Thermochemical scanning probe lithography of protein gradients at the nanoscale.

Patterning nanoscale protein gradients is crucial for studying a variety of cellular processes in vitro. Despite the recent development in nano-fabrication technology, combining nanometric resolution and fine control of protein concentrations is still an open challenge. Here, we demonstrate the use of thermochemical scanning probe lithography (tc-SPL) for defining micro- and nano-sized patterns with precisely controlled protein concentration. First, tc-SPL is performed by scanning a heatable atomic force microscopy tip on a polymeric substrate, for locally exposing reactive amino groups on the surface, then the substrate is functionalized with streptavidin and laminin proteins. We show, by fluorescence microscopy on the patterned gradients, that it is possible to precisely tune the concentration of the immobilized proteins by varying the patterning parameters during tc-SPL. This paves the way to the use of tc-SPL for defining protein gradients at the nanoscale, to be used as chemical cues e.g. for studying and regulating cellular processes in vitro.

Characterization of a multilayer heparin coating for biomolecule presentation to human mesenchymal stem cell spheroids.

Mesenchymal stem cells therapies have the potential to treat many pathologies, however, controlling cell fate after implantation remains challenging. We have used a multilayer technology to graft a range of 5 µg/mL - 5 mg/mL heparin onto the surface of MSC aggregates. Heparin coating does not affect cell viability (seen through LIVE/DEAD staining), cell anti-inflammatory properties (seen through co-culture with activated monocytes)and facilitates sequestration by coated cells of a growth factor (TGF-ß1) that remains bioactive. This system can maximize therapeutic potential of MSC-based treatments because the cell surface-loaded protein could both signal to the cells to influence transplanted cell fate and be released into the surrounding environment to help repair injured tissue.

Influence of sorbitol on protein crowding in solution and freeze-concentrated phases.

Small-angle neutron scattering was employed to study protein crowding under freezing conditions that mimic those used in pharmaceutical processing. The results demonstrate that, although there is an increase in heterogeneity as the temperature is reduced, sorbitol reduces protein crowding in both solution and freeze-concentrated phases, thus protecting the protein from forming oligomers or irreversible aggregates.

Nanosecond relaxation dynamics of hydrated proteins: water versus protein contributions.

We have studied picosecond to nanosecond dynamics of hydrated protein powders using dielectric spectroscopy and molecular dynamics (MD) simulations. Our analysis of hydrogen-atom single particle dynamics from MD simulations focused on "main" (τ(main) ≈ tens of picoseconds) and "slow" (τ(slow) ≈ nanosecond) relaxation processes that were observed in dielectric spectra of similar hydrated protein samples. Traditionally, the interpretation of these processes observed in dielectric spectra has been ascribed to the relaxation behavior of hydration water tightly bounded to a protein and not to protein atoms. Detailed analysis of the MD simulations and comparison to dielectric data indicate that the observed relaxation process in the nanosecond time range of hydrated protein spectra is mainly due to protein atoms. The relaxation processes involve the entire structure of protein including atoms in the protein backbone, side chains, and turns. Both surface and buried protein atoms contribute to the slow processes; however, surface atoms demonstrate slightly faster relaxation dynamics. Analysis of the water molecule residence and dipolar relaxation correlation behavior indicates that the hydration water relaxes at much shorter time scales.

Atomistic ensemble modeling and small-angle neutron scattering of intrinsically disordered protein complexes: applied to minichromosome maintenance protein.

The minichromosome maintenance (MCM) proteins are thought to function as the replicative helicases in archaea and eukarya. In this work we determined the solution structure of the N-terminal portion of the MCM complex from the archaeon Methanothermobacter thermautotrophicus (N-mtMCM) in the presence and absence of DNA using small-angle neutron scattering (SANS). N-mtMCM is a multimeric protein complex that consists of 12 monomers, each of which contains three distinct domains and two unstructured regions. Using an all-atom approach incorporating modern force field and Monte Carlo methods to allow the unstructured regions of each monomer to be varied independently, we generated an ensemble of biologically relevant structures for the complex. An examination of the subsets of structures that were most consistent with the SANS data revealed that large movements between the three domains of N-mtMCM can occur in solution. Furthermore, changes in the SANS curves upon DNA binding could be correlated to the motion of a particular N-mtMCM domain. These results provide structural support to the previously reported biochemical observations that large domain motions are required for the activation of the MCM helicase in archaea and eukarya. The methods developed here for N-mtMCM solution structure modeling should be suitable for other large protein complexes with unstructured flexible regions.

