ALBERT

Description: Epstein Barr Virus (EBV) associated Lymphoproliferative Disease (LPD) is a complication of allogeneic haemopoietic stem cell tranplantation (HSCT). In certain groups (congenital immunodeficiency, unrelated and mismatched donor transplants, T cell depletion) the risk may be as high as 25% with significant morbidity and mortality. Strategies to predict such illlness and allow early intervention have therefore assumed importance. We have routinely screened peripheral blood of paediatric, transplanted patients by quantitiative PCR. We report the results of such analysis of 28 successive patients and the EBV serial quantitation in 4 patients with EBV LPD. The median age at time of transplant was 6.5 years. 17 patients received an unrelated donor transplant and one patient received a haplo-identical transplant. The remainder (n=9) received a matched family donor transplant. 23 patients received either Alemtuzumab (n=19) or ATG (n=4). 13 patients had leukaemia, 5 had mucopolysaccharide syndrome, 4 for congential immune deficiency and 6 for non malignant haeamtological conditions. 9 (32%) patients showed no evidence of EBV reactivation using serial PCR monitoring. 10 patients had low level EBV reactivation as defined with a PCR log[copy number] 6, whilst the highest level without disease was 5.2). All 4 patients responded to therapy for EBV LPD, with a combination of rituximab, withdrawal of immune suppression or administration of donor lymphocytes - DLI). At higher EBV levels the quantitative PCR had increasing positive predictive value for clinical LPD. We therefore conclude that EBV serial quantitative PCR is useful in discriminating those who will develop LPD from those that will not. Our data suggest that it is possible to use EBV PCR quantitation further to discriminate asymptomatic EBV reactivation that will resolve without therapy from EBV reactivation that will require intervention. This prevents over exposure of patients to treatments (rituximab, DLI, immune suppression withdrawal) with significant associated toxicity (prolonged B cell aplasia, graft versus host disease).

Description: We have investigated the role of the Rho and Rac family small guanine triphosphate (GTP) exchange factors (RhoGEFs), Vav1 and Vav2, in the activation of platelets by the immunoreceptor tyrosine-based activation motif (ITAM)–coupled collagen receptor GPVI and by the G protein–coupled receptor agonist thrombin. The glycoprotein VI (GPVI)–specific agonist collagen-related peptide (CRP) and thrombin stimulated tyrosine phosphorylation of Vav1 but not Vav2 in human platelets. Surprisingly, however, CRP did not activate the low-molecular-weight G protein Rac and stimulated only a small increase in activity of p21-associated kinase 2 (PAK2), despite the fact that both proteins are regulated downstream of Vav1 in other cells. Further, activation of Rac and PAK2 by thrombin was maintained in platelets from mice deficient in Vav1. Activation of phospholipase C (PLC) by GPVI and thrombin was unaltered in Vav1-, Vav2-, and Vav1/Vav2-deficient platelets. A weak inhibition of late-stage aggregation to CRP and thrombin was observed in platelets deficient in Vav1 but not Vav2, whereas spreading on fibrinogen was not changed. The present results demonstrate that neither Vav1 nor Vav2 lie upstream of PLC or Rac in platelets, highlighting an important difference in their role in signaling by ITAM-coupled receptors in other cell types. The present study has provided evidence for a possible role of Vav1 but not Vav2 in the later stages of platelet aggregation.

