ALBERT

Description: Complex pertubations of hemostasis occur in sickle cell disease (SCD). Although the procoagulant property of sickle erythrocytes in vitro is tied to exposure of phosphatidylserine (PS), no study has directly linked this PS positivity to in vivo thrombin generation. This study was designed to determine if thrombin generation in SCD correlates with erythrocyte PS, or whether platelets play a significant role. PS was quantified on erythrocytes and platelets from 40 patients with SCD (SS genotype = 25; SC genotype = 15) and 11 controls. Markers of thrombin generation (prothrombin fragment F1.2; thrombin-antithrombin or TAT complexes) and fibrin dissolution (D-dimer; plasmin-antiplasmin or PAP complexes) were also evaluated. Thrombin generation and activation of fibrinolysis occurred with elevations in F1.2, TAT, and D-dimer. Although numbers of both PS-positive erythrocytes and platelets were elevated, there was no correlation between PS-positive platelets and any hemostatic markers. In contrast, correlations were noted between PS-positive erythrocytes and F1.2 (P 

Description: Juvenile myelomonocytic leukemia (JMML) is a rare pediatric malignancy. Hematopoietic stem cell transplantation (SCT) is the only curative approach. However, relapse after SCT remains the major cause of treatment failure. Unlike most other pediatric malignancies, JMML may be susceptible to a graft-versus-leukemia (GVL) effect, although, unlike chronic myeloid leukemia, reports of response to donor lymphocyte infusions (DLIs) remain scanty. This is the first report that describes the successful treatment of relapsed JMML with DLI in the absence of further chemotherapy and provides definite proof of a GVL effect in JMML.

Description: The most prominent cell-surface integrin α4β1 partner, a 70-kDa protein, was isolated from MOLT-4 T leukemia cells, using anti–α4β1 integrin antibody-coated beads. By mass spectrometry, this protein was identified as EWI-2, a previously described cell-surface partner for tetraspanin proteins CD9 and CD81. Wild-type EWI-2 overexpression had no effect on MOLT-4 cell tethering and adhesion strengthening on the α4β1 ligand, vascular cell adhesion molecule-1 (VCAM-1), in shear flow assays. However, EWI-2 markedly impaired spreading and ruffling on VCAM-1. In contrast, a mutant EWI-2 molecule, with a different cytoplasmic tail, neither impaired cell spreading nor associated with α4β1 and CD81. The endogenous wild-type EWI-2–CD81–α4β1 complex was fully soluble, and highly specific as seen by the absence of other MOLT-4 cell-surface proteins. Also, it was relatively small in size (0.5 × 106 Da to 4 × 106 Da), as estimated by size exclusion chromatography. Overexpression of EWI-2 in MOLT-4 cells caused reorganization of cell-surface CD81, increased the extent of CD81-CD81, CD81-α4β1, and α4β1-α4β1 associations, and increased the apparent size of CD81-α4β1 complexes. We suggest that EWI-2–dependent reorganization of α4β1-CD81 complexes on the cell surface is responsible for EWI-2 effects on integrin-dependent morphology and motility functions. (Blood. 2004;103: 3013-3019)

Description: Transcription factors of the nuclear factor of activated T cells (NFAT) family are thought to regulate the expression of a variety of inducible genes such as interleukin-2 (IL-2), IL-4, and tumor necrosis factor-α. However, it remains unresolved whether NFAT proteins play a role in regulating transcription of the interferon- γ (IFN-γ) gene. Here it is shown that the transcription factor NFAT1 (NFATc2) is a major regulator of IFN-γ production in vivo. Compared with T cells expressing NFAT1, T cells lacking NFAT1 display a substantial IL-4–independent defect in expression of IFN-γ mRNA and protein. Reduced IFN-γ production by NFAT1−/−× IL-4−/− T cells is observed after primary in vitro stimulation of naive CD4+ T cells, is conserved through at least 2 rounds of T-helper cell differentiation, and occurs by a cell-intrinsic mechanism that does not depend on overexpression of the Th2-specific factors GATA-3 and c-Maf. Concomitantly, NFAT1−/−× IL-4−/− mice show increased susceptibility to infection with the intracellular parasiteLeishmania major. Moreover, IFN-γ production in a murine T-cell clone is sensitive to the selective peptide inhibitor of NFAT, VIVIT. These results suggest that IFN-γ production by T cells is regulated by NFAT1, most likely at the level of gene transcription.