Influence of hydration on the dynamics of lysozyme.

Quasielastic neutron and light-scattering techniques along with molecular dynamics simulations were employed to study the influence of hydration on the internal dynamics of lysozyme. We identified three major relaxation processes that contribute to the observed dynamics in the picosecond to nanosecond time range: 1), fluctuations of methyl groups; 2), fast picosecond relaxation; and 3), a slow relaxation process. A low-temperature onset of anharmonicity at T approximately 100 K is ascribed to methyl-group dynamics that is not sensitive to hydration level. The increase of hydration level seems to first increase the fast relaxation process and then activate the slow relaxation process at h approximately 0.2. The quasielastic scattering intensity associated with the slow process increases sharply with an increase of hydration to above h approximately 0.2. Activation of the slow process is responsible for the dynamical transition at T approximately 200 K. The dependence of the slow process on hydration correlates with the hydration dependence of the enzymatic activity of lysozyme, whereas the dependence of the fast process seems to correlate with the hydration dependence of hydrogen exchange of lysozyme.

Onsets of anharmonicity in protein dynamics.

Two onsets of anharmonicity are observed in the dynamics of the protein lysozyme. One at T approximately 100 K appears in all samples regardless of hydration level and is consistent with methyl group rotation. The second, the well-known dynamical transition at T approximately 200-230 K, is only observed at a hydration level h greater than approximately 0.2 and is ascribed to the activation of an additional relaxation process. Its variation with hydration correlates well with variations of catalytic activity suggesting that the relaxation process is directly related to the activation of modes required for protein function.

Acute Myeloblastic Leukemia: Management with High-Dose Cytosine Arabinoside, Daunorubicin and Marrow Transplantation; Malignancy; Current Clinical Practice.

Combination high-dose cytosine arabinoside (ARA-C) and daunorubicin (DNR) for primary remission induction of patients with acute myeloblastic leukemia (AML) was evaluated in a single institution study. Patients aged 55 or less with an HLA-sibling received an allogeneic bone marrow transplant (alloBMT) in first remission; other responders were offered autologous BMT (autoBMT). For remission induction 93 patients aged less than 60 received DNR 45 mg/m(2) BSA x 3 and ARA-C 2 gm/m(2) BSA every 12 hours for 12 doses; 53 aged 60 or older DNR 25 mg/m(2) daily x 3 and ARA-C 1.5-2.0 gm/m(2) BSA every 12 hours for 12 doses. Consolidation doses of DNR were the same but ARA-C 100 mg/m(2) BSA/day x 5 was given by continuous intravenous infusion. The complete remission rate for patients less than 60 years was 69.9% (95% CI: 59.5-79.0%) and 47.2% (95% CI: 33.3-61.4%) for the older patients. The median duration of first remission for the younger patients was 13.0 months and of overall survival 17.9 months; for patients over 60 years 5.6 and 10.0 months respectively. Disease-free survival and overall survival of the 19 patients receiving alloBMT and the 13 patients undergoing autoBMT aged less than 55 years and in first or second complete remission were significantly increased compared with 22 patients in remission but not having BMT (p < 0.001 and p < 0.013). The results support the effectiveness of high-dose ARA-C for remission induction, a need for intensive consolidation therapy and a role for BMT in the management of AML.

Three decades of allogeneic bone marrow transplants at the Princess Margaret Hospital.