Description: Intravenous BU allows more predictable exposure and is more convenient to administer once daily than the conventional four times daily regimen. Low-dose pretransplant ATG may reduce morbidity and mortality from graft-versus-host disease (GVHD). We have investigated a myeloablative regimen incorporating these agents in 140 adults aged 18–65 (median 47) receiving a first MRD SCT between 05/99 and 01/04. Conditioning comprised fludarabine (FLU) 50mg/m2on days −6 to −2 and IV BU (Busulfex, ESP Pharma) 3.2 mg/kg daily days −5 to −2 inclusive (FLUBUP) in 116 patients (pts) with additional TBI 200cGy x 2 in 24 (14 ALL, 8AML, 2 biphenotypic). Forty-three pts had standard-risk (SR) acute leukemia in CR1 (35 AML, 8 ALL), and 23 had active acute leukemia (HA) (18 AML±MDS, 3 ALL, 2 biphenotypic). A third group of 74 other (OTH) pts comprised 2 AML CR2, 2 ALL CR2, 2 CML (1 AP, 1CP2), 13 MDS, 11 MM, 24 NHL, 6 HD (2 with CLL), 8 CLL, 5 other. All pts were given cyclosporine A, “short course” methotrexate with folinic acid and Thymoglobulin (Genzyme) (ATG) 4.5 mg/kg in divided doses over 3 consecutive days pretransplant finishing D0. Followup of survivors is 6–61 months, median 26. Incidence of acute GVHD (aGVHD) grade II–IV was 19±4%, grade III–IV 6±2% and chronic GVHD (cGVHD) 66±5% (at 2 years). Projected TRM is 4±2% at 100 days, 10±3% at 1 year and 14±4% at 5 years. For acute leukemia pts projected TRM at one year is 5±5% vs 12±5% with (n = 24) and without (n = 47) TBI respectively (p = ns). Four non-relapse deaths occurred before day 100 one each from myocardial infarct (MI), acute GVHD, candidiasis and VOD. Ten deaths between days 102–569 were related to aGVHD in one and complications in pts with cGVHD in 9 (occurring in 1 pt after 2nd BCT for graft failure) including 1 VZV, 3 other infection, 1 PE, 1 MI. Of 11 follicular NHL pts 3 died of cGVHD and its complications after 1st BCT compared with 5 of 129 others (p = 0.02). Twelve of the transplant-related deaths were in 76 pts ≥ 46 years old compared with 2 in 64 younger pts (TRM = 23±6%, vs 4±3%, p = 0.01). Of 55 relapsed pts 17 are alive, 8 of these are in remission after more treatment and/or onset of GVHD. Projected 5-year disease-free survival and survival respectively is 46±5% and 57±5% for all pts, 62±8% and 76±7% for SR, 51±7% and 59±9% for OTH, and 9±6% and 16±8% for HA. This study indicates that: 1) regimen related mortality is low in MRD BCT recipients given this regimen 2) cGVHD is the main cause of TRM 3) pts with follicular NHL seem to be comparatively susceptible to death related to cGVHD 4) addition of 400cGy TBI does not increase risk of TRM and 5) older age remains a risk factor for TRM.

Description: It has been demonstrated that in a proportion of patients with Acute Myeloid Leukaemia (AML) their leukaemic blasts can be induced to differentiate into Dendritic Like Leukaemic Cells (DLLC) in vitro under the influence of GMCSF, IL-4 and TNF alpha. DLLC hold promise as cellular vaccines aimed at eradicating Minimal Residual Disease following successful induction of remission by chemotherapy. In A Phase I/II Multi-Centre Study we assessed leukaemic blasts of 21 patients with AML for their cytokine driven potential to differentiate into DLLC in vitro. 7 out of 21 (33%) patients showed DLLC conversion of their AML blasts based on morphological appearances and immunophenotype. 5 patients have completed chemotherapy and were then eligible to receive 4 escalating doses of subcutaneous DLLC vaccine. Immune responses to the administration of the vaccine are monitored by a combination of methods. The emergence of leukaemia specific T-cells following vaccination is demonstrated using Elispot assaying of Interferon gamma release in an in vitro re-stimulation assay. In WT1 expressing patients HLA-Tetramers allow to determine the frequency of WT1 specific T-cells. Regulatory T-cells (CD4/CD25 positive) are monitored by flow-cytometry. Monitoring of Minimal Residual Disease (MRD) is undertaken by means of real-time quantitative RT-PCR for leukaemia specific fusion transcripts or WT1 gene expression. 4 patients have so far completed the DLLC vaccination course. Vaccination was well tolerated with minimal side effects. A more than two-fold increase of leukaemia specific cytotoxicity was demonstrated following DLLC vaccination in one patient, while reduction of MRD was seen in several patients during the vaccination. The increase in CD4 /CD25 positive regulatory T-cells observed in several patients post vaccination may serve to dampen the induced anti-leukaemic immunity.

