping regime. In the uncapped MoS2 structures, the excitonic g-factor varies from -1.15 to -1.39 depending on the thickness of the bottom hBN layer and decreases as a function of rising temperature.

Correlation of the in vitro biotransformation of H3B-6527 in dog and human hepatocytes with the in vivo metabolic profile of 14C-H3B-6527 in a dog mass balance study.

1. H3B-6527 is an orally available covalent small molecule inhibitor of FGFR4 undergoing evaluation in adults with hepatocellular carcinoma. Absorption, metabolism, transport and elimination of H3B-6527 were investigated in vitro and in a 14C-H3B-6527 beagle dog mass balance study.2. Following intravenous dosing in dogs, unchanged 14C-H3B-6527 represents only 1.6% of the total dose in excreta. The low amount of radioactivity in the dog urine (4.9% of the administered dose), suggests that renal elimination is a minor pathway of clearance for H3B-6527. A majority of the radioactivity was observed in the feces up to 5 days after dose administration, suggesting that drug-related material was secreted in the bile, and that H3B-6527 clearance was mostly driven by metabolism.3. In vitro, H3B-6527 is a substrate of GSTs, CYP3A and P-glycoprotein.4. The major pathways of metabolism were similar in human and dog hepatocytes, and occurred via glutathione (GSH) conjugations and sequential hydrolysis, N-deethylation and hydroxylation.5. The metabolic profile of H3B-6527 was qualitatively similar in dog hepatocytes and plasma/excreta.

Neutral, heteroleptic copper(i)-4H-imidazolate complexes: synthesis and characterization of their structural, spectral and redox properties.

Facile synthetic access to four novel, neutral, heteroleptic copper(i)-complexes, incorporating 4H-imidazolates as well as the phosphane ligands XantPhos and DPEPhos is reported. The complexes were characterized in the solid state as well as in solution by means of single crystal X-ray diffraction as well as NMR spectroscopy, mass spectrometry and elemental analysis. The copper(i)-4H-imidazolate complexes show a broad intense absorption that spans almost the entire visible range. TD-DFT calculations revealed the charge transfer character of the underlying transitions. NMR as well as electrochemical investigations and UV-Vis absorption suggest a polarization of the complexes with the negative charge pushed towards the 4H-imidazolate moiety.

Evaluation for Blunt Cerebrovascular Injury: Review of the Literature and a Cost-Effectiveness Analysis.

BACKGROUND AND PURPOSE: Evaluation for blunt cerebrovascular injury has generated immense controversy with wide variations in recommendations regarding the need for evaluation and the optimal imaging technique. We review the literature and determine the most cost-effective strategy for evaluating blunt cerebrovascular injury in trauma patients. MATERIALS AND METHODS: A comprehensive literature review was performed with data extracted to create a decision-tree analysis for 5 different strategies: anticoagulation for high-risk (based on the Denver screening criteria) patients, selective DSA or CTA (only high-risk patients), and DSA or CTA for all trauma patients. The economic evaluation was based on a health care payer perspective during a 1-year horizon. Statistical analyses were performed. The cost-effectiveness was compared through 2 main indicators: the incremental cost-effectiveness ratio and net monetary benefit. RESULTS: Selective anticoagulation in high-risk patients was shown to be the most cost-effective strategy, with the lowest cost and greatest effectiveness (an average cost of $21.08 and average quality-adjusted life year of 0.7231). Selective CTA has comparable utility and only a slightly higher cost (an average cost of $48.84 and average quality-adjusted life year of 0.7229). DSA, whether performed selectively or for all patients, was not optimal from both the cost and utility perspectives. Sensitivity analyses demonstrated these results to be robust for a wide range of parameter values. CONCLUSIONS: Selective CTA in high-risk patients is the optimal and cost-effective imaging strategy. It remains the dominant strategy over DSA, even assuming a low CTA sensitivity and irrespective of the proportion of patients at high-risk and the incidence of blunt cerebrovascular injury in high-risk patients.