A total of 1,122 patients with various hemopoietic disorders were transplanted at the Princess Margaret Hospital since 1970. The majority suffered from acute or chronic myeloid leukemia. Improvements in support strategies permitted a gradual escalation of the upper age limit for transplant candidates and resulted in better survival. The overall survival at 10 years of all patients transplanted consecutively either before or after 1986 increased from 30 to 50%. This change was observed independent of other transplant related risk factors and is predominantly attributable to the use of cyclosporine and ganciclovir. An improvement of similar magnitude was seen for transplant recipients presenting with good risk features. The 10-year survival of patients with acute and chronic myeloid leukemia transplanted from a fully matched sibling donor in first complete remission or first chronic phase increased from 40 to approximately 70%. The quality of life of surviving patients may not return to normal but appears to improve with time after the transplant.

A role for paclitaxel in the combination chemotherapy of acute myeloblastic leukaemia: preclinical cell culture studies.

Paclitaxel dose responses in culture have been investigated alone and in association with cytosine arabinoside (ARA-C) and all-trans retinoic acid (ATRA), with the objective of identifying a role for paclitaxel in the treatment of acute myeloblastic leukaemia (AML). Initial studies were done to determine if paclitaxel dose responses of AML blast cell precursors were altered by regulatory compounds known to modify the dose responses of ARA-C. In contrast to ARA-C, paclitaxel dose responses were independent of cell culture method, the growth factors G-CSF and GM-CSF, and the ligands all-trans retinoic acid (ATRA) and hydrocortisone. Most blast cell populations were sensitive to paclitaxel; compared with normal marrow progenitors the dose responses were markedly heterogenous with some more, and others less, sensitive. Remission marrow progenitor paclitaxel responses resembled those of AML blasts in heterogeneity. The cell culture model tested the effect of pacliataxel and ATRA on the ARA-C dose responses of OCI/ AML-5; paclitaxel exposure was either before or after ARA-C to test for an effect of schedule; ATRA was added to the MEC cultures after paclitaxel and ARA-C. Repeat experiments were done to test three dose levels each of paclitaxel and ATRA. When paclitaxel was given after ARA-C, synergism was found for all but one of the dose combinations tested; only three examples of synergy were seen when paclitaxel preceded ARA-C. The studies justify trials combining ARA-C, paclitaxel and ATRA using a schedule suggested by the cell culture findings.

Intensive remission induction therapy for chronic myeloid leukemia in blast phase with a goal of post-remission bone marrow transplant--a pilot study.

An intensive protocol utilizing mitoxantrone, high-dose cytarabine, vincristine, etoposide and methylprednisolone as induction therapy for chronic myeloid leukemia in blast transformation is described. Fourteen patients were treated, with a remission/second chronic phase achieved in 64%. None of the 3 patients older than 50 yr responded. Complete hematological responses were seen in 9 of the 11 younger patients, 4 of whom also became BCR-ABL negative by Southern Blot analysis. Four patients went on to allogeneic bone marrow transplant. Median remission durations were 4.5 (1-5) and 8.5 (5-16) months in the non-transplanted and transplanted cohorts, respectively. Median survival is 1.5 (0.5-3), 9.5 (7-14) and 17 (14-61+) months in the non-responding, responding non-transplanted and transplanted cohorts, respectively. Toxicity, particularly gastrointestinal, was significant. This represents an aggressive protocol that should be reserved for patients who are potential transplant candidates.

Response of the blast stem cells of acute myeloblastic leukemia to G-CSF, GM-CSF, or the ligand for C-KIT, alone or in combination.

Growth factors are commonly included in protocols for the treatment of acute myeloblastic leukemia (AML). Because the response of blast stem cells in culture to growth factors might influence the contribution of factor to clinical outcome, we studied 42 patients with AML or severe myelodysplasia. Peripheral blood blast cells were cultured in a clonogenic assay at three cell concentrations and with the following combinations of growth factors: no added growth factor (NF), G-CSF, GM-CSF, Kit ligand (KL), G-CSF + KL, GM-CSF + KL, and G-CSF + GM-CSF + KL. The slope of the line relating cell number plated to colony formation was calculated by least squares. The slopes were used to form three equally sized groups of patients. Marked heterogeneity was found in response of the blast populations to factor. A few general conclusions emerged: (1) autonomous blast populations are very rare; (2) although usually a population responds better to one of the growth factors than to others, seldom is the response exclusively to one factor; (3) when more than one factor is included in the cultures, synergism is usually seen. Significant associations were seen between successful remission induction for low slope values in cultures with NF or KL alone. For remission, but not survival, associations were found with intermediate values of slope in cultures with G-CSF + KL and GM-CSF + KL. We conclude that measurements of growth factor response are feasible and yield clinically useful data.