Description: Although current vaccine strategies have achieved anti-myeloma immune responses, meaningful clinical responses have not been observed. This may be related with the significant immune dysfunction observed in myeloma that may interfere with the development of effective anti-myeloma immune responses. The initiation of immune response is controlled by CD4+CD25+ T regulatory (Treg) cells, which can suppress anti-tumor immune responses. This property of Treg cells to modulate anti-tumor immune responses may function as a barrier to cancer immunotherapy. In order to overcome such suppression, signaling through Toll-like receptors (TLRs) can induce adjuvant effect by increasing local production of chemokines, and pro-inflammatory cytokines, and by enhancing antigen-presentation by APCs. Here, we have evaluated the role of Treg cells and the effects of TLRs in myeloma. We observed significant increase in CD4+CD25+ Treg cells in MM patient samples compared to normal donors (23±4% vs 6±3%). Proliferation of T cells depleted of Treg cells with anti-CD3 antibody was significantly lower in MGUS (n=9, SI=12±2) and MM (n=9, SI=28±8) compared with normal donors (n=9, SI=74±9, p

Description: The failure to overcome drug resistance leads to a high rate of relapse in elderly patients with acute myeloid leukemia. We evaluated, in a Phase I study the feasibility of a dose dense regimen of HiDAC, and MylotargTM therapy for newly diagnosed elderly (≥60 years) patients with AML in terms of toxicity with two cycles of this regimen as the sole induction and consolidation therapy. HiDAC was administered in a dose escalation pattern: 3000mg/m2 intravenously given for 6, and 9 doses, and MylotargTM was administered at a dose of 6mg/m2 intravenously on days 1 and 8 of each cycle. Patients without unacceptable toxicity, defined as failure to recover counts to a minimum of ANC ≥ 500/ μl, platelets ≥ 30K and hematocrit ≥ 25%, received a second cycle of therapy, though not before day 28 following day 1 of induction. In addition, death within the first 30 days of induction (not related to disease progression) and life-threatening non-hematologic toxicity (such as cardiac or pulmonary arrest) was also considered dose-limiting. Patients with persistent disease but at least a 50% decrease in the marrow or peripheral blood blast count, or those with low blood counts and patients achieving CR without platelet recovery (CRp) at the 4–6 week examination received cycle 2 with a de-escalation of the Mylotarg dose (from 6 mg/m2 to 4 mg/m2). All patients received G-CSF 5mcg/kg/day subcutaneously from days 11–14. Eight patients (five male, three female) with a median age of 68 years (range 60–74) were enrolled. In cohort one (6 doses of HiDAC), four of six patients were able to complete both cycles of therapy and two of these have achieved CR. Two of the six patients achieved CRp with persistent thrombocytopenia and thus received a second cycle of chemotherapy off protocol. One patient in this cohort had progressive disease and persistent pancytopenia requiring transfusions and subsequently received chemotherapy using Etoposide and Cyclophosphamide. Five out of six patients are alive and remain disease free. In cohort two (9 doses of HiDAC), two patients have been enrolled thus far. One patient developed neurotoxicity after six doses of HiDAC and thus completed both cycles of therapy with six doses of HiDAC along with protocol dose of MylotargTM. The other patient was able to get all nine doses of HiDAC and both patients have achieved a CR. No unexpected hematologic toxicity was observed. All patients developed grade IV thrombocytopenia requiring platelet transfusions. One patient in cohort one died after developing aspergillus infection and multiorgan failure before he could be evaluated for response. Two patients in cohort one developed uncomplicated gram-positive bacteremia requiring antibiotics. In cohort two, one patient developed neurotoxicity and the other developed uncomplicated gram-positive bacteremia. At the time of submission of this abstract seven out of eight patients are alive with four CR and two CRp. No veno-occlusive disease was seen in these eight patients treated with two cycles of HiDAC and MylotargTM back to back. The high rate of CR and relatively good tolerance of this regimen remains encouraging.