Skeletal muscle homeostasis and plasticity in youth and ageing: impact of nutrition and exercise.

Skeletal muscles comprise a substantial portion of whole body mass and are integral for locomotion and metabolic health. Increasing age is associated with declines in both muscle mass and function (e.g. strength-related performance, power) with declines in muscle function quantitatively outweighing those in muscle volume. The mechanisms behind these declines are multi-faceted involving both intrinsic age-related metabolic dysregulation and environmental influences such as nutritional and physical activity. Ageing is associated with a degree of 'anabolic resistance' to these key environmental inputs, which likely accelerates the intrinsic processes driving ageing. On this basis, strategies to sensitize and/or promote anabolic responses to nutrition and physical activity are likely to be imperative in alleviating the progression and trajectory of sarcopenia. Both resistance- and aerobic-type exercises are likely to confer functional and health benefits in older age, and a clutch of research suggests that enhancement of anabolic responsiveness to exercise and/or nutrition may be achieved by optimizing modifications of muscle-loading paradigms (workload, volume, blood flow restriction) or nutritional support (e.g. essential amino acid/leucine) patterns. Nonetheless, more work is needed in which a more holistic view in ageing studies is taken into account. This should include improved characterization of older study recruits, that is physical activity/nutritional behaviours, to limit confounding variables influencing whether findings are attributable to age, or other environmental influences. Nonetheless, on balance, ageing is associated with declines in muscle mass and function and a partially related decline in aerobic capacity. There is also good evidence that metabolic flexibility is impaired in older age.

Density of states in graphene with vacancies: midgap power law and frozen multifractality.

The density of states ϱ(E) of graphene is investigated numerically and within the self-consistent T-matrix approximation in the presence of vacancies within the tight binding model. The focus is on compensated disorder, where the concentration of vacancies n(A) and n(B) in both sublattices is the same. Formally, this model belongs to the chiral symmetry class BDI. The onlinear sigma model predicts for BDI a Gade-type singularity ϱ(E)∼|E|(-1)exp[-|log(E)|(-1/x)]. Our numerical data are comparable to this result in a preasymptotic regime that gives way, however, at even lower energies to ϱ(E)∼E(-1)|log(E)|(-x̃), 1≤x̃<2. We take this finding as evidence that, similar to the case of dirty d-wave superconductors, generic bipartite random hopping models may also exhibit unconventional (strong-coupling) fixed points for certain kinds of randomly placed scatterers if these are strong enough. Our research suggests that graphene with (effective) vacancy disorder is a physical representative of such systems.

A cost analysis of postoperative management in endometrial cancer patients treated by robotics versus laparoscopic approach.

OBJECTIVE: The purpose of this study is to compare postoperative pain management and costs in endometrial cancer patients who had a robotic-assisted or laparoscopic-assisted hysterectomy. METHODS: This is a retrospective cohort study of all endometrial cancer patients from 9/2005 to 6/2010 who had a completed robotic-assisted or laparoscopic-assisted hysterectomy. All surgeries were performed by gynecologic oncologists on the da Vinci S surgical system. Demographic data, patient-recorded pain scores, pain-management interventions, and postoperative pain medication costs were compared. Data was analyzed using Student's t-tests and Pearson's χ(2) tests in SPSS. RESULTS: Two-hundred fifteen (101 robotic and 114 laparoscopic) patients met the inclusion criteria. There were no significant differences between the groups in age, BMI, clinical stage, comorbidities, lymph nodes retrieved, and the number of narcotic vs. non-narcotic drug interventions administered. Robotic patients had a lower number of initial drug interventions (21 vs. 52; P<.001) and total drug interventions (162 vs. 219; P<.001) than laparoscopic patients. Robotics had a lower initial pain score (2.1 vs. 3.0; P=.012). There was a 50% reduction in the pain medication cost on the day of surgery for robotic patients ($12.24 vs. $24.45; P<.01), and a 56% cost reduction for the rest of their length of stay ($3.63 vs. $8.17; P<.01). CONCLUSION: Endometrial cancer patients who have robotic surgery experience less initial postoperative pain and have fewer drug interventions. The cost associated for their pain management represents a savings of greater than 50%. These factors demonstrate the value of robotic surgery in regard to postoperative pain management by delivering higher quality care at a lower cost.

