Activation of the LMO2 oncogene through a somatically acquired neomorphic promoter in T-cell acute lymphoblastic leukemia.

Somatic mutations within noncoding genomic regions that aberrantly activate oncogenes have remained poorly characterized. Here we describe recurrent activating intronic mutations of LMO2, a prominent oncogene in T-cell acute lymphoblastic leukemia (T-ALL). Heterozygous mutations were identified in PF-382 and DU.528 T-ALL cell lines in addition to 3.7% of pediatric (6 of 160) and 5.5% of adult (9 of 163) T-ALL patient samples. The majority of indels harbor putative de novo MYB, ETS1, or RUNX1 consensus binding sites. Analysis of 5'-capped RNA transcripts in mutant cell lines identified the usage of an intermediate promoter site, with consequential monoallelic LMO2 overexpression. CRISPR/Cas9-mediated disruption of the mutant allele in PF-382 cells markedly downregulated LMO2 expression, establishing clear causality between the mutation and oncogene dysregulation. Furthermore, the spectrum of CRISPR/Cas9-derived mutations provides important insights into the interconnected contributions of functional transcription factor binding. Finally, these mutations occur in the same intron as retroviral integration sites in gene therapy-induced T-ALL, suggesting that such events occur at preferential sites in the noncoding genome.

Flow-Based Single Cell Deposition for High-Throughput Screening of Protein Libraries.

The identification and engineering of proteins having refined or novel characteristics is an important area of research in many scientific fields. Protein modelling has enabled the rational design of unique proteins, but high-throughput screening of large libraries is still required to identify proteins with potentially valuable properties. Here we report on the development and evaluation of a novel fluorescent activated cell sorting based screening platform. Single bacterial cells, expressing a protein library to be screened, are electronically sorted and deposited onto plates containing solid nutrient growth media in a dense matrix format of between 44 and 195 colonies/cm2. We show that this matrix format is readily applicable to machine interrogation (<30 seconds per plate) and subsequent bioinformatic analysis (~60 seconds per plate) thus enabling the high-throughput screening of the protein library. We evaluate this platform and show that bacteria containing a bioluminescent protein can be spectrally analysed using an optical imager, and a rare clone (0.5% population) can successfully be identified, picked and further characterised. To further enhance this screening platform, we have developed a prototype electronic sort stream multiplexer, that when integrated into a commercial flow cytometric sorter, increases the rate of colony deposition by 89.2% to 24 colonies per second. We believe that the screening platform described here is potentially the foundation of a new generation of high-throughput screening technologies for proteins.

Successful outcomes achieved in assisted reproduction cycles using sperm with high levels of high DNA stainability.

The sperm chromatin structure assay (SCSA) has been proposed as a useful addition to the battery of tests routinely used to explore semen quality and hence to give an indication of the likelihood of a successful pregnancy. As usually performed at present, the assay yields two main sperm variables, the DNA fragmentation index (DFI) and the high DNA stainability (HDS). In the present study 275 patients undergoing 215 in vitro fertilization (IVF) and 215 intracytoplasmic sperm injection (ICSI) cycles were studied with the purpose of defining the clinical significance of HDS in IVF and ICSI cycles. Using the Spearman correlation test there were no significant statistical relationships between %HDS and fertilization rate, rate of embryo growth, blastocyst rate, implantation rate, or live birth rate. Rate of pregnancy loss showed a negative relationship significant at the 0.05 level which is unexplained. It is not known whether the normal practice of using processed sperm for fertilization plays any part in this lack of a negative effect of HDS level upon the stages of the cycle. A total of 16 patients with HDS levels >28% had an average live birth rate of 47.8% and an average pregnancy loss of 8.7%, which compared favourably with the group of patients as a whole.

Procoagulant activity in patients with sickle cell trait.