Description: Stem cell transplantation (SCT), indicated for many non-malignant disorders, is limited by donor availability, graft rejection (GR), toxicities of conditioning, morbidity and mortality (TRM), and graft versus host disease (GVHD). To overcome these barriers, we tested a novel conditioning for SCT. It was designed to support engraftment by deleting host immune reactive lymphocytes and macrophages. Campath -1H (anti-CD52 mab) was given on days -21, -20, and -19 (total dose 48 mg), fludarabine (day -8 to -4) (total 150 mg/m2) and melphalan on day -3 (140 [n=15] or 70 [n=1] mg/m2). Stem cell sources were related/unrelated bone marrow (BM) (8), peripheral blood (PB) (5) and umbilical cord blood (UCB) (3). GVHD prophylaxis was cyclosporine (tapered after 3 months), methylprednisone (tapered after day +28) and methotrexate on days +1, +3, and +6. Methotrxate was not used in UCBT. End points of the study included engraftment and TRM. Sixteen patients (1.5–40 yrs) diagnosed with aplastic anemia (5), Hurler’s (2), sickle cell anemia, XLAAD, histiocytosis (3), thalassemia, adrenoleukodystrophy, Evan’s syndrome and dyserythropoietic anemia were transplanted. Median follow-up was 219 days (66–845). The regimen was well tolerated. All patients that survived 〉1 month engrafted. Neutrophils (ANC 〉500/dL) engrafted at a median of 12.5 (10–36) days and platelets (〉50,000/dL) at a median of 21 (12 –63) days. Skin GVHD developed in 3 patients and resolved early. Four are tapering immune suppression; 8 are successfully off immune suppression. All survivors have either stable disease or are cured. One recipient had a normal pregnancy and delivered twins. Two patients died prior to engraftment from previously acquired Pseudomonas infection. A third patient died of CMV disease (day +112) and another recipient died of intracranial hemorrhage/refractory thrombocytopenia (day +43) after engraftment. Other complications were bacterial and viral infections occurring within the first 100 days post-transplant. All but 1 CMV+ recipient reactivated CMV as assessed by PCR. Profound lymphopenia was present in all recipients on day +30. NK cells recovered by day +100, CD8+ cells by day +180, CD4+ and B cells between days 180–270 after transplant. Immunoglobulin (Ig M and A) levels dropped post transplant and normalized by 9 months. Ig A recovered later than IgM. In summary, successful engraftment despite varied stem cell sources, was achieved without significant toxicity or GVHD. Lymphopenia resulted in a significant infection risk within the first 100 days post transplant, requiring close surveillance and early intervention. This transplant regimen is well tolerated and may preserve fertility, making it a promising alternative to conventional SCT.