Variability in the magnitude of response of metabolic enzymes reveals patterns of co-ordinated expression following endurance training in women.

Skeletal muscles improve their oxidative fatty acid and glucose metabolism following endurance training, but the magnitude of response varies considerably from person to person. In 20 untrained young women we examined interindividual variability in training responses of metabolic enzymes following 6 weeks of endurance training, sufficient to increase maximal oxygen uptake by 10 ± 8% (mean ± SD). Training led to increases in mitochondrial enzymes [succinate dehydrogenase (SDH; 47 ± 78%), cytochrome c oxidase (52 ± 70%) and ATP synthase (63 ± 69%)] and proteins involved in fatty acid metabolism [3-hydroxyacyl CoA dehydrogenase (69 ± 92%) and fatty acid transporter CD36 (86 ± 31%)]. Increases in enzymes of glucose metabolism [phosphofructokinase (29 ± 94%) and glucose transporter 4 (18 ± 65%)] were not significant. There was no relationship between changes in maximal oxygen uptake and the changes in the metabolic proteins. Considerable interindividual variability was seen in the magnitude of responses. The response of each enzyme was proportional to the change in SDH; individuals with a large increase in SDH also showed high gains in all other enzymes, and vice versa. Peroxisome proliferator-activated receptor γ coactivator 1α protein content increased after training, but was not correlated with changes in the metabolic proteins. In conclusion, the results revealed co-ordinated adaptation of several metabolic enzymes following endurance training, despite differences between people in the magnitude of response. Differences between individuals in the magnitude of response might reflect the influence of environmental and genetic factors that govern training adaptations.

HIF1A P582S gene association with endurance training responses in young women.

Sequence variations in the gene encoding the hypoxia-inducible factor-1alpha, HIF1A, have been associated with physiologic function and could be associated with exercise responses. In the HIF1A P582S gene polymorphism (C1772T; rs 11549465 C/T), a single nucleotide transition from C → T alters the codon sequence from the usual amino acid; proline (C-allele), to serine (T-allele). This polymorphism was examined for association with endurance training responses in 58 untrained young women who completed a 6-week laboratory-based endurance training programme. Participant groups were defined as CC homozygotes versus carriers of a T-allele (CC vs. CT genotypes). Adaptations were examined at the systemic-level, by measuring [Formula: see text] and the molecular-level by measuring enzymes determined from vastus lateralis (n = 20): 3-hydroacyl-CoA-dehydrogenase (HAD), which regulates mitochondrial fatty acid oxidation; cytochrome C oxidase (COX-1), a marker of mitochondrial density; and phosphofructokinase (PFK), a marker of glycolytic capacity. CT genotypes showed 45% higher training-induced gains in [Formula: see text] compared with CC genotypes (P < 0.05). At the molecular level, CT increased the ratios PFK/HAD and PFK/COX-1 (47 and 3%, respectively), while in the CC genotypes these ratios were decreased (-26 and -54%, respectively). In conclusion, the T-allele of HIF1A P582S was associated with greater gains in [Formula: see text] following endurance training in young women. In a sub-group we also provide preliminary evidence of differential muscle metabolic adaptations between genotypes.

Boosting enrollment in neurology trials with Local Identification and Outreach Networks (LIONs).

