Small molecule inhibition of cAMP response element binding protein in human acute myeloid leukemia cells.

The transcription factor CREB (cAMP Response-Element Binding Protein) is overexpressed in the majority of acute myeloid leukemia (AML) patients, and this is associated with a worse prognosis. Previous work revealed that CREB overexpression augmented AML cell growth, while CREB knockdown disrupted key AML cell functions in vitro. In contrast, CREB knockdown had no effect on long-term hematopoietic stem cell activity in mouse transduction/transplantation assays. Together, these studies position CREB as a promising drug target for AML. To test this concept, a small molecule inhibitor of CREB, XX-650-23, was developed. This molecule blocks a critical interaction between CREB and its required co-activator CBP (CREB Binding Protein), leading to disruption of CREB-driven gene expression. Inhibition of CBP-CREB interaction induced apoptosis and cell-cycle arrest in AML cells, and prolonged survival in vivo in mice injected with human AML cells. XX-650-23 had little toxicity on normal human hematopoietic cells and tissues in mice. To understand the mechanism of XX-650-23, we performed RNA-seq, ChIP-seq and Cytometry Time of Flight with human AML cells. Our results demonstrate that small molecule inhibition of CBP-CREB interaction mostly affects apoptotic, cell-cycle and survival pathways, which may represent a novel approach for AML therapy.

Renal clear-cell carcinoma: an ultrastructural study on the junctional complexes.

Junctional complexes such as tight junctions, adherens junctions, and desmosomes play crucial roles in the structure and function of epithelial cells. These junctions are involved in increasing cell-cell contact and as well serve as signaling centers regulating multiple functions in epithelial cells. Carcinoma cell lines cultured in the laboratory generally lack junctional complexes. However, studies directed towards understanding the distribution of junctional complexes in human cancer tissues are lacking. In this study, we analyzed by electron microscopy the distribution of junctional complexes in patients diagnosed with renal clear-cell carcinoma. We found that both tight junctions and adherens junctions were drastically reduced in patients with cancer compared to normal tissues. Desmosomes were not detected in normal proximal tubules while distinctly present in cancer tissues. These results suggest that analysis of junctional complexes in human tumors should provide valuable information that might have prognostic and diagnostic value.

Acute upregulation of blood-brain barrier glucose transporter activity in seizures.

Brain extraction of (18)F-labeled 2-fluoro-2-deoxy-D-glucose (FDG) was significantly higher in pentylene tetrazole (PTZ)-treated rats (32 +/- 4%) than controls (25 +/- 4%). The FDG permeability-surface area product (PS) was also significantly higher with PTZ treatment (0.36 +/- 0.05 ml. min(-1). g(-1)) than in controls (0.20 +/- 0.06 ml. min(-1). g(-1)). Cerebral blood flow rates were also elevated by 50% in seizures. The internal carotid artery perfusion technique indicated mean [(14)C]glucose clearance (and extraction) was increased with PTZ treatment, and seizures increased the PS by 37 +/- 16% (P < 0.05) in cortical regions. Because kinetic analyses suggested the glucose transporter half-saturation constant (K(m)) was unchanged by PTZ, we derived estimates of 1) treated and 2) control maximal transporter velocities (V(max)) and 3) a single K(m). In cortex, the glucose transporter V(max) was 42 +/- 11% higher (P < 0.05) in PTZ-treated animals (2.46 +/- 0.34 micromol. min(-1). g(-1)) than in control animals (1.74 +/- 0.26 micromol. min(-1). g(-1)), and the K(m) = 9.5 +/- 1.6 mM. Blood-brain barrier (BBB) V(max) was 31 +/- 10% greater (P < 0.05) in PTZ-treated (2.36 +/- 0. 30 micromol. min(-1). g(-1)) than control subcortex (1.80 +/- 0.25 micromol. min(-1). g(-1)). We conclude acute upregulation of BBB glucose transport occurs within 3 min of an initial seizure. Transporter V(max) and BBB glucose permeability increase by 30-40%.

Transport across the primate blood-brain barrier of a genetically engineered chimeric monoclonal antibody to the human insulin receptor.

