A murine cytokine fusion toxin specifically targeting the murine granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor on normal committed bone marrow progenitor cells and GM-CSF-dependent tumor cells

A fusion protein was synthesized consisting of the murine granulocyte- macrophage colony-stimulating factor (mGM-CSF) gene spliced to a truncated form of the diphtheria toxin (DT390) gene coding for a molecule that retained full enzymatic activity, but excluded the native binding domain. The DT390-mGM-CSF hybrid gene was cloned into a vector under the control of an inducible promoter and the protein expressed in Escherichia coli. After induction, a protein was purified from inclusion bodies in accord with the deduced molecular weight of DT390 mGM-CSF. Cell-free studies of the adenosine diphosphate-ribosylating activity of DT390 mGM-CSF showed results that were similar to those of native DT. The DT390 mGM-CSF immunotoxin inhibited FDCP2.1d, a murine myelomonocytic tumor line expressing the GM-CSF receptor with an IC50 (concentration inhibiting 50% activity) of 5 x 10(-11) mol/L. The fusion toxin was specifically cytotoxic and directed by the GM-CSF portion of the molecule because addition of a monoclonal antibody directed against GM-CSF inhibited its ability to kill the cell line. Cell lines that do not express GM-CSF receptor were not inhibited by the fusion toxin. DT390 mGM-CSF was also able to specifically inhibit normal committed bone marrow (BM) progenitor cells as measured in clonal colony-forming unit granulocyte-macrophage assays. Together, these findings indicate that DT390 mGM-CSF may be useful as a novel tool for purging BM of contaminating leukemia cells or in vivo for eliminating residual leukemia cells. Also, it can be used to determine whether committed and/or noncommitted BM progenitor cells express the GM-CSF receptor.     

Sonographic Evaluation of Gallbladder Motility in Children with Chronic Functional Constipation

Background/Aims

Studies in adults suggest that constipation may not be a purely colonic pathology and may be a component of a generalized gastrointestinal (GI) motor disorder in which proximal GI motility can be impaired. Pediatric data are scarce, and the natural history of the disorder remains undefined. We aimed to evaluate gallbladder motility in a subset of Asian children with chronic functional constipation.

Methods

Abdominal ultrasound was performed on 105 children, including 55 patients (aged 3 to 13 years) with chronic functional constipation who met the inclusion criteria and 50 age- and gender-matched controls. The gallbladder contractility index was calculated based on the preprandial and postprandial gallbladder areas. Preprandial and postprandial values for gallbladder volume and wall thickness were evaluated.

Results

The mean value of the contractility index for the patients (15.77±24.68) was significantly lower than the mean value for the controls (43.66±11.58) (p=0.001). The mean postprandial gallbladder volumes and areas were larger in children with gallbladder hypomotility (p<0.05). The mean duration of constipation (4.8 months) was significantly higher (p=0.004) in the children with gall-bladder hypomotility.

Conclusions

Gallbladder motility is significantly impaired in children with chronic functional constipation. This study contributes to the understanding of the underlying pathophysiology, which will enable advancement in and improved management of children with chronic constipation and associated gallbladder hypomotility.     

Real-time analysis of shear-dependent thrombus formation and its blockade by inhibitors of von Willebrand factor binding to platelets