OBJECTIVE: Our purpose was to develop a geographically localized, multi-institution strategy for improving enrolment in a trial of secondary stroke prevention. METHODS: We invited 11 Connecticut hospitals to participate in a project named the Local Identification and Outreach Network (LION). Each hospital provided the names of patients with stroke or TIA, identified from electronic admission or discharge logs, to researchers at a central coordinating center. After obtaining permission from personal physicians, researchers contacted each patient to describe the study, screen for eligibility, and set up a home visit for consent. Researchers traveled throughout the state to enroll and follow participants. Outside the LION, investigators identified trial participants using conventional recruitment strategies. We compared recruitment success for the LION and other sites using data from January 1, 2005, through June 30, 2007. RESULTS: The average monthly randomization rate from the LION was 4.0 participants, compared with 0.46 at 104 other Insulin Resistance Intervention after Stroke (IRIS) sites. The LION randomized on average 1.52/1,000 beds/month, compared with 0.76/1,000 beds/month at other IRIS sites (p = 0.03). The average cost to randomize and follow one participant was $8,697 for the LION, compared with $7,198 for other sites. CONCLUSION: A geographically based network of institutions, served by a central coordinating center, randomized substantially more patients per month compared with sites outside of the network. The high enrollment rate was a result of surveillance at multiple institutions and greater productivity at each institution. Although the cost per patient was higher for the network, compared with nonnetwork sites, cost savings could result from more rapid completion of research.

p38 pathway kinases as anti-inflammatory drug targets.

Mitogen-activated protein kinases (MAPK) are intracellular signaling molecules involved in cytokine synthesis. Several classes of mammalian MAPK have been identified, including extracellular signal-regulated kinase, c-jun N-terminal kinase, and p38 MAP kinase. p38alpha is a key MAPK involved in tumor necrosis factor alpha and other cytokine production, as well as enzyme induction (cyclooxygenase-2, inducible nitric oxide synthase, and matrix metalloproteinases) and adhesion molecule expression. An understanding of the broad biologic and pathophysiological roles of p38 MAPK family members has grown significantly over the past decade, as has the complexity of the signaling network leading to their activation. Downstream substrates of MAPK include other kinases (e.g., mitogen-activated protein-kinase-activated protein kinase 2) and factors that regulate transcription, nuclear export, and mRNA stability and translation. The high-resolution crystal structure of p38alpha has led to the design of selective inhibitors that have good pharmacological activity. Despite the strong rationale for MAPK inhibitors in human disease, direct proof of concept in the clinic has yet to be demonstrated, with most compounds demonstrating dose-limiting adverse effects. The role of MAPK in inflammation makes them attractive targets for new therapies, and efforts are continuing to identify newer, more selective inhibitors for inflammatory diseases.

Identification and quantification of high fluorescence-stained lymphocytes as antibody synthesizing/secreting cells using the automated routine hematology analyzer XE-2100.

OBJECTIVES: The aim of this study was to classify and quantify the high fluorescence lymphocytes area (HFL-count) from the SYSMEX XE-2100 leucocyte differential channel as antibody-synthesizing or -secreting cells (ASC, plasma cells or lymphoplasmacytoid cells) in reactive diseases. To unequivocally identify the HFL cells, all possibly eligible cell populations have been investigated: activated B-lymphocytes, activated T-lymphocytes, large granular lymphocytes (LGL), activated monocytes, and immature granulocytes. METHODS: In total, 85 patients were analyzed on the XE-2100 and compared with the automated image analysis system Cellavision Diffmaster 96 based on artificial neural network and immunophenotyping method with the BD FACSCalibur. RESULTS: Reproducibility tests for HFL demonstrated a mean coefficient of variation of 13.9% for very low results and 1.5% for high results. The linearity data showed a good correlation (R(2) = 0.99) between expected and measured HFL. The comparison with possibly eligible cell populations showed no significant correlation between activated monocytes and immature granulocytes, with most immature granulocytes (promyelocyte I or II), natural killer cells or LGLs, activated T-lymphocytes, and sub-T-lymphocytes populations. However, for activated B-lymphocytes an excellent significant correlation with the peripheral blood smear, and the immunophenotyping method has been found with R(2) = 0.900, P < 0.001 and R(2) = 0.897, P < 0.001, respectively. The slope of 1.1 and intercept of minus 5 cells/microL of the regression equation between HFL-count and ASC (smear) do indicate an excellent quantification of the HFL-count, as well. CONCLUSION: The fully automated SYSMEX XE-2100 HFL-count identifies and quantifies the ASC cells (activated B-lymphocytes) with high precision and reliability in patients without hematology system diseases, thus providing a potential screening and monitoring tool for any patient with suspected infection. Additional studies are required to comprehend in more detail the full clinical utility of an HFL (ASC) count as a potential diagnostic indicator of inflammation, infection, or sepsis.