Sensitivities of AML blast stem cells to idarubicin and daunorubicin: a comparison with normal hematopoietic progenitors.

The objective of this study was to determine whether or not cell culture studies could contribute to the correct choice between idarubicin (IDA) and daunorubicin (DNR) for combination with ara-C in remission induction therapy of AML. Two growth factor-sensitive AML cell lines and the peripheral blood blast cells from 10 patients with AML were studied in culture for sensitivity to IDA and DNR under four culture conditions; cells were grown either in methylcellulose or suspension culture in the presence of G-CSF or GM-CSF. Normal bone marrow cells were cultured in methylcellulose in the presence of IDA or DNR under conditions suitable for the detection of BFU-E and CFU-C. Dose-response curves for AML blast cells were characterized by an initial shoulder and then an exponential decrease in survival. Marked patient-to-patient variation was observed for both portions of the survival curves. IDA was significantly more toxic to blast cells than DNR, especially for more sensitive cell populations. Consistent differences in drug sensitivity in the four culture conditions were not observed. BFU-E and CFU-C dose-response curves of normal marrow progenitors resembled those of AML blast cells but in contrast fell within a narrow range. The culture studies support the clinical finding in AML of modest superiority of IDA over DNR. The heterogeneity in sensitivity of AML blasts in culture suggests an opportunity to individualize treatment. Preclinical studies may help in developing such a therapeutic approach.

Inhibition of human factor VIIIa by anti-A2 subunit antibodies.

Human inhibitory alloantibodies and autoantibodies to Factor VIII (FVIII) are usually directed toward the A2 and/or C2 domains of the FVIII molecule. Anti-C2 antibodies block the binding of FVIII to phospholipid, but the mechanism of action of anti-A2 antibodies is not known. We investigated the properties of a patient autoantibody, RC, and a monoclonal antibody, 413, that bind to the region which contains the epitopes of all anti-A2 alloantibodies or autoantibodies studied to date. mAb 413 and RC were noncompetitive inhibitors of a model intrinsic Factor X activation complex (intrinsic FXase) consisting of Factor IXa, activated FVIII (FVIIIa), and synthetic phospholipid vesicles, since they decreased the Vmax of intrinsic FXase by > 95% at saturating concentrations without altering the Km. This indicates that RC and mAb 413 either block the binding of FVIIIa to FIXa or phospholipid or interfere with the catalytic function of fully assembled intrinsic FXase, but they do not inhibit the binding of the substrate Factor X. mAb 413 did not inhibit the increase in fluorescence anisotropy that results from the binding of Factor VIIIa to fluorescein-5-maleimidyl-D-phenylalanyl-prolyl-arginyl-FIXa (Fl-M-FPR-FIXa) on phospholipid vesicles in the absence of Factor X, indicating it does not inhibit assembly of intrinsic FXase. Addition of Factor X to Fl-M-FPR-FIXa, FVIIIa, and phospholipid vesicles produced a further increase in fluorescence anisotropy and a decrease in fluorescence intensity. This effect was blocked completely by mAb 413. We conclude that anti-A2 antibodies inhibit FVIIIa function by blocking the conversion of intrinsic FXase/FX complex to the transition state, rather than by interfering with formation of the ground state Michaelis complex.

Isolation and characterization of thrombin-activated human factor VIII.