Patients with sickle cell trait (STr) are usually considered to be asymptomatic. However, complications, including hypercoagulability, increased risk of venous thromboembolism and the exertional exercise syndrome with rhabdomyolysis and sudden death, have been described. The exact cause of these adverse events is unclear. We have investigated two patients, a set of monozygotic twins with STr, to establish their procoagulant activity status as a potential indicator of thrombotic risk. In-vivo thrombin generation was assessed by the measurement of prothrombin fragment 1 + 2 (F1 + 2) and thrombin-antithrombin complexes (TAT). D-dimer was used as a marker of fibrinolytic activity. The potential to generate thrombin was determined using an ex-vivo thrombin generation test (TGT). The impact of red blood cell (RBC)-derived microparticle shedding and RBC rheology were examined. TAT (>60 µg/l) and F1 + 2 (948 pmol/l) were markedly elevated in patient 2 but within the normal reference range in patient 1 (TAT = 2.5 µg/l; F1 + 2 = 138 pmol/l). D-dimer levels (0.9 mg/l FEU) were similarly elevated in both patients. TGT peak thrombin and endogenous thrombin potential (ETP) were elevated to similar degrees in both patients. Flow cytometric analysis for RBC-derived microparticles showed that both patients had elevated levels on two occasions. RBC deformability, blood viscosity and RBC aggregation were normal and similar in both patients. The results demonstrated different coagulation activity in the patients with one patient in a prothrombotic state, suggesting that there may be two levels of hypercoagulability in STr. Measurement of such differences would allow for separation of high and low-risk patients from serious complications.

Pro- and antiinflammatory cytokines in threatened miscarriages.

OBJECTIVE: The purpose of this study was to evaluate circulating and intracellular levels of Th1 and Th2 cytokines in women with threatened miscarriage (TM) and subsequent outcome. STUDY DESIGN: Plasma levels of tumor necrosis factor (TNF)-receptors 1 and 2, TNFα, interferon gamma (IFNγ), and interleukins (IL) -6 and -10 were measured by flow cytometric bead assays in 80 women with TM: 53 women with normal outcome and 27 women who miscarried. Fluorescent antibody labeling was also performed on whole blood in a subgroup of 27 women of TM: 16 women with normal outcome and 11 women who miscarried. RESULTS: Monocyte expression of TNFα and circulating levels of TNFα, IFNγ, IL-10, IL-6, and TNF-R1 were significantly lower, whereas circulating levels of TNFα/IL-10, IFNγ/IL-10, and TNFα/IL-6 ratios were significantly higher, in women with TM who subsequently miscarried, compared with the women with normal outcome. CONCLUSION: An increased Th1 type of immune response, which was similar to that observed in preterm delivery, was found in TM cases that were complicated by a subsequent miscarriage.

Cytomegalovirus-specific T cell immunotherapy promotes restoration of durable functional antiviral immunity following allogeneic stem cell transplantation.

BACKGROUND: The profound immunodeficiency associated with allogeneic hematopoietic stem cell transplantation is permissive to uncontrolled replication of latent human herpesviridae such as cytomegalovirus. Morbidity and mortality associated with viral dissemination or its treatment are significant. Although adoptive cellular therapy with virus-specific T cells offers the potential for accelerating pathogen-specific immune reconstitution, the risk of induction of graft-versus-host disease and the logistics of production of clonal T cell populations restrict application. METHODS: We investigated the ability of cytomegalovirus-specific mixed CD4(+) and CD8(+) T cell lines, generated by short-term ex vivo culture of donor lymphocytes with donor monocyte-derived dendritic cells pulsed with virus lysate, to restore antiviral immunity in 30 allogeneic transplant recipients at high risk of both uncontrolled viral replication and of graft-versus-host disease. RESULTS: There were no immediate toxicities and no excess of graft-versus-host disease. Massive in vivo expansions of cytomegalovirus-specific T lymphocytes occurred, temporally associating with periods of viral replication, suggesting that antigen exposure was necessary for optimal cytomegalovirus-specific immune reconstitution. The expanding populations maintained functional competence in ex vivo re-stimulation assays, promoting reconstitution of durable functional cytomegalovirus-specific immunity and effectively preventing recurrent viral infection and late cytomegalovirus disease. CONCLUSIONS: These data confirm the ability of cellular immunotherapy to hasten reconstitution of antiviral immunity following allogeneic transplantation, indicating that significant clinical benefits may be conferred in terms of reduction of secondary viral infection episodes, potentially reducing exposure to the toxicities of antiviral drugs.

KSHV manipulates Notch signaling by DLL4 and JAG1 to alter cell cycle genes in lymphatic endothelia.