Enrichment of integral membrane proteins from small amounts of brain tissue.

Subcellular fractionation represents an essential technique for functional proteome analysis. Recently, we provided a subcellular fractionation protocol for minute amounts of tissue that yielded a nuclear fraction, a membrane and organelle fraction, and a cytosolic fraction. In the current study, we attempted to improve the protocol for the isolation of integral membrane proteins, as these are particularly important for brain function. In the membrane and organelle fraction, we increased the yield of membranes and organelles by about 50% by introducing a single re-extraction step. We then tested two protocols towards their capacity to enrich membrane proteins present in the membrane and organelle fraction. One protocol is based on sequential solubilization using subsequent increases of chaotropic conditions such as urea, thereby partitioning hydrophobic proteins from hydrophilic ones. The alternative protocol applies high-salt and high-pH washes to remove non-membrane proteins. The enrichment of membrane proteins by these procedures, as compared to the original membrane and organelle fraction, was evaluated by 16-BAC-SDS-PAGE followed by mass spectrometry of randomly selected spots. In the original membrane and organelle fraction, 7 of 50 (14%) identified proteins represented integral membrane proteins, and 15 (30%) were peripheral membrane proteins. In the urea-soluble fraction, 4 of 33 (12%) identified proteins represented integral membrane proteins, and 10 (30%) were peripheral membrane proteins. In the high-salt/high-pH resistant sediment, 12 of 45 (27%) identified proteins were integral membrane proteins and 13 (29%) represented peripheral membrane proteins. During the analysis, several proteins involved in neuroexocytosis were detected, including syntaxin, NSF, and Rab3-interaction protein 2. Taken together, differential centrifugation in combination with high-salt and high-pH washes resulted in the highest enrichment of integral membrane proteins and, therefore, represents an adequate technique for region-specific profiling of membrane proteins in the brain.

Add-back of allodepleted donor T cells to improve immune reconstitution after haplo-identical stem cell transplantation.

Poor immune reconstitution after haplo-identical stem cell transplantation results in high mortality from viral infections and relapse. One approach to overcome this problem is to deplete alloreactive cells selectively by deleting T cells activated by recipient stimulators, using an immunotoxin directed against the activation marker CD25. However, the degree of depletion of alloreactive cells is variable following stimulation with recipient PBMC, and this can result in GvHD. We have shown that using recipient EBV-transformed LCL as stimulators to activate donor alloreactive T cells results in more consistent depletion of in vitro alloreactivity while preserving T-cell responses to viral and potential myeloid tumor Ag. Based on these data, we have embarked on a phase I clinical dose escalation study of add-back of allo-LCL-depleted donor T cells in the haplo-identical setting, to determine if the allodepletion we achieve to allow infusion of sufficient T cells to restore useful antiviral/anti-leukemic responses without causing GvHD. Fifteen patients have so far been treated. The incidence of significant acute or chronic GvHD has been low (2/15), as has mortality from infection (1/15). Preliminary data show accelerated immune reconstitution in dose level 2 patients. Infused allodepleted donor T cells appear able to expand significantly in the face of viral reactivations, and doses as low as 3 x 10(5)/kg may be sufficient to confer useful antiviral immunity in this setting. At a median follow-up of 19.5 months, nine of 15 patients are alive and disease-free. Five patients have relapsed, all of whom have died.