Recombinant human factor VIII (fVIII) was activated by thrombin at pH 7.4, followed by CM-Sepharose chromatography at pH values ranging from 3.5 to 7.4. Optimal coagulant activity was recovered at pH 5.5 and was associated with the isolation of an A1/A2/A3-C1-C2 heterotrimer. The activity was stable at -80 degrees C, but decayed slowly (t1/2 approximately 1 week) and nonproteolytically at room temperature or 4 degrees C. The coagulant activity of the pH 5.5 fVIIIa preparation assayed in human hemophilia A plasma was only 20% that of porcine factor VIIIa. However, its activity was approximately 75% that of porcine fVIIIa in a plasma-free assay, indicating that human fVIIIa is unstable relative to porcine fVIIIa during the coagulation assay. The first-order rate constant for spontaneous, nonproteolytic loss of activity of human fVIIIa at pH 7.4 was decreased 8-fold by fIXa and phospholipid, indicating that human fVIIIa is stabilized when incorporated into the intrinsic pathway factor X activation complex.

Expression of the Kit and KitA receptor isoforms in human acute myelogenous leukemia.

Genetic and biologic evidence suggests that the Kit receptor tyrosine kinase is important in early events in hematopoietic stem cell differentiation. Two naturally occurring isoforms of the Kit receptor, termed Kit and KitA, were originally described in mouse cells and, subsequently, in human cells. These isoforms differ by the presence (KitA) or absence (Kit) of four amino acids (Gly-Asn-Asn-Lys) that lie immediately outside the transmembrane domain. RNase protection was used to measure the levels of Kit and KitA mRNA in normal bone marrow and the blast cells from individuals with acute myelogenous leukemia (AML). Although both isoforms were present in all the AML samples tested, there was considerable heterogeneity in the relative levels of the two transcripts, with Kit to KitA RNA ratios varying from as low as 1.3 to as high as 12. In contrast, the ratio of Kit to KitA transcripts in normal bone marrow was tightly clustered between 4.4 and 5.5. Because alterations in the relative levels of expression of Kit and KitA may affect the ability of a cell to respond to the Kit ligand, Steel factor, we examined the Kit/KitA RNA ratio in AML patients that differed with respect to a number of diagnostic, prognostic, and biologic parameters. The relative levels of Kit to KitA RNA was independent of French-American-British subtype, response to therapy, and primary and secondary plating efficiencies in vitro. Thus, these data suggest that the relative levels of the two isoforms of the Kit receptor in AML are not associated with any obvious biologic or clinical parameters and, therefore, may reflect naturally occurring changes in splicing mechanisms as stem cells differentiate.

High-dose cytosine arabinoside remission induction for acute myelogenous leukemia: comparison of two regimens of remission maintenance.

We report a single institution sequential trial of two maintenance treatment regimens for patients with acute myelogenous leukemia (AML). A total of 175 consecutive patients with AML received initial remission induction therapy with high-dose cytosine arabinoside (ara-C) and glucocorticoids. For the initial 63 patients (group A), the control population, planned maintenance treatment was with conventional-dose ara-C given over 4 days for up to 18 months. The subsequent 107 patients (group B) had planned maintenance therapy of up to 6 courses of daunorubicin, ara-C and prednisone and daily cis-retinoic acid for up to two years. The presenting features of group A and B patients were similar as were the response to remission induction, 60 and 52%, respectively. Severe neurological toxicity was encountered once after high-dose ara-C; no drug-related deaths occurred during maintenance treatment. Median duration of remission for group B patients was 9.9 months compared with 5.5 for group A (p = 0.0685). Median survival duration for the two groups was similar, 9.1 months for group A and 10.4 for group B. Survival of patients in group B who attained a complete remission was significantly better than that of patients in group A (p = 0.0439). The studies confirm our initial experience with remission induction using single agent high-dose ara-C and suggest a positive role for maintenance therapy in AML.

Comparison of the effects of all-trans and cis-retinoic acid on the blast stem cells of acute myeloblastic leukemia in culture.