Description: Cholesterol in the membrane not only regulates flexibility and mechanical stability of the membrane but also plays a critical role in differentiating and maintaining cell surface microdomains of differing lipid composition, particularly sphingolipid rafts. Cholesterol- and sphingolipid-rich rafts in association with a structural protein, caveolin, form caveolae, flask-shaped invaginations in the plasma membrane. Lipid rafts and caveolae are shown to regulate various cellular functions, including receptor function, endocytosis, intracellular trafficking of receptors and signaling pathways. In the present study, we investigated the role of membrane cholesterol and caveolae in modulating coagulant and signaling functions of tissue factor (TF)-factor VIIa (VIIa) complexes on tumor cells. Breast carcinoma cells, MDA-MB-231, were treated with ß-methyl cyclodextrin (CD), which depletes membrane cholesterol and thereby disrupts caveolae, or with filipin, which disrupts caveolae without depleting the membrane cholesterol. TF-VIIa coagulant function was measured in factor X activation assay and the signaling function was evaluated in IP3 hydrolysis and IL-8 gene induction. As expected, CD (10 mM) treatment of tumor cells depleted cellular cholesterol level by more than 70% whereas filipin (5 μg/ml) treatment did not reduce the cellular cholesterol level. Both the treatments had no effect on the cell viability as measured in trypan blue exclusion method and MTT assay. CD treatment, in a dose-pendent manner (1 to 10 mM CD), impaired both TF-VIIa coagulant function and the signaling function (both IP3 hydrolysis and IL-8 gene expression). In contrast, varying effects were observed with filipin treatment. Filipin treatment (5 μg/ml) increased TF-VIIa coagulant function, reduced the TF-VIIa-induced IP3 hydrolysis and no effect on TF-VIIa-induced IL-8 gene expression. Detergent (Triton X-100) extraction of cells followed by fractionation on sucrose gradient centrifugation showed that TF was distributed both in lipid rafts and soluble fractions. CD and filipin treatments slightly reduced TF association with lipid rafts. Overall these data suggest that the membrane cholesterol modulates TF-VIIa proteolytic function and hence the TF-VIIa protease-induced signaling. In contrast, disruption of caveolae uncouples selective signaling pathways activated by TF-VIIa without impairing TF-VIIa protease activity.

Description: Binding of factor VIIa (FVIIa) to its cellular receptor tissue factor (TF) was previously shown to induce various intracellular signaling events, which were thought to be responsible for TF-mediated biologic effects, including angiogenesis, tumor metastasis, and restenosis. To understand the mechanisms behind these processes, we have examined the effect of FVIIa on apoptosis. Serum deprivation–induced apoptosis of BHK(+TF) cells was characterized by apoptotic blebs, nuclei with chromatin-condensed bodies, DNA degradation, and activation of caspase 3. FVIIa markedly decreased the number of cells with apoptotic morphology and prevented the DNA degradation as measured by means of TdT-mediated dUTP nick end labeling (TUNEL). The antiapoptotic effect of FVIIa was confirmed by the observation that FVIIa attenuated caspase 3 activation. FVIIa-induced antiapoptotic effect was dependent on its proteolytic activity and TF but independent of factor Xa and thrombin. FVIIa-induced cell survival correlated with the activation of Akt and was inhibited markedly by the specific PI3-kinase inhibitor, LY294002. Blocking the activation of p44/42 mitogen-activated protein kinase (MAPK) by the specific mitogen-induced extracellular kinase (MEK) inhibitor, U0126, impaired modestly the ability of FVIIa to promote cell survival. In conclusion, FVIIa binding to TF provided protection against apoptosis induced by growth factor deprivation, primarily through activation of PI3-kinase/Akt pathway, and to a lesser extent, p44/42 MAPK pathway.

Description: Human embryonic stem (huES) cells have the ability to differentiate into a variety of cell lineages and potentially provide a source of differentiated cells for many therapeutic uses. However, little is known about the mechanism of differentiation of huES cells and factors regulating cell development. We have used high-quality microarrays containing 16 659 seventy–base pair oligonucleotides to examine gene expression in 6 of the 11 available huES cell lines. Expression was compared against pooled RNA from multiple tissues (universal RNA) and genes enriched in huES cells were identified. All 6 cell lines expressed multiple markers of the undifferentiated state and shared significant homology in gene expression (overall similarity coefficient 〉 0.85).A common subset of 92 genes was identified that included Nanog, GTCM-1, connexin 43 (GJA1), oct-4, and TDGF1 (cripto). Gene expression was confirmed by a variety of techniques including comparison with databases, reverse transcriptase–polymerase chain reaction, focused cDNA microarrays, and immunocytochemistry. Comparison with published “stemness” genes revealed a limited overlap, suggesting little similarity with other stem cell populations. Several novel ES cell–specific expressed sequence tags were identified and mapped to the human genome. These results represent the first detailed characterization of undifferentiated huES cells and provide a unique set of markers to profile and better understand the biology of huES cells. (Blood. 2004;103: 2956-2964)