Increased expression of Notch signaling pathway components is observed in Kaposi sarcoma (KS) but the mechanism underlying the manipulation of the canonical Notch pathway by the causative agent of KS, Kaposi sarcoma herpesvirus (KSHV), has not been fully elucidated. Here, we describe the mechanism through which KSHV directly modulates the expression of the Notch ligands JAG1 and DLL4 in lymphatic endothelial cells. Expression of KSHV-encoded vFLIP induces JAG1 through an NFkappaB-dependent mechanism, while vGPCR upregulates DLL4 through a mechanism dependent on ERK. Both vFLIP and vGPCR instigate functional Notch signalling through NOTCH4. Gene expression profiling showed that JAG1- or DLL4-stimulated signaling results in the suppression of genes associated with the cell cycle in adjacent lymphatic endothelial cells, indicating a role for Notch signaling in inducing cellular quiescence in these cells. Upregulation of JAG1 and DLL4 by KSHV could therefore alter the expression of cell cycle components in neighbouring uninfected cells during latent and lytic phases of viral infection, influencing cellular quiescence and plasticity. In addition, differences in signaling potency between these ligands suggest a possible complementary role for JAG1 and DLL4 in the context of KS.

Insulin uptake across the luminal membrane of the rat proximal tubule in vivo and in vitro.

We visualized insulin uptake in vivo across the apical membrane of the rat proximal tubule (PT) by confocal microscopy; we compared it with in vitro findings in a rat PT cell line (WKPT) using fluorescence microscopy and flow cytometry. Surface tubules were observed in vivo with a 633-nm single laser-illuminated real-time video-rate confocal scanning microscope in upright configuration for optical sectioning below the renal capsule. Fields were selected containing proximal and distal tubules; Cy5-labeled insulin was injected twice (the second time after approximately 140 min) into the right jugular vein, and the fluorescence signal (at 650-670 nm) was recorded. Fluorescence was detected almost immediately at the brush-border membrane (BBM) of PT cells only, moving inside cells within 30-40 min. As a measure of insulin uptake, the ratio of the fluorescence signal after the second injection to the first doubled (ratio: 2.11 +/- 0.26, mean +/- SE, n = 10), indicating a "priming," or stimulating, effect of insulin on its uptake mechanism at the BBM. This effect did not occur after pretreatment with intravenous lysine (ratio: 1.03 +/- 0.07, n = 6; P < 0.01). Cy2- or Cy3-labeled insulin uptake in a PT cell line in vitro was monitored by 488-nm excitation fluorescence microscopy using an inverted microscope. Insulin localized toward the apical membrane of these cells. Semiquantitative analysis of insulin uptake by flow cytometry also demonstrated a priming effect (upregulation) on insulin internalization in the presence of increasing amounts of insulin, as was observed in vivo; moreover, this effect was not seen with, or affected by, the similarly endocytosed ligand beta2-glycoprotein.

Homozygous HAX1 mutations in severe congenital neutropenia patients with sporadic disease: a novel mutation in two unrelated British kindreds.

Patients with autosomal dominant (AD), sporadic and X-linked severe congenital neutropenia (SCN) may have mutations in the elastase 2 (ELA2) or Wiskott-Aldrich syndrome (WAS) genes. Homozygous mutations in the HAX1 gene have recently been reported in autosomal recessive (AR) cases of primarily Middle-Eastern descent and the original Kostmann family. We screened 109 predominantly Caucasian SCN kindreds for mutations in these genes; 33 (30%) had 24 different ELA2 mutations, five of them novel, two kindreds (2%) had WAS mutations and four kindreds (4%) had three different HAX1 mutations, two of them novel. One HAX1 mutation (p.Ser43LeufsX11) was found in an AR Ashkenazi Jewish kindred, the other (p.Glu31LysfsX54) in two unrelated British patients with sporadic disease. Microsatellite analysis of the HAX1 locus revealed a common haplotype (maximum distance 4.1 Megabases) for the p.Glu31LysfsX54 patients, suggesting a possible ancestral founder. In functional assays, the level of spontaneous and staurosporine-induced apoptosis was increased in neutrophils from both p.Ser43LeufsX11 patients but not a p.Glu31LysfsX54 patient, suggesting the possible presence of modifying factors. The low incidence of HAX1 mutations in our study suggests that the frequency may vary between racial groups but suggests that irrespective of inheritance or racial origin, SCN patients should be screened for HAX1 mutations.

