Arnold Pizzey

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.

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.

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

Human mesenchymal stem cells are potential agents for tissue regeneration, enhancing hematopoietic stem cell transplantation and delivering genes of therapeutic interest. This study shows that tissue homing of systemically administered mesenchymal stem cells can be increased by enforced expression of CXCR4, at least in irradiated hosts. 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 (1.5–2.0×106/animal) were injected via the tail vein into unconditioned β2m/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 β2m/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.

Fall in implantation rates following ICSI with sperm with high DNA fragmentation

BACKGROUND There is considerable uncertainty as to the significance of a high sperm DNA fragmentation index (DFI) for achieving a successful pregnancy. METHODS The sperm DFI of 124 patients undergoing 192 IVF cycles and of 96 patients undergoing 155 ICSI cycles was determined using the sperm chromatin structure assay on neat sperm. RESULTS The rate of continuing pregnancies in ICSI cycles (but not in IVF cycles) showed significant negative correlation (r = -0.184, P = 0.022) with the DFI value. A threshold value of DFI which showed a significant difference (P = 0.005) in rate of continuing pregnancies between higher and lower DFI levels was found for ICSI cycles to be > or = 19%, but no such threshold was found for IVF cycles. However, if the threshold of > or = 30% was used for IVF cycles there was a non-significant lowering of the rates of continuing pregnancy and implantation at the higher DFI levels. DFI level had no effect on fertilization rate or on the percentage of embryos having more than 4 cells at Day 3 after fertilization. A high DFI level had a marked significant effect (P = 0.001) on implantation rate in ICSI cycles but not in IVF cycles. A significant positive correlation (r = 0.268, P = 0.001) between DFI and sperm midpiece defects was also noted in the ICSI patients. CONCLUSIONS These observations may help to resolve the issues about how, and to what extent, sperm DNA damage impacts upon the success of IVF and ICSI procedures.

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 NFκB-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.

Relationship between male reproductive hormones, sperm DNA damage and markers of oxidative stress in infertility

This study investigated the relationship between male reproductive hormones and sperm DNA damage and markers of oxidative stress in men undergoing infertility evaluation for male factor (n = 66) and non-male factor (n = 63) infertility. Semen samples were analysed for DNA fragmentation index (DFI). Serum samples were analysed for FSH, inhibin B, anti-Müllerian hormone (AMH), testosterone and total antioxidant capacity (TAC). Serum inhibin B was significantly lower in the male factor group compared with the non-male factor group. Inhibin B showed a positive correlation with sperm concentration and motility, and serum AMH showed a positive correlation with sperm concentration and semen volume. DFI was 3-fold higher in the male factor group and showed a negative correlation with sperm motility. Blood plasma TAC was negatively related to sperm concentration. The results confirm that AMH and inhibin B are markers of Sertoli cell function. Sperm DNA damage is moderately increased in male factor infertility, and is negatively associated with sperm motility. A negative association between antioxidant activity and sperm concentration suggests that even minimal oxidative stress may influence sperm concentration. However, there was no significant relationship between hormone concentrations, sperm DNA damage and total antioxidant capacity, suggesting other mechanisms for sperm dysfunction.

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.

An activating mutation in the transmembrane domain of the granulocyte colony-stimulating factor receptor in patients with acute myeloid leukemia.

To date, constitutively activating point mutations reported in hematopoietic growth factor receptors in patients with acute myeloid leukemia (AML) have been restricted to receptors with intrinsic tyrosine kinase activity such as c-kit and FLT3. We describe here a Thr617Asn mutation in the transmembrane domain of the non-tyrosine kinase receptor for granulocyte colony-stimulating factor (G-CSF) in the blast cells of two out of 555 AML patients examined. The mutant receptor conferred growth factor independence on factor-dependent Ba/F3 cells. In the absence of ligand, immunoblotting showed weak phosphorylation of JAK2, STAT3, ERKs 1 and 2 and the receptor itself, and there was approximately 70% of maximal growth in a proliferation assay. All signals were significantly enhanced in the presence of G-CSF. Retroviral transduction of mutant receptor into primary hematopoietic CD34+ cells induced G-CSF independent myeloid differentiation as assessed by the development of neutrophils and surface expression of CD11b and CD14. These results confirm the importance of the transmembrane domain for receptor function and suggest that introduction of an asparagine residue can cause sufficient stabilization of helix–helix interactions in the absence of ligand to activate downstream signaling pathways involved in directing proliferation and differentiation.

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

Summary. 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.