Molecular characterization of breakpoints in patients with holoprosencephaly and definition of the HPE2 critical region 2p21

Holoprosencephaly (HPE) is a common developmental defect involving the brain and face in humans. Cytogenetic deletions in patients with HPE have localized one of the HPE genes (HPE2) to the chromosomal region 2p21. Here we report the molecular genetic characterization of nine HPE patients with cytogenetic deletions or translocations involving 2p21. We have determined the parental origin of the deleted chromosomes and defined the HPE2 critical region between D2S119 and D2S88/D2S391. As a first step towards cloning the HPE2 gene which is crucial for normal brain development we have constructed a YAC contig which spans the smallest region of deletion overlap. Several of these YACs could be identified which span three different 2p21 breakpoints in HPE patients. These YACs narrow the HPE2 critical region to less than 1 Mb and are now being further analyzed to identify the gene causing holoprosencephaly on chromosome 2.      

Allogeneic bone marrow transplantation for acute nonlymphocytic leukemia in first remission

Seventy-three patients with acute nonlymphocytic leukemia in first complete remission (CR) have received allogeneic bone marrow transplantation (BMT) with non-T-lymphocyte-depleted marrow obtained from matched sibling donors. The first 36 patients received a preparative regimen consisting of cyclophosphamide, 60 mg/kg/d (days -6 and -5), and 750 cGy single-dose total-body irradiation (TBI) (day -1). Subsequently, 37 patients received cyclophosphamide 60 mg/kg/d (days -6 and -5), and 165 cGy fractionated TBI administered twice daily for a total dose of 1,320 cGy (days -4, -3, -2, and -1). Survivors have been followed from 9 to 124 months (median, 40 months). The 61% (95% confidence interval [CI], 45% to 77%) projected disease-free survival (DFS) of 41 children less than 18 years old does not differ significantly from the 62% (95% CI, 49% to 73%) projected DFS of 32 adults at 84 months (P = .89). Similarly, the 15% (95% CI, 1% to 29%) projected relapse rate seen in children does not differ from the 9% (95% CI, 0% to 21%) seen in adults (P = .69). Multivariate Cox regression analysis of presenting features demonstrates that a presenting WBC count greater than 20,000/m3 is associated with decreased DFS (P = .01). When compared with other French-American- British (FAB) subtypes, presentation with FAB M4 or M5 morphology is significantly associated with relapse in multivariate analysis (P = .014). Other presenting features such as preparation with single-dose or fractionated TBI, interval from diagnosis to CR or CR to BMT, donor or recipient sex, and donor or recipient cytomegalovirus serology do not correlate independently with either DFS or relapse. When included in the stepwise multivariate analysis of presenting patient features, two posttransplant events, development of grades 2 to 4 acute graft-v- host disease (GVHD) (P less than .03) and development of interstitial pneumonitis (P less than .001), also correlate independently with poor DFS. Allogeneic BMT provides equivalent, prolonged DFS in both children and young adults when performed in first CR and should be considered the therapy of choice for all first CR patients under 45 years of age with a suitable donor. Continued efforts to prevent and treat acute GVHD and pneumonitis as well as efforts designed to prevent relapse in patients presenting with FAB M4 and M5 morphology should further improve outcome.     

(2S, 4R)-4-[18F]Fluoroglutamine for In vivo PET Imaging of Glioma Xenografts in Mice: an Evaluation of Multiple Pharmacokinetic Models

Purpose

The glutamine analogue (2S, 4R)-4-[¹⁸F]fluoroglutamine ([¹⁸F]FGln) was investigated to further characterize its pharmacokinetics and acquire in vivo positron emission tomography (PET) images of separate orthotopic and subcutaneous glioma xenografts in mice.

Procedures

[¹⁸F]FGln was synthesized at a high radiochemical purity as analyzed by high-performance liquid chromatography. An orthotopic model was created by injecting luciferase-expressing patient-derived BT3 glioma cells into the right hemisphere of BALB/cOlaHsd-Foxn1^nu mouse brains (tumor growth monitored via in vivo bioluminescence), the subcutaneous model by injecting rat BT4C glioma cells into the flank and neck regions of Foxn1^nu/nu mice. Dynamic PET images were acquired after injecting 10–12 MBq of the tracer into mouse tail veins. Animals were sacrificed 63 min after tracer injection, and ex vivo biodistributions were measured. Tumors and whole brains (with tumors) were cryosectioned, autoradiographed, and stained with hematoxylin-eosin. All images were analyzed with CARIMAS software. Blood sampling of 6 Foxn1^nu/nu and 6 C57BL/6J mice was performed after 9–14 MBq of tracer was injected at time points between 5 and 60 min then assayed for erythrocyte uptake, plasma protein binding, and plasma parent-fraction of radioactivity to correct PET image-derived whole-blood radioactivity and apply the data to multiple pharmacokinetic models.