PURPOSE: Brain drug targeting may be achieved by conjugating drugs, that normally do not cross the blood-brain barrier (BBB), to brain drug delivery vectors. The murine 83-14 MAb to the human insulin receptor (HIR) is a potential brain drug targeting vector that could be used in humans, if this MAb was genetically engineered to form a chimeric antibody. where most of the immunogenic murine sequences are replaced by human antibody sequence. METHODS: The present studies describe the production of the gene for the chimeric HIRMAb, expression and characterization of the protein, radiolabeling of the chimeric HIRMAb with 111-indium and 125-iodine, and quantitative autoradiography of living primate brain taken 2 hours after intravenous administration of the [111In]chimeric HIRMAb. RESULTS: The chimeric HIRMAb had identical affinity to the target antigen as the murine HIRMAb based on Western blotting and immunoradiometric assay using partially purified HIR affinity purified from serum free conditioned media produced by a CHO cell line secreting soluble HIR. The [125I]chimeric HIRMAb was avidly bound to isolated human brain capillaries, and this binding was blocked by the murine HIRMAb. The [111In]chimeric HIRMAb was administered intravenously to an anesthetized Rhesus monkey, and the 2 hour brain scan showed robust uptake of the chimeric antibody by the living primate brain. CONCLUSIONS: A genetically engineered chimeric HIRMAb has been produced, and the chimeric antibody has identical reactivity to the human and primate BBB HIR as the original murine antibody. This chimeric HIRMAb may be used in humans for drug targeting through the BBB of neurodiagnostic or neurotherapeutic drugs that normally do not cross the BBB.

Selecting donors of platelets for refractory patients on the basis of HLA antibody specificity.

BACKGROUND: Patients who are refractory to platelet transfusion as a result of HLA alloimmunization are generally given HLA-matched or crossmatched platelets. However, HLA-matched platelets that are matched at HLA-A and -B loci (A-matched) or those without any mismatched or cross-reactive antigens (BU-matched) are frequently unavailable. A disadvantage of crossmatching is that crossmatched platelets have a shelf life of only 5 days, so that crossmatch tests must be performed frequently for patients requiring long-term platelet transfusions. An alternative method is the selection of platelets according to the patient's HLA antibody specificity, called the antibody specificity prediction (ASP) method. STUDY DESIGN AND METHODS: An anti-human globulin-enhanced microlymphocytotoxicity test modified by a double addition of serum and a computer program were used to determine the specificity of patients' HLA antibodies. Platelet crossmatching was performed with a solid-phase adherence assay. The percentage of platelet recovery (PPR) was determined in 1621 platelet transfusions in an observational study in 114 patients, and the PPR of platelets selected by the ASP method was compared with the PPR of those that were HLA-matched, crossmatched, or randomly selected. The numbers of potential donors in files of HLA-typed donors as identified by HLA matching vs. the ASP method were determined. RESULTS: After adjustments for covariates, the mean +/- SEM PPR was similar for HLA-matched (21 +/-4%), cross-matched (23+/-4%), and ASP-selected (24+/-3%) platelets and was significantly lower for randomly selected (15+/-1.4%) platelets. For 29 alloimmunized HLA-typed patients, the mean number of potential donors found in a file of 7247 HLA-typed donors was 6 who were an HLA-A match (median = 1), 33 who were an HLA-BU match (median = 20), and 1426 who were identified by the ASP method (median = 1365). CONCLUSION: The ASP method of donor selection for refractory alloimmunized patients appears as effective as HLA matching or crossmatching. Far more donors are identified in a file of HLA-typed donors by the ASP method than by HLA matching, and this indicates that the ASP method provides important advantages regarding the availability of compatible platelet components.

A model-based approach for assessing in vivo combination therapy interactions.

We present an approach for evaluating the efficacy of combination antitumor agent schedules that accounts for order and timing of drug administration. Our model-based approach compares in vivo tumor volume data over a time course and offers a quantitative definition for additivity of drug effects, relative to which synergism and antagonism are interpreted. We begin by fitting data from individual mice receiving at most one drug to a differential equation tumor growth/drug effect model and combine individual parameter estimates to obtain population statistics. Using two null hypotheses: (i) combination therapy is consistent with additivity or (ii) combination therapy is equivalent to treating with the more effective single agent alone, we compute predicted tumor growth trajectories and their distribution for combination treated animals. We illustrate this approach by comparing entire observed and expected tumor volume trajectories for a data set in which HER-2/neu-overexpressing MCF-7 human breast cancer xenografts are treated with a humanized, anti-HER-2 monoclonal antibody (rhuMAb HER-2), doxorubicin, or one of five proposed combination therapy schedules.