Recent work has shown that acute promyelocytic leukemia (APL) cells have a characteristic translocation involving the retinoic acid receptor on chromosome 17 and the myl protein on chromosome 15. Patients with APL respond to the administration of all-trans-retinoic acid. A cell line with t15;17 (NB4) has recently been reported; this line responds to all-trans-retinoic acid with differentiation. There is also a recent report showing that all-trans-retinoic acid is more active than cis-retinoic acid in inducing differentiation in freshly obtained APL cells. All-trans-retinoic and cis-retinoic acid are compared for their effects on growth in culture of freshly obtained AML cells, cell lines without t15;17, and NB4 cells. While all of these AML populations responded to both forms of retinoic acid, NB4 cells only were much more sensitive to all-trans-retinoic acid compared to cis-retinoic acid. The difference was seen when the NB4 cells were exposed in suspension and not when colony-formation in methylcellulose was used as an end point. Both forms of retinoic acid increased the sensitivity of blast cells to cytosine arabinoside; for NB4 cells, the sensitization was much greater when all-trans-retinoic acid was used rather than cis-retinoic acid. We conclude that the increased effects of all-trans-retinoic acid are specific for APL cells, and that a major effect of retinoic acid is on blast stem cell self-renewal.

Treatment of adults with severe aplastic anemia: primary therapy with antithymocyte globulin (ATG) and rescue of ATG failures with bone marrow transplantation.

PURPOSE: To evaluate a policy of immunosuppression with antithymocyte globulin (ATG) as primary therapy for adults with severe aplastic anemia (SAA) regardless of the availability of an HLA-identical bone marrow donor. PATIENTS AND METHODS: Thirty-one consecutive adults with SAA who satisfied the age criteria for allogeneic bone marrow transplantation (BMT) (age less than 51 years) were treated with ATG 20 mg/kg/day for 10 days along with high-dose corticosteroids. Patients with an HLA-identical donor received a transplant if they did not respond to ATG or if they developed life-threatening complications during or soon after ATG administration. Eight patients with no response to ATG were also treated with oral cyclosporine 12.5 mg/kg/day. RESULTS: Eleven patients had a complete and five a partial response to ATG; two patients improved with cyclosporine treatment, resulting in an overall response rate of 58% to immunosuppression. Nine of 14 patients with donors received a BMT: seven because they did not respond to ATG and two because of serious infections. Seven grafts were obtained from related and two from unrelated donors. There was no significant difference in survival between those with and without a related HLA-identical donor (log-rank p value = 0.969). At a median follow-up of 58 months, 26 of 31 are alive with an actuarial survival of 80% at 5 years. Two patients died of infection, two died from complications of BMT, and one remains transfusion-dependent. One patient died of refractory leukemia at 30 months; one patient relapsed with hypoplasia 95 months after initial therapy with ATG. He showed a complete response to treatment with cyclosporine. No other late hematologic events have occurred. CONCLUSIONS: This treatment approach resulted in the restoration of hematopoiesis and independence from transfusion in 80% of patients with SAA entered into the study. The efficacy of allogeneic BMT in salvaging cases in which ATG failed does not appear to be compromised. Follow-up for the development of clonal hematologic disorders remains an important part of this treatment policy.

Long-term results of bone marrow transplantation for patients with AML, ALL and CML prepared with single dose total body irradiation of 500 cGy delivered with a high dose rate.

One hundred and sixty-six patients between the ages of 12 and 48 years with acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL) or chronic myelogenous leukemia (CML) underwent allogeneic bone marrow transplantation following single fraction total body irradiation (TBI) of 500 cGy from a cobalt source. Patients also received one of three chemotherapeutic regimens according to their diagnosis or disease status at time of transplant. The median follow-up was 67 months with a range of 33-120 months. The actuarial 5-year event-free survival (EFS) for the subgroup of patients with good risk disease (first complete remission AML and ALL or first chronic phase CML) was 43% with an actuarial relapse rate at 5 years of 26%. Patients with poor risk disease (other than first remission AML and ALL or other than first chronic phase CML) had an EFS at 5 years of 15% with a relapse rate of 62%. Disease status at the time of transplantation was the most important factor predicting outcome in this patient population. We conclude that preparation of good risk patients with chemotherapy and single fraction TBI of 500 cGy at a dose rate of 42-91 cGy/min resulted in EFS and relapse rates similar to those observed by centers using fractionated radiotherapy schedules, without a concomitant increase in toxicity, in particular interstitial pneumonitis and cataracts.