Results

Orthotopic human glioma xenografts displayed PET image tumor-to-healthy brain region ratio of 3.6 and 4.8 while subcutaneously xenografted BT4C gliomas displayed (n?=?12) a tumor-to-muscle (flank) ratio of 1.9?±?0.7 (range 1.3–3.4). Using PET image-derived blood radioactivity corrected by population-based stability analyses, tumor uptake pharmacokinetics fit Logan and Yokoi modeling for reversible uptake.

Conclusions

The results reinforce that [¹⁸F]FGln has preferential uptake in glioma tissue versus that of corresponding healthy tissue and fits well with reversible uptake models.

     

Responder analyses in randomised controlled trials for chronic low back pain: an overview of currently used methods

Purpose

To provide an overview and a critical appraisal of the use of responder analyses in published randomised controlled trials (RCTs) of interventions for chronic low back pain (LBP). The methodology used for the analyses, including the justification, as well as the implications of responder analyses on the conclusions was explored.

Methods

A convenience sample of four systematic reviews evaluating 162 RCTs of interventions for chronic LBP was used to identify individual trials. Randomised trials were screened by two reviewers and included if they performed and reported a responder analysis (i.e. the proportion of participants achieving a pre-defined level of improvement). The cutoff value for responders, the period of follow-up, and the outcome measure used were extracted. Information on how RCT authors justified the methodology of their responder analyses was also appraised.

Results

Twenty-eight articles (17 %) using 20 different definitions of responders were included in this appraisal. Justification for the definition of responders was absent in 80 % of the articles. Pain was the most frequently used domain for the definition of response (50 %), followed by back-specific function (30 %) and a combination of pain and function (20 %). A reduction in pain intensity ≥50 % was the most common threshold used to define responders (IQR 33–60 %).

Conclusions

Few RCTs of interventions for chronic LBP report responder analyses. Where responder analyses are used, the methods are inconsistent. When performing responder analyses authors are encouraged to follow the recommended guidelines, using empirically derived cutoffs, and present results alongside mean differences.     

Uncoupling fibrin from integrin receptors hastens fibrinolysis at the platelet-fibrin interface

A well-characterized in vitro model system composed of thrombin- stimulated gel-filtered human platelets, fibrin-(ogen), plasminogen, and recombinant tissue plasminogen activator (rt-PA) was used to examine the relationship between platelet-fibrin adhesive interactions and the lytic resistance of a platelet-rich thrombus. Laser light scattering kinetic experiments demonstrated that the ligand-mimetic peptide D-RGDW and an anti-alpha IIb beta 3 monoclonal antibody both inhibited clot retraction, but neither integrin-targeted reagent affected the overall delay in lysis of "bulk" fibrin caused by thrombin- stimulated platelets. However, lysis of the model platelet-rich thrombus did proceed some 30% more quickly when treated with a plasminogen activator inhibitor (PAI)-resistant t-PA variant. Taken together, these results confirm that platelet-released PAI-1 is a major determinant of global lytic resistance. Next events occurring during fibrinolysis in the unique microenvironment near the platelet surface were monitored by scanning electron microscopy and quantitative fluorescence microscopy. Scanning electron micrographs of the partially lysed model thrombus in the presence of 200 mumol/L of D-RGDW showed no platelet aggregates, and fibrin was attached by fewer strands to the platelets. Quantitative fluorescence microscopy, using fluorescein- labeled fibrin, showed that fibrin adherent to the surface of thrombin- stimulated platelets lysed 20% to 50% more slowly than bulk fibrin (monitored in parallel by laser light scattering). Furthermore, this microspectroscopic technique showed that D-RGDW reduced the quantity of platelet-bound fibrin, and accelerated lysis near the platelet surface with both native rt-PA and the PAI-resistant variant. These observations suggest that the dense network of fibrin bound to the platelet surface is protected from fibrinolysis by tissue-type plasminogen activators. Further, uncoupling fibrin from its platelet receptors uniquely hastens fibrinolysis at the cell/fibrin interface.     