Testing the fit of a quantal model of neurotransmission.

Many studies of synaptic transmission have assumed a parametric model to estimate the mean quantal content and size or the effect upon them of manipulations such as the induction of long-term potentiation. Classical tests of fit usually assume that model parameters have been selected independently of the data. Therefore, their use is problematic after parameters have been estimated. We hypothesized that Monte Carlo (MC) simulations of a quantal model could provide a table of parameter-independent critical values with which to test the fit after parameter estimation, emulating Lilliefors's tests. However, when we tested this hypothesis within a conventional quantal model, the empirical distributions of two conventional goodness-of-fit statistics were affected by the values of the quantal parameters, falsifying the hypothesis. Notably, the tests' critical values increased when the combined variances of the noise and quantal-size distributions were reduced, increasing the distinctness of quantal peaks. Our results support two conclusions. First, tests that use a predetermined critical value to assess the fit of a quantal model after parameter estimation may operate at a differing unknown level of significance for each experiment. Second, a MC test enables a valid assessment of the fit of a quantal model after parameter estimation.

An analysis of a class of DNA sequence reading molecules.

Linked polyamides are a class of designed molecules that bind in the minor groove of double-stranded DNA in a partially sequence-specific manner but have limited sequence discriminatory abilities. This suggests a need for design alternatives to create molecules with enhanced sequence specificity. In this report we present formal proofs of the theoretical limits of the DNA sequence specificity of hypothetical sequence reading molecules as a function of their base recognition properties and sequence content and length of their target sequence. We prove that molecules containing nonspecific readers at critical positions within the molecule may have enhanced sequence specificity over molecules composed entirely of base specific reading elements. We also determine optimal patterns of base recognition for molecules in order to optimize their target sequence specificity. We also examine the effect of the length of a polyamide (i.e., the number of base pairs it binds) on its sequence discriminatory ability and determine necessary concentration dependent constraints on the binding free energies in order for longer polyamides to have greater sequence specificity than shorter ones. We show that unless the discriminatory ability of a ring for its preferred base is very strong, longer polyamides do not necessarily have greater sequence specificity over shorter ones when compared at the same molar concentration.

Thrombotic microangiopathy following allogeneic bone marrow transplantation is associated with intensive graft-versus-host disease prophylaxis.

Thrombotic microangiopathy (TM), manifesting clinically as thrombotic thrombocytopenic purpura or hemolytic uremic syndrome, is an uncommon complication after bone marrow transplantation (BMT). A retrospective analysis of potential risk factors for TM following allogeneic BMT was performed. Clinical data were analyzed from seven patients diagnosed with severe TM and 409 patients who underwent BMT during the same time period and who survived for at least 100 days afterwards. Six of the seven patients with TM received intensive GVHD prophylaxis consisting of cyclosporine, methotrexate and glucocorticoids, whereas only 66 of the 409 patients without TM received this regimen (P < 0.001, Fisher's exact test). This regimen was administered to patients older than 40 years, or recipients of a mismatched or unrelated allograft. Univariate analysis also revealed an increased risk of TM associated with the use of an unrelated bone marrow donor (P = 0.02), but no significant association with patient age or gender, diagnosis, amount of prior chemotherapy, transplant conditioning regimen or severity of GVHD. A multivariate exact logistic regression analysis revealed that only the type of GVHD prophylaxis had a significant impact on the risk for TM. The combined use of cyclosporine, methotrexate and glucocorticoids as GVHD prophylaxis may predispose to the development of TM following BMT.

Dynamic [18F]fluorodeoxyglucose positron emission tomography and hypometabolic zones in seizures: reduced capillary influx.