Pseudosubarachnoid haemorrhage in subdural haematoma.

Two patients with large bilateral subdural haematomas with patterns of non-enhanced brain computed tomography (CT) falsely suggesting coexistent subarachnoid haemorrhage are presented. The CT images showed marked effacement of the basal cisterns with hyperdense signal along the tentorium, sylvian fissure, and the perimesencephalic cisterns. In both cases, the suspicion of subarachnoid haemorrhage led to the performance of angiographic studies to rule out vascular lesions. Thus, recognition of this radiological feature is important to avoid unnecessary testing and treatment delay.

Clinical evaluation of ricin A-chain immunotoxins in patients with Hodgkin's lymphoma.

BACKGROUND: Immunotoxins (ITs) consist of cell binding ligands coupled to toxins or their subunits. Hodgkin's lymphoma (HL) is an excellent target for ITs since lymphocyte activation markers such as CD25 and CD30 are expressed in large numbers. The ITs RFT5.dgA (anti CD25) and Ki-4.dgA (anti CD30) were constructed by linking the monoclonal antibodies RFT5 and Ki-4 to deglycosylated ricin A-chain (dgA). Both ITs showed potent specific activity against HL cells in vitro and in vivo in animal models, and were subsequently evaluated in phase I/II clinical trials in humans. PATIENTS AND METHODS: In two separate trials, the ITs were administered i.v. four times every other day over 4 h. The objectives of the phase I trials included the determination of the maximum tolerated dose (MTD), dose-limiting toxicities (DLTs), pharmacokinetics, antitumor activity and immune response against the IT. RESULTS: Twenty-seven patients with refractory HL were included in the phase I/II study of RFT5.dgA and 17 patients were included in the phase I study of Ki-4.dgA. The MTD of RFT5.dgA was 15 mg/m(2), whereas that of Ki-4.dgA was 5 mg/m(2). DLTs were related to vascular leak syndrome, consisting of edema, tachycardia, dyspnea, weakness and myalgia. Measurement of serum levels of RFT5.dgA demonstrated a C(max) of 0.2-9.7 micro g/ml with a half-life (t()) varying from 4 to 10.5 h. Peak serum concentration of Ki-4.dgA ranged from 0.23 to 1.7 micro g/ml. In both trials approximately 60% of patients developed human anti-mouse and/or anti-dgA antibodies. Seventeen of 18 patients treated at the MTD of RFT5.dgA were evaluable for clinical response. Responses included two partial remissions (PR), one minor response (MR) and five stable diseases (SD). Fifteen of 17 patients treated with Ki-4.dgA were evaluable for clinical response. Responses included one PR, one MR and two SD. CONCLUSIONS: RFT5.dgA and Ki-4.dgA showed moderate efficacy in heavily pretreated refractory patients with HL. Ki-4.dgA was less well tolerated than RFT5.dgA. This might be due, at least in part, to the formation of Ki-4.dgA/sCD30 complexes.

Optimized clinical-scale culture conditions for ex vivo selective depletion of host-reactive donor lymphocytes: a strategy for GvHD prophylaxis in allogeneic PBSC transplantation.