Description: The authors studied the role that interleukin (IL)-11 plays during the early stages of megakaryocyte (MK) development by investigating its in vitro effects on cell subpopulations enriched for bone marrow primitive progenitor cells and early and late committed progenitor cells. Progenitor subpopulations were isolated from bone marrow of normal or 5-fluorouracil (5FU)-treated mice and separated by sorting based on the surface antigens Sca-1, c-kit, and CD34. Functional analysis of the cell subpopulations, 5FU Lin−Sca-1+c-kit+ or normal bone marrow (NBM) Lin−Sca-1+c-kit+CD34−cells, indicated that exposure of these cells to recombinant human (rh)IL-11 in combination with steel factor (SF) stimulates the formation of colonies in methylcellulose and their proliferation in single cell-containing liquid cultures. Kinetic studies of MK progenitor generation, in response to SF and rhIL-11, demonstrated that a significant number of the progenitors produced are committed to the MK lineage. RhIL-11 also synergized with both SF and IL-3 to stimulate MK colony growth from NBM Lin−Sca-1+c-kit+ cells (early progenitors) and NBM Lin−Sca-1−c-kit+ cells (committed late progenitors). In the presence of IL-3, NBM, Lin−Sca-1−c-kit+ cells responded more strongly to rhIL-11 than SF. Consistent with these results is the observation that IL-11 receptor  chain mRNA is present in all the progenitor cells from which the MKs are derived. This cell culture and RNA analysis suggest that murine bone marrow primitive progenitor cells and early and late progenitor cells are direct targets of rhIL-11 and that rhIL-11 has the potential to promote megakaryocyte development at several very early stages. (Blood, 2000;95:503-509)

Description: Depsipeptide, FR901228, has demonstrated potent in vitro and in vivo cytotoxic activity against murine and human tumor cell lines. In the laboratory, it has been shown to be a histone deacetylase (HDAC) inhibitor. In a phase I trial of depsipeptide conducted at the National Cancer Institute, 3 patients with cutaneous T-cell lymphoma had a partial response, and 1 patient with peripheral T-cell lymphoma, unspecified, had a complete response. Sézary cells isolated from patients after treatment had increased histone acetylation. These results suggest that inhibition of HDAC is a novel and potentially effective therapy for patients with T-cell lymphoma.

Description: The phosphoinositide 3-kinase (PI3K) catalytic subunit p110δ is expressed in neutrophils and is thought to play a role in their accumulation at sites of inflammation by contributing to chemoattractant-directed migration. We report here that p110δ is present in endothelial cells and participates in neutrophil trafficking by modulating the proadhesive state of these cells in response to tumor necrosis factor α (TNFα). Specifically, administration of the selective inhibitor of PI3Kδ, IC87114, to animals reduced neutrophil tethering to and increased rolling velocities on cytokine-activated microvessels in a manner similar to that observed in mice deficient in p110δ. These results were confirmed in vitro as inhibition of this isoform in endothelium, but not neutrophils, diminished cell attachment in flow. A role for PI3Kδ in TNFα-induced signaling is demonstrated by a reduction in Akt-phosphorylation and phosphatidylinositol-dependent kinase 1 (PDK1) enzyme activity upon treatment of this cell type with IC87114. p110δ expressed in neutrophils also contributes to trafficking as demonstrated by the impaired movement of these cells across inflamed venules in animals in which this catalytic subunit was blocked or genetically deleted, results corroborated in transwell migration assays. Thus, PI3Kδ may be a reasonable therapeutic target in specific inflammatory conditions as blockade of its activity reduces neutrophil influx into tissues by diminishing their attachment to and migration across vascular endothelium. (Blood. 2004;103:3448-3456)