We performed dynamic [18F]fluorodeoxyglucose ([18F]FDG) positron emission tomographic (PET) analyses in 8 patients. Rate constants of influx (K1*), efflux (k2*), phosphorylation (k3*), and dephosphorylation (k4*) were derived for the regions of interest (ROIs), which included (1) the hypometabolic epileptogenic regions and (2) the homologous regions in the contralateral hemispheres. In addition, the four constants were determined from at least one clearly defined (control) ROI from the same plane and its homologous contralateral ROI. Influx (K1*) in the epileptogenic region was reduced in comparison with the contralateral ROI. Reductions in influx (K1*), which averaged 18 +/- 13% (mean +/- SD), [18F]FDG phosphorylation (k3*) (25 +/- 20%), and brain glucose utilization rates (26 +/- 10%) were observed in the epileptogenic region. Reductions in efflux were not statistically significant (k2* = 13 +/- 28%) but were comparable in magnitude to the average reduction in K1*. No ipsilateral versus contralateral differences were seen for any rate constants measured outside the epileptogenic region. Influx correlated highly with phosphorylation in the epileptogenic region. Our data suggest that the hypometabolic epileptogenic focus seen in [18F]FDG-PET studies is also a region of reduced blood-brain barrier glucose transporter activity and that reductions in phosphorylation are proportional to reductions in [18]FDG influx.

The theoretical limits of DNA sequence discrimination by linked polyamides.

Linked polyamides bind in the minor groove of double-stranded DNA in a partially sequence-specific manner. This report analyzes the theoretical limits of DNA sequence discrimination by linked polyamides composed of two to four different types of heterocyclic rings, determining (i) the optimal choice of base-binding specificity for each ring and (ii) the optimal design for a polyamide composed of these rings to target a given DNA sequence and designed to maximize the fraction of the total polyamide binding to the specified target sequence relative to all other sequences. The results show that, fortuitously, polyamides composed of pyrrole, a naturally occurring G-excluding element, and imidazole, a rationally designed G-favoring element, have features similar to the theoretical optimum design for polyamides composed of two different rings. The results also show that, in polyamides composed of two or three types of heterocyclic rings, choosing a nonspecific "placeholder" ring, which binds equally strongly to each of the four bases, along with one or two base-specific rings will often enhance sequence specificity over a polyamide composed entirely of base-specific rings.

Interictal seizure resections show two configurations of endothelial Glut1 glucose transporter in the human blood-brain barrier.

Immunogold electron microscopy was used to analyze and quantify the Glut1 glucose transporter in brain tissue from five patients undergoing surgery for treatment of seizures. Samples were prepared from two different regions of each resection: (1) the most actively spiking epileptogenic site, and (2) the least actively spiking region, as indicated by intraoperative EEG monitoring. Two configurations of endothelial cell Glut1 were observed. About one half of the capillary profiles examined displayed abundant Glut1 immunoreactivity on both luminal and abluminal endothelial membranes. In the remainder of the profiles, reduced Glut1 labeling was seen, but adjacent erythrocyte membranes remained highly Glut1 immunoreactive, suggesting that reduced endothelial Glut1 reactivity was not attributable to method artifacts. Immunogold studies using antisera to human glial fibrillary acidic protein and human serum albumin demonstrated increased quantities of these two epitopes in the extravascular regions in which more EEG spiking activity had been demonstrated. These observations were consistent with the hypotheses that capillary integrity was more compromised, and gliosis was quantitatively increased, in the more actively spiking region of the resection. Altered glucose transporter activity in the blood-brain barrier was characterized by a bimodal Glut1 distribution in which the smaller (type B) endothelial cells displayed low Glut1 immunoreactivity, whereas adjacent (and even contiguous) larger (type A) endothelial cells showed 5- to 10-fold greater expression of membrane Glut1 transporter protein. Because this transporter facilitates glucose entry to the brain, small pericapillary volumes of brain tissue may have quite different concentrations of glucose. We hypothesize that in complex partial seizures and other forms of brain insult, an alteration of blood-brain barrier Glut1 glucose transporter activity is indicated by the appearance of these two subpopulations of endothelial cells. In comparison with previous studies of human brain capillaries in hemangioblastoma and brain injury, endothelial Glut1 density was apparently reduced (interictally) in affected temporal lobes of patients with complex partial seizures.

Estimation of the DNA sequence discriminatory ability of hairpin-linked lexitropsins.