Factors that influence short-term homing of human bone marrow-derived mesenchymal stem cells in a xenogeneic animal model.

BACKGROUND: Human mesenchymal stem cells are potential agents for tissue regeneration, enhancing hematopoietic stem cell transplantation and delivering genes of therapeutic interest. To implement any of these strategies successfully, we need a better understanding of factors that influence the tissue distribution of systemically administered mesenchymal stem cells. DESIGN AND METHODS: The present study was designed to investigate the short-term tissue homing of mesenchymal stem cells in immunodeficient mouse models, exploring the effects of animal age, duration of ex vivo expansion of mesenchymal stem cells, lentiviral transduction and CXCR4 over-expression. Dye-labeled mesenchymal stem cells (1.5-2.0 x 10(6)/animal) were injected via the tail vein into unconditioned beta2m/NOD/SCID animals. Animals were sacrificed 20-24 hours later and cell suspensions from tissues were examined by flow cytometry for the presence of PKH-positive cells. RESULTS: PKH-positive cells were readily detected in the bone marrow, spleen, liver and lungs at 20-24 hours after infusion. The homing of systemically infused mesenchymal stem cells to the bone marrow and spleen of unconditioned beta2m/NOD/SCID animals was significantly (>2-fold, p<0.001) higher in younger (<10 weeks) animals, and was reduced with increasing passage number. Despite low surface CXCR4 expression, human mesenchymal stem cells migrated to SDF-1 in vitro, and this was enhanced by over-expression of CXCR4 using lentiviral transduction. Over-expression of CXCR4 by lentiviral transduction (>80%) did not alter the bone marrow homing of mesenchymal stem cells in unconditioned animals, but caused a significant (p<0.05) increase in homing to bone marrow and spleen of animals that had received prior irradiation. CONCLUSIONS: Tissue homing of systemically administered mesenchymal stem cells is influenced by host factors such as age, is diminished by prolonged in vitro culture, and can be increased by enforced expression of CXCR4, at least in irradiated hosts.

Microvesicles in haemoglobinopathies offer insights into mechanisms of hypercoagulability, haemolysis and the effects of therapy.

Levels of circulating red blood cell (RBC)-derived vesicles are increased in sickle cell anaemia (SCA) and thalassaemia intermedia (TI) but the mechanisms, effects and controlling factors may differ. This study found that levels of vesicles and intravascular haemolysis were linked as shown by the correlation between levels of vesicles and plasma Hb. Vesicle levels were 6-fold greater in SCA and 4-fold greater in TI than in controls. The proportion of plasma Hb within vesicles was increased in SCA and TI with a significantly higher proportion in TI. We examined whether subpopulations of RBC expressing phosphatidylserine (PS) were a source of PS(+) vesicles and observed a significant association. Thrombin generation was promoted by the vesicles in which 40-50% expressed PS. In TI, markers of thrombin generation were significantly related to PS(+) RBC. Splenectomy in TI had significant effects including greater increases in vesicle levels, plasma Hb, PS(+) RBCs and thrombin generation markers than in unsplenectomised patients. In hydroxycarbamide (HC)-treated SCA patients these measures were decreased compared with untreated controls. The relationship between vesicle levels and plasma Hb suggests a mechanism linking vesiculation to haemolysis and consequently nitric oxide (NO) bioavailability and suggests a means by which HC treatment improves NO bioavailability.

Human mesenchymal stem cells (hMSCs) expressing truncated soluble vascular endothelial growth factor receptor (tsFlk-1) following lentiviral-mediated gene transfer inhibit growth of Burkitt's lymphoma in a murine model.