Long-term human bone marrow cultures

Pronlonged in vitro growth of murine bone marrow has been achieved using a flask culture system. We report the adaption of the technique to the growth of human bone marrow. In this system, CFU-GM and CFU-E are maintained for 4-9 wk. Morphologically recognizable granulopoiesis occurs for 4-6 wk and erythropoiesis for 1.5-2.5 wk. Functional T lymphocytes are maintained for at least 5 wk. The adherent population is composed of macrophages, fibroblast-like cells, and flat pavement- like cells. Fat-containing cells are not prominant. This culture system provides a means to study human hematopoietic cell proliferation and differentiation in vitro, as well as to examine interactions between stromal cells and hematopoietic and lymphoid progenitors.     

Inhibition of leukemic HL60 cell growth by transferrin-gallium: effects on ribonucleotide reductase and demonstration of drug synergy with hydroxyurea [see comments]

Cellular requirements for iron during DNA synthesis are related to the increased activity of the iron-containing M2 subunit of ribonucleotide reductase, the enzyme responsible for the reduction of ribonucleotides to deoxyribonucleotides. We have previously shown that transferrin- gallium (Tf-Ga) inhibits cellular iron incorporation. In the present study, Tf-Ga-induced inhibition of HL60 cell growth and upregulation of Tf receptor density was reversed with hemin. Cells exposed to 2 mumol/L Tf-Ga for six hours or longer displayed a diminution in the electron spin resonance (ESR) spectroscopy signal of the tyrosyl radical of the M2 subunit of ribonucleotide reductase. The effect of Tf-Ga on the ESR signal was reversed by hemin. Tf-Ga decreased the incorporation of 14C- adenosine into DNA and decreased intracellular deoxyribonucleotide pools, with the maximum diminution seen in deoxyadenosine triphosphate (dATP) and deoxycytidine triphosphate (dCTP) pools. Exposure of cells to combinations of Tf-Ga and hydroxyurea (a known inhibitor of ribonucleotide reductase) resulted in a marked inhibition of cell growth that was consistent with drug synergy. Our studies suggest that Tf-Ga inhibits DNA synthesis through action on the M2 subunit of ribonucleotide reductase and that combinations of Ga and hydroxyurea should be further evaluated in in vivo tumor models.     

Cell surface expression of c-kit receptors by childhood acute myeloid leukemia blasts is not of prognostic value: a report from the Childrens Cancer Group

The prognostic significance of c-kit receptor expression on leukemic blast cells was determined in 122 children with acute myeloid leukemia (AML) entered onto Childrens Cancer Group protocol 213. Clinical and laboratory characteristics as well as outcome were analyzed according to the percentage of blast cells expressing c-kit receptors and the relative number of c-kit receptors per cell as determined by indirect immunofluorescence. c-kit receptor expression was strongly associated with the expression of the CD34 antigen. However, contrary to findings in adult patients with AML, c-kit receptor expression by childhood AML blast cells was not predictive of a poor response to therapy.     

Regulation of fibrinolysis by platelet-released plasminogen activator inhibitor 1: light scattering and ultrastructural examination of lysis of a model platelet-fibrin thrombus

We have investigated the role of plasminogen activator inhibitor 1 (PAI- 1) in the regulation of fibrinolysis using a model thrombus composed of thrombin-stimulated platelets, fibrin(ogen), plasminogen, and recombinant tissue-type plasminogen activator. Laser light scattering kinetic measurements showed that clot lysis was significantly delayed both by thrombin-stimulated platelets and their cell-free releasate. This delay in lysis was almost fully reversed by the addition of a PAI- 1-specific monoclonal antibody that blocks the ability of PAI-1 to inhibit plasminogen activators. Lysis half-times exhibited a linear dependence on the concentration of PAI-1 antigen present, as determined by enzyme-linked immunosorbent assay (ELISA). Sodium dodecylsulfate- polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting confirmed the presence of PAI-1 antigen in the platelet releasates. Scanning electron micrographs of the model thrombus components sampled late in lysis showed considerable unproteolyzed fibrin still attached to platelets. Immunogold cytochemistry detected large amounts of PAI-1 antigen in the partially lysed platelet-fibrin thrombi. This PAI-1 appeared to be bound to the fibrin network rather than to the platelet surface itself. We conclude that the residual clots observed late in lysis represent platelet-associated fibrin to which platelet-released PAI-1 has bound, rendering it less susceptible to degradation.