BACKGROUND: Ex vivo selective depletion (SD) is a strategy to prevent GvHD, in which host-reactive donor lymphocytes are selectively eliminated from a PBSC allograft while conserving useful donor immune function. Prior to testing this strategy in patients, our goal was to develop a clinical-scale SD process, which involves co-culture of donor lymphocytes and irradiated recipient cells, followed by the addition of an immunotoxin (IT) directed against the alpha-chain of the IL-2 receptor (CD25), expressed on activated donor T cells. METHODS: Stimulator cells were generated from immunomagnetically selected and expanded recipient T lymphocytes. Donor PBMCs from G-CSF-mobilized peripheral blood were co-cultured for 72 h with irradiated stimulator cells. Alloreactive T cells were targeted for elimination by the addition of the anti-CD25 IT, RFT5-SMPT-dgA, and the IT enhancer, NH(4)Cl. RESULTS: Stimulator-cell selection/expansion yielded > 2 x 10(10) highly enriched CD3(+) cells (98.9 +/- 2.2%). After SD, cell recovery was 68.5 +/- 23.3% and viability was 84.6 +/- 6.4%. This permitted a potential T-cell dose >/= 1 x 10(8) CD3(+) cells kg(-1) to transplant recipients. Although SD donor lymphocytes retained little proliferative capacity against the original stimulator cells (2.6 +/- 0.6%), responses were conserved against third party cells (107.6 +/- 18.6%), the bacterial superantigen staphylococcus enterotoxin B (108.2 +/- 4.2%), and CMV Ag (72.1 +/- 3.8%). DISCUSSION: We have demonstrated that ex vivo SD is feasible in clinical-scale culture conditions. The ability of this strategy to prevent GvHD is the subject of an ongoing clinical trial, in which the SD lymphocyte product is transplanted in conjunction with a T cell-depleted PBSC allograft.

[T-cell-depleted HLA non-identical bone marrow transplantation in the child: prevention of graft-versus-host reaction by administration of donor T lymphocytes alloreactive against the recipient]. / Greffe de moelle osseuse T déplétée HLA non géno-identique chez l'enfant: prévention de la réaction du greffon contre l'hôte par adjonction de lymphocytes T du donneur dépourvus d'alloréactivité vis-à-vis du receveur.

The success of HSCT from HLA partially disparate donors depends on the development of new strategies able to efficiently prevent GVHD and to protect patients from infections and relapse. Using an immunotoxin (IT) directed against the alpha-chain (p55) of the human IL-2r (RFT5-SMPT-dgA), we have previously shown that it is possible to kill mature T cells activated towards a specific HLA complex by a one-way MLR. We designed a clinical trial assessing the effect of infusing increasing doses of T lymphocytes in the setting of children recipients of non HLA genetically identical HSCT. Thirteen patients have been enrolled from September 1998 to April 2000 and fourteen HSCT have been realized in 13 patients (pts). Donors were MUD in 3 cases and familial HLA partially disparate in the remaining cases. Allodepleted donor T cells were injected between day +14 and day +30 provided that ATG was undetectable in the serum and blood PMN counts was > 500/microliter. The mean age of these patients was 17 months (range 1 to 42). Diagnosis included immune deficient and malignant hemopathies. Three patients received 1 x 10(5) allodepleted T cell/kg, 7 patients received 4 x 10(5)/kg and 4 patients received 6 x 10(5)/kg allodepleted T cells. Full inhibition of MLR was achieved in 12 out of 14 cases. In two cases, a residual T cell reactivity to the recipient was observed (4 to 5%) and patients developed grade II aGVHD. aGVHD occurred in 4 out of 11 grafted patients (all grade II). No chronic GVHD has developed, so far. Three patients died from severe VOD or PHT at day +34, day 51 and day +166, while one infected patient by VZV, CMV and EBV before HSCT died 6 months after transplantation from meningoencephalitis and another patient died from relapse at day +291. The patient for which there was no engraftment died at day +48 from staphylococcus infection. Overall survival is 54%, with a median follow up of 8 months; the mean time to reach a blood lymphocyte count > 500 was 41 days, to reach a CD3 count > 300 microliters 63 days (20-111), CD4 > 200 microliters 97 days and positive mitogen-induced proliferation 90 days. In three patients, a tetanus-toxoid positive proliferation was detected before immunization. From this intermediate analysis, we conclude that 1) specific allodepletion is an effective approach to prevent aGVHD in a haploincompatible setting, 2) data on immunological reconstitution suggest that infused T cells do survive and expand. A higher number of patients must be enrolled to determine the optimal number of T cells to infuse.