Three- and four-ring polyamides containing N-methylimidazole and N-methylpyrrole, and their hairpin-linked derivatives, bind side-by-side in the minor groove of DNA in a sequence-specific manner. The sequences recognized by side-by-side molecules are dependent on the pairings of the polyamide rings to the bases. In this study we report a mathematical model for estimating the free energies of binding for gamma-aminobutyric acid-linked polyamides to 5- and 6-bp DNA sequences. The model parameters are calibrated by a least-squares fit to 35 experimental binding constants. The model performs well in cross-validation experiments and the parameters are consistent with previously proposed empirical rules of polyamide-DNA binding. We apply the model to the design of targeted polyamides, evaluating the ability of the proposed polyamides to bind to a DNA sequence of interest while minimizing binding to the remaining DNA sequences.

Suramin in hormone-refractory metastatic prostate cancer: a drug with limited efficacy.

PURPOSE: To confirm the previously reported high response rates and prolonged survival in hormone-refractory prostate cancer treated with suramin. PATIENTS AND METHODS: Thirty-six eligible patients with hormone-refractory prostate cancer with either measurable disease or bone disease only and a prostate-specific antigen (PSA) level greater than 50 ng/mL were enrolled. Treatment consisted of two 8-week courses of outpatient-based therapy with an interposed rest period. A bayesian adaptive control strategy and a three-compartment pharmacokinetic model that accommodates clearance changes was used to guide individual dosing. A rapid infusion of 1,000 mg/m2 suramin was followed by five daily infusions that targeted 285 micrograms/mL peak plasma levels during the first week. All patients received concomitant hydrocortisone. For the next 7 weeks, patients received one to two doses per week that targeted levels in the 150 to 285 micrograms/mL range and integrated weekly averages of 200 ug/mL. RESULTS: Nine patients (28%) had a partial response to suramin based on a > or = 50% decrease in PSA levels coupled with either relief of bone pain or by a 50% decrease in measurable disease. The median overall survival time for all patients is 31 weeks (95% confidence interval [CI], 23 to 51). Treatment was generally well tolerated, with fatigue being the most common significant toxicity, but fatal idiosyncratic myelosuppression (grade V) was observed in one patient. CONCLUSION: Using this dosing schedule, suramin has limited activity against hormone-refractory metastatic prostate cancer. Recent data suggest that hydrocortisone administered with suramin may be partly responsible for the benefit attributed to the drug. Although a small cohort of patients appeared to benefit, we were unable to confirm the previously reported high rate of activity and durability of remission using this agent.

Developmental modulation of blood-brain-barrier glucose transport in the rabbit.

Blood-brain barrier (BBB) glucose transport rates were measured using the intracarotid injection method in newborn, 14-day-old suckling, 28-day-old weanling and adult rabbits, and compared with membrane transporter density. Light microscope immunochemistry confirmed the presence of the GLUT1 glucose transporter isoform in these rabbits. Quantitative electron microscopic immunogold analyses of GLUT1-immunoreactive sites per micrometer of capillary membrane indicated GLUT1 density increased with age, and correlated with in vivo measurements of Vmax. Maximal transport velocities (Vmax) of glucose transfer (an indicator of the activity and relative number of transporter proteins) increased significantly (P = 0.05) with age: in neonates Vmax = 0.61 mumol.min-1.g-1, in sucklings Vmax = 0.68 mumol.min-1.g-1, in weanlings Vmax = 0.88 mumol.min-1.g-1, and in adults Vmax = 1.01 mumol.min-1 g-1. Cerebral blood flow (CBF) rates, increased with age from 0.19 and 0.26 ml.min-1.g-1 in neonates and sucklings to 0.51 (weanlings) and 0.70 (adults) ml.min-1.g-1. Non-linear regression analyses indicated the half-saturation constant (Km) for glucose transport ranged from 13 mM in adult rabbits to 19 mM in 14-day-old sucklings: differences in Km were not significant. Age-related changes in the Permeability-Surface Area product (PS +/- S.E.) of both water and glucose were also seen. At all ages studied, the diffusion component (Kd) of glucose uptake was not distinguishable from zero. We conclude developmental up-regulation of the rabbit BBB glucose transporter is characterized by no changes in transporter affinity, and provide the first demonstration of increased membrane transporter proteins correlating with an age-related increase (65%) in glucose transporter maximal velocity.

Unrelated donor bone marrow transplants in children.