BACKGROUND: Efficient gene transfer to bone marrow derived mesenchymal stem cells (MSC) would provide an important opportunity to express potent anticancer agents in the tumour microenvironment because of their contribution to the tumour stroma. METHODS: HIV-based lentiviral vectors were pseudotyped with four different envelope proteins; amphotropic murine leukaemia virus (ampho), murine leukaemia virus (10A1), feline endogenous virus (RD114), and the vesicular stomatitis virus glycoprotein (VSVG). These pseudotypes were examined for transduction efficiency in human bone marrow derived MSC. The effect of lentiviral expression of truncated soluble vascular endothelial growth factor decoy receptor (tsFlk-1) in MSC on growth of Raji cells was determined, both in vitro and in vivo. RESULTS: All lentiviral vectors produced significant levels of transduction at an multiplicity of infection (MOI) of 1, those bearing the RD114 envelope glycoprotein consistently produced higher transduction levels (mean 70 +/- 6%) compared with the other pseudotyped lentiviral vectors, although there was significant inter-donor variation. Stable transgene expression was achieved after multiple rounds of transduction with VSVG-pseudotyped particles, without alteration in the differentiative capacity of transduced cells. Co-injection of MSC stably expressing tsFlk-1 with Raji Burkitt's lymphoma cells significantly impaired subcutaneous tumour growth in immunodeficient mice when compared to controls where either unmanipulated MSC or GFP-expressing MSC were used. CONCLUSIONS: Human MSC are easily transduced by pseudotyped lentiviral particles but there is inter-donor variation in transduction efficiency. Gene-modified MSC expressing a gene of therapeutic potential can moderate growth of haematological malignancies.

Relationship between FLT3 mutation status, biologic characteristics, and response to targeted therapy in acute promyelocytic leukemia.

The prognostic significance of FLT3 mutations in acute promyelocytic leukemia (APL) is not firmly established and is of particular interest given the opportunities for targeted therapies using FLT3 inhibitors. We studied 203 patients with PML-RARA-positive APL; 43% of the patients had an FLT3 mutation (65 internal tandem duplications [ITDs], 19 D835/I836, 4 ITD+D835/I836). Both mutations were associated with higher white blood cell (WBC) count at presentation; 75% of the patients with WBC counts of 10 x 10(9)/L or greater had mutant FLT3. FLT3/ITDs were correlated with M3v subtype (P < .001), bcr3 PML breakpoint (P < .001), and expression of reciprocal RARA-PML transcripts (P = .01). Microarray analysis revealed differences in expression profiles among patients with FLT3/ITD, D835/I836, and wild-type FLT3. Patients with mutant FLT3 had a higher rate of induction death (19% vs 9%; P = .04, but no significant difference in relapse risk (28% vs 23%; P = .5) or overall survival (59% vs 67%; P = .2) at 5 years. In in vitro differentiation assays using primary APL blasts (n = 6), the FLT3 inhibitor CEP-701 had a greater effect on cell survival/proliferation in FLT3/ITD+ cells, but this inhibition was reduced in the presence of ATRA. Furthermore, in the presence of CEP-701, ATRA-induced differentiation was reduced in FLT3/ITD+ cells. These data carry implications for the use of FLT3 inhibitors as frontline therapy for APL.

Generation of potent antitumor CTL from patients with multiple myeloma directed against HM1.24.

PURPOSE: The purpose of this work was to test the suitability of the HM1.24 antigen as a CTL target for immunotherapy of patients with multiple myeloma. EXPERIMENTAL DESIGN: Antigen-specific T cells were generated from patients with multiple myeloma using stimulation with protein-pulsed dendritic cells and tested in ELISPOT and CTL assays. RESULTS: HM1.24-primed T cells responded selectively to HM1.24-loaded autologous peripheral blood mononuclear cells (PBMC) in an IFN-gamma ELISPOT assay (median, 342; range, 198-495 IFN-gamma-producing cells/10(5) cf. unloaded PBMC median, 98; range, 7-137; P < 0.05, n = 5) and also to autologous malignant plasma cells (MPC; median, 227; range, 153-335; P < 0.05 when compared with the response to allogeneic MPC median, 57; range, 22-158; n = 5). HM1.24-primed T cells lysed autologous MPC (at 20:1 E/T ratio: median, 48% specific killing; range, 23-88%; at 10:1 E/T ratio: median, 43%; range, 15-80%; n = 12) but not allogeneic MPC. Lysis of autologous MPC was inhibited by anti-MHC class I but not anti-MHC class II antibodies and was blocked by Concanamycin A. Lysis of autologous MPC was blocked by competition with autologous HM1.24-transfected dendritic cells (10:1 ratio with autologous MPC). Unmanipulated, or control plasmid-transfected dendritic cells had no effect on lysis of autologous MPC. CONCLUSION: Our results indicate that HM1.24 is a promising target for immunotherapy of multiple myeloma.