Description: von Willebrand factor (VWF) released from endothelium is ultralarge (UL) and hyperreactive. If released directly into plasma, it can spontaneously aggregate platelets, resulting in systemic thrombosis. This disastrous consequence is prevented by the ADAMTS13 (ADisintegrin and Metalloprotease with ThromboSpondin motif) cleavage of ULVWF into smaller, less active forms. We previously showed that ULVWF, on release, forms extremely long stringlike structures. ADAMTS13 cleaves these strings under flow significantly faster than it does under static conditions. As ULVWF tethering to endothelium is important for its rapid proteolysis, we investigated 2 molecules for their potential to anchor the ULVWF strings: P-selectin and integrin αvβ3. We demonstrated that P-selectin anchors ULVWF to endothelium by several means. First, Chinese hamster ovary (CHO) cells expressing P-selectin specifically adhered to immobilized ULVWF and ULVWF-coated beads to immobilized P-selectin. Second, an anti-VWF antibody coimmunoprecipitates P-selectin from the histamine-activated endothelial cells. Third, P-selectin antibody or soluble P-selectin, but not a αvβ3 antibody, RGDS peptide, or heparin, blocked the formation of ULVWF strings. Fourth, P-selectin expression was in clusters predominantly along the ULVWF strings. Finally, the strength of the minimal ULVWF–P-selectin bond was measured to be 7.2 pN. We, therefore, conclude that P-selectin may anchor ULVWF strings to endothelial cells and facilitate their cleavage by ADAMTS13.

Description: Intraperitoneal injections of specific toxoid induce a prolonged eosinophilia in the peritoneal exudate of BDF1 mice previously immunized with tetanus toxoid combined with an adjuvant of pertussis vaccine. An injection of diphtheria toxoid into tetanus-primed mice induces a transient local eosinophilia which peaks at 24 hours. A challenging injection of tetanus toxoid produced a greater 24 hour eosinophil response and, in addition, a persisting accumulation of eosinophils peaking at 3 days and still evident at days 5 and 7. Injections of heterologous antitetanus globulins or isologous immune serum did not affect the transient eosinophilia, but did suppress the accumulation of eosinophils during the second or prolonged phase of the response to challenge. Since humoral antibody prevented the antigen from inititating the eosinophil response in animals primed to respond to a challenging injection, it was concluded that antigen-antibody reactions per se could not be responsible for the prolonged phase of the eosinophil response. It was suggested that antibody may block the antigen from complexing with sensitized cells, thereby preventing the release of eosinophilotactic factors.

Description: Because human CD34+ and murine Sca-1+hematopoietic stem–progenitor cells (HSPCs) express platelet-binding sialomucin P-selectin (CD162) and integrin Mac-1 (CD11b–CD18) antigen, it was inferred that these cells might interact with platelets. As a result of this interaction, microparticles derived from platelets (PMPs) may transfer many platelet antigens (CD41, CD61, CD62, CXCR4, PAR-1) to the surfaces of HSPCs. To determine the biologic significance of the presence of PMPs on human CD34+ and murine Sca-1+ cells, their expressions on mobilized peripheral blood (mPB) and on nonmobilized PB- and bone marrow (BM)–derived CD34+ cells were compared. In addition, the effects of PMPs on the proliferation of CD34+ and Sca-1+ cells and on adhesion of HSPCs to endothelium and immobilized SDF-1 were studied. Finally, the hematopoietic reconstitution of lethally irradiated mice receiving transplanted BM mononuclear cells covered or not covered with PMPs was examined. It was found that PMPs are more numerous on mPB than on BM CD34+cells, do not affect the clonogenicity of human and murine HSPCs, and increase adhesion of these cells to endothelium and immobilized SDF-1. Moreover, murine BM cells covered with PMPs engrafted lethally irradiated mice significantly faster than those not covered, indicating that PMPs play an important role in the homing of HSPCs. This could explain why in a clinical setting human mPB HSPCs (densely covered with PMPs) engraft more rapidly than BM HSPCs (covered with fewer PMPs). These findings indicate a new role for PMPs in stem cell transplantation and may have clinical implications for the optimization of transplantations.