Only a small proportion of children who might benefit from bone marrow transplant (BMT) have an HLA-identical sibling. To provide this potentially curative therapy to patients without a matched related donor, marrow transplants using less well matched related donors or unrelated donors (identified through computerized donor registries) have been performed. We report the outcome of 24 consecutive unrelated donor BMT's performed on children. Eligible diagnosis included acute leukemia (AL) (n = 15), chronic myelogenous leukemia (CML) (n = 4), myelodysplastic syndrome (MDS) (n = 3), and severe aplastic anemia (SAA) (n = 2). All donor/recipient pairs were sero-matched at 5 or 6 of the 6 HLA A, B, and DR antigens. Several different preparative regimens were used, but fractionated total body irradiation (TBI) was used in 20 patients. All recipients received graft-versus-host-disease (GVHD) prophylaxis with cyclosporine-A (CSA), four with short course methotrexate (MTX), 14 in combination with short course MTX and methylprednisolone (MPS), and five in combination with a mouse monoclonal antibody to CD5, coupled to the A-chain of ricin (Xomazyme-65). One patient received CSA and MPS alone after a T-cell depleted marrow transplant. Twenty of 23 evaluable recipients engrafted (87%). Two patients with CML never engrafted and had autologous marrow recovery, one patient with SAA died at 128 days without evidence of engraftment, and there was one early death at day + 9. Fourteen of 20 patients (70%) with stable donor-derived hematopoiesis developed significant acute GVHD > or = grade II). Eleven of 15 engrafted patients who survived > 100 days after BMT developed chronic GVHD (73%).(ABSTRACT TRUNCATED AT 250 WORDS)

Exchange of CO2 and bicarbonate between the circulation and cerebrospinal fluid in piglets.

The dynamic exchange of CO2 and HCO-3 between the central circulation and peripheral sites such as the cerebrospinal fluid (CSF) is not completely understood. To examine this we administered a tracer dose of NaHCO3 labeled with the stable isotope 13C into the central circulation of nine 3- to 4-wk-old anesthetized, instrumented piglets. Serial samples of arterial and venous blood, breath, and CSF were obtained to determine the 13C/12C ratio by isotope-ratio mass spectrometry. Gas-exchange and hemodynamic parameters were obtained through standard techniques. The patterns of tracer washout in venous blood, arterial blood, and breath were nearly indistinguishable from each other, and the mean residence time was 113 +/- 24 min (average +/- SD). In contrast, the CSF 13C/12C ratio was not equivalent to that in venous blood, arterial blood, or breath until 20 min after tracer administration. Tracer washout data were used to determine the best-fit three-exponential-term equation and to calculate compartmental parameters of a three-compartment mammillary system in which CO2 and HCO3- residing in the central compartment (compartment 1) exchanges relatively rapidly with one peripheral compartment (compartment 2) and slowly with the other (compartment 3). The turnover time for the CSF was 6.8 +/- 2.5 min and the estimate for compartment 2 was 7.1 +/- 3.7 min. Accordingly, CSF appears to be one of the rapidly interchanging peripheral compartments.

N-ras mutations are associated with poor prognosis and increased risk of leukemia in myelodysplastic syndrome.

To evaluate the clinical significance of N-ras mutations in the myelodysplastic syndrome (MDS) archival bone marrow samples from 252 patients were studied for the presence of N-ras exon I mutations using polymerase chain reaction amplification and differential oligonucleotide hybridization. Subsequently, clinical information about these patients was obtained and analyzed. Of 220 evaluable patients, 20 (9%) had point mutation of N-ras involving codon 12. Individuals with N-ras mutation had a significantly shorter survival period than those who were N-ras negative (P = .02). An increased risk of acute myelogenous leukemia (AML) was also found in patients with N-ras mutations (P = .005). N-ras mutations were not associated with any French-American-British (FAB) subtype, with the presence of increased myeloblasts, or with chromosomal aberrations in the bone marrow. However, the presence of increased bone marrow blasts was strongly associated with poor survival rate and risk of AML (P < .001 for each). After stratifying for the percentage of blasts, N-ras mutations remained significantly associated with shorter survival period (P = .04) and increased risk of AML (P = .02). Bone marrow cytogenetic abnormalities, particularly when multiple abnormalities were present, were significantly associated with a poor prognosis (P < .001). In conclusion, N-ras mutation, although relatively infrequent in MDS, is associated with short survival period and increased probability of developing AML.