Four new cases of stomatin-deficient hereditary stomatocytosis syndrome: association of the stomatin-deficient cryohydrocytosis variant with neurological dysfunction.

This report concerns congenitally Na(+)-K(+) leaky red cells of the 'hereditary stomatocytosis' class. Three new isolated cases and one new pedigree are described, and one previously reported case is expanded. In all cases, Western blotting of red cell membranes revealed a deficiency in the 32 kDa membrane protein, stomatin. All showed pronounced cation leaks at 37 degrees C with markedly abnormal intracellular Na(+) and K(+) concentrations, like all other such stomatin-deficient cases. Consistent with recent findings in two previously described British pedigrees, immunocytochemistry demonstrated that the deficiency of stomatin was not complete. On typical blood films, some red cells showed positive stomatin immunoreactivity, while most were negative, although in one case only a minority were negative. All platelets and neutrophils were stomatin positive. The cases differed markedly between themselves with regard to the temperature dependence of the passive leak to K(+). Three showed a simple monotonic temperature dependence, while two showed a minimum at around 20-25 degrees C, such that the cells were extremely leaky at 0 degrees C, giving the phenotype known as 'cryohydrocytosis'. These patients are the only two known cases of stomatin-deficient cryohydrocytosis. Both showed a congenital syndrome of mental retardation, seizures, cataracts and massive hepatosplenomegaly, probably defining a new haemato-neurological syndrome.

Impaired bone marrow homing of cytokine-activated CD34+ cells in the NOD/SCID model.

The reduced engraftment potential of hematopoietic stem/progenitor cells (HSPCs) after exposure to cytokines may be related to the impaired homing ability of actively cycling cells. We tested this hypothesis by quantifying the short-term homing of human adult CD34+ cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) animals. We show that the loss of engraftment ability of cytokine-activated CD34+ cells is associated with a reduction in homing of colony-forming cells (CFCs) to bone marrow (BM) at 24 hours after transplantation (from median 2.8% [range, 1.9%-6.1%] to 0.3% [0.0%-0.7%]; n = 3; P < .01), coincident with an increase in CFC accumulation in the lungs (P < .01). Impaired BM homing of cytokine-activated cells was not restored by using sorted cells in G0G1 or by inducing cell cycle arrest at the G1/S border. Blocking Fas ligation in vivo did not increase the BM homing of cultured cells. Finally, we tested cytokine combinations or culture conditions previously reported to restore the engraftment of cultured cells but did not find that any of these was able to reverse the changes in homing behavior of cytokine-exposed cells. We suggest that these changes in homing and, as a consequence, engraftment result from the increased migratory capacity of infused activated cells, leading to the loss of selectivity of the homing process.

T- and B-cell immune reconstitution and clinical outcome in patients with multiple myeloma receiving T-cell-depleted, reduced-intensity allogeneic stem cell transplantation with an alemtuzumab-containing conditioning regimen followed by escalated donor lymphocyte infusions.

Immune reconstitution after conventional allogeneic transplantation is a major determinant of survival. We conducted a detailed investigation of T- and B-cell immune reconstitution and clinical outcome in 19 patients with multiple myeloma undergoing reduced-intensity stem cell transplantation using in vivo T-cell depletion with alemtuzumab. These patients experienced delayed T-cell recovery, particularly in the naïve (CD45 RA+) CD4 compartment. T-cell receptor spectratype analysis showed a reduced repertoire diversity, which improved rapidly after the administration of donor leucocyte infusions and subsequent conversion to full donor T-cell chimaerism. Post-transplant recovery of CD19+ B cells was also delayed for up to 18 months. Spectratype analysis of IgH CDR3 repertoire revealed a gradual normalization in IgM spectratype complexity by 6-12 months after transplant. There was a high incidence of viral infection, particularly cytomegalovirus reactivation, but the regimen-related mortality was low, perhaps because of the very low incidence of acute graft-versus-host disease (GVHD; grade I-II skin GVHD was seen in 5/19 patients). Over 80% of all patients have relapsed at a median of 283 (range 153-895) d after transplant, suggesting that the initially low rate of GVHD comes at a high price with regard to the desired graft-versus-myeloma effect.

Adoptive cellular therapy for early cytomegalovirus infection after allogeneic stem-cell transplantation with virus-specific T-cell lines.

Adoptive transfer of CMV-specific T cells offers the potential for reconstitution of viral immunity after allogeneic transplantation. However, the logistics of producing virus-specific T-cell clones has limited the application of cellular therapies. We treated 16 patients for CMV infection with polyclonal CMV-specific T-cell lines generated by short-term culture. Massive in-vivo expansions of CMV-specific cytotoxic T lymphocytes were observed, resulting in reconstitution of viral immunity. In eight cases antiviral drugs were not required, and subsequent episodes of reactivation occurred in only two patients. Our findings indicate that application of CMV-specific cell lines is both feasible and effective in a clinical environment.

The "stomatin" gene and protein in overhydrated hereditary stomatocytosis.

In overhydrated hereditary stomatocytosis (OHSt), Coomassie- and silver-stained polyacrylamide gels show an apparently complete deficit of the 32-kDa membrane protein, stomatin. We have used an antistomatin antibody to examine peripheral blood films, bone marrow, splenic tissue, and hepatic tissue from these patients by immunocytochemistry. This technique revealed that, in fact, some red cells did show positive stomatin immunoreactivity; and consistent with this result, Western blot analysis of the red cell membranes confirmed that about one twentieth to one fiftieth of the normal amount of stomatin was in fact present. Flow cytometry, combining immunoreactive quantitation of stomatin expression with thiazole orange staining for reticulocytes, showed that in OHSt, it was the young cells that had more stomatin. Magnetic-activated cell separation studies, using beads to which an anti-transferrin receptor antibody was conjugated, confirmed that in OHSt there was a correspondence between expression of stomatin and the transferrin receptor. Immunocytochemistry and Western blotting revealed that in OHSt patients, the protein was present in spleen, liver, neutrophils, platelets, monocytes, and about 50% of the peripheral lymphocytes, with the same distribution as in healthy controls. Neither Southern blots, nor direct sequencing of multiple subclones of the cDNA, nor sequencing of amplicons from genomic DNA revealed any significant abnormality in stomatin gene sequence in these patients. The deficiency of stomatin from red cells appears to be due to a loss of stomatin from these red cells on maturation in the bone marrow and in the circulation.

Reconstitution of T-cell repertoire after autologous stem cell transplantation: influence of CD34 selection and cytomegalovirus infection.

The period of immunodeficiency following autologous hematopoietic stem cell transplantation is characterized by transient expansions of CD8+CD45RO+CD57+ T lymphocytes, displaying markers of an activated phenotype. Most evidence suggests that this early reconstitution results from proliferation of mature T cells that have survived conditioning or were transferred with the graft. Although homeostatic mechanisms are thought to act in maintaining total T-cell numbers, the degree to which antigen-driven expansions contribute and the nature of the stimulating antigens remain unclear. CD34 selection of stem cell grafts reduces the available T-cell pool, potentially delaying immune reconstitution and resulting in increased infective complications. In the allogeneic transplantation setting, lymphopenia has been associated with cytomegalovirus (CMV) infection risk and, if persistent, with adverse outcome. We prospectively studied patients undergoing CD34-selected (n = 13) or unselected (n = 13) autologous hematopoeitic stem cell transplantation for immune reconstitution and CMV infection. No significant differences were demonstrated between graft types with respect to lymphocyte subset recovery, T-cell receptor beta-chain variable region spectratype diversity, or CMV DNA detection rates (45% versus 40%). CMV infection was associated with a trend toward higher rather than lower CD8+ counts at 6 weeks posttransplantation (P =.08) that became significant by 3 months (P=.007), and that was associated with decreased T-cell receptor beta-chain variable region spectratype diversity (P =.01). CMV-specific HLA-tetramer analysis demonstrated transient expansions with CDR3 lengths corresponding to those of some of the major posttransplantation T-cell expansions demonstrated by spectratype analysis suggesting that CMV-specific T cells contribute to the pattern of immune reconstitution.