Two likely mechanisms for the initiation of arterial platelet thrombus formation under conditions of elevated fluid shear stresses are: (1) excessive adhesion and aggregation of platelets from rapidly flowing blood onto the exposed sub-endothelium of injured, atherosclerotic arteries; or (2) direct, fluid shear stress-induced aggregation of platelets in constricted arteries with intact endothelial cells. Mechanism (1) was simulated using a parallel plate flow chamber, fibrillar collagen type I-coated slides, and mepacrine-labeled (fluorescent) platelets in whole blood anticoagulated with citrate, hirudin, unfractionated porcine heparin, or low molecular weight heparin flowing for 1 to 2 minutes at wall shear rates of 100 to 3,000 seconds-1 (4 to 120 dynes/cm2). The precise sequence of interactions among von Willebrand factor (vWF), glycoprotein (GP)Ib, and GPIIb-IIIa during platelet adhesion and subsequent aggregation were resolved by direct real-time observation using a computerized epifluorescence video microscopy system. Adhesion at high shear rates was the result of the adsorption of large vWF multimers onto collagen and the binding of platelet GPIb to the insolubilized vWF. Aggregation occurred subsequently and required the binding of ligands, including vWF via its RGD binding domain, to GPIIb-IIIa. Mechanism (2) was modeled by producing shear stresses of 90 to 180 dynes/cm2 in a rotational cone and plate viscometer, which aggregates platelets from platelet-rich- plasma (PRP) anti-coagulated with citrate, hirudin, or either type of heparin in reactions that require large vWF multimers, Ca2+, adenosine diphosphate, and both GPIb and GPIIb-IIIa. Both vWF-mediated shear- aggregation in PRP and platelet-collagen adhesion in flowing whole blood (anticoagulated with citrate and hirudin) are inhibited by two potentially useful anti-arterial thrombotic agents: polymeric aurin tricarboxylic acid (ATA; 28.5 to 114 micrograms/mL), which binds to vWF and inhibits its attachment of GPIb, and a recombinant vWF fragment (rvWF445-733; 30 to 200 micrograms/mL) that binds to platelet GPIb (in the absence of any modulator) and blocks attachment of vWF multimers. Unfractionated heparin, but not low molecular weight heparin, apparently binds to rvWF445-733 and counteracts the inhibitory effects of the vWF fragment in vitro on shear-aggregation and platelet-collagen adhesion.     

Unrelated donor bone marrow transplantation: influence of HLA A and B incompatibility on outcome

We have studied the outcome of 211 consecutive unrelated donor (URD) bone marrow transplants (BMT) performed at the University of Minnesota (Minneapolis, MN) between May 1985 and December 1992. Ninety patients (43%) received marrow matched serologically at HLA A, B, and DR loci; 86 (41%) received marrow with a major and 32 (15%) marrow with a minor serologic mismatch at the HLA A or B locus. Multivariate analysis revealed that older age had an adverse effect on survival. In younger (age less than 18 years) recipients, survival after fully matched (A, B, and DR sub-type) or major mismatched (A or B locus), DR subtype- matched donor BMT was not significantly different (P = .4; survival: 53% v 41%, respectively, at 3 years). For adults, survival after matched donor BMT was significantly better than that with mismatched donors (P < .01; survival: 30% v 10%, respectively, at 3 years). Formal quality of life assessment by telephone interview demonstrated similar functional status in survivors of URD and related donor (RD) BMT at least 2 years post-BMT. URD BMT provides effective therapy for a variety of lethal hematopoietic diseases that rivals outcome of RD transplant in some cases. Use of URD marrow with a major mismatch at one HLA A or B locus is well tolerated in young, but not in older, recipients. These observations should be used to improve donor selection and counseling for URD BMT candidates.     

Immunologic abnormalities in myelofibrosis with activation of the complement system

Eighteen patients with agnogenic myeloid metaplasia with myelofibrosis were studied for clinical and laboratory evidence of immunologic dysfunction. Clinical findings included the presence of arthritis, vasculitis, and erythema nodosum. Laboratory abnormalities included the presence of circulating immune complexes, antinuclear antibodies, positive direct Coombs tests, elevated latex fixations, and a circulating lupus type anticoagulant. Total hemolytic complement was markedly depressed in four patients. Analysis of complement (C) components C1-C9 and factor B demonstrated significant reduction of only C3 and factor B. By crossed-immunoelectrophoresis, both C3 and factor B, but not C4, were cleaved, indicating that C activation was occurring predominantly via the alternative pathway. The control proteins beta 1H and C3b inactivator were decreased in three of four patients with hypocomplementemia. These data suggest that immunologic mechanisms associated with activation of the complement system play an important role in the disease process of some patients with agnogenic myeloid metaplasia with myelofibrosis.     

Increased radiosensitivity of a subpopulation ot T-lymphocyte progenitors from patients with Fanconi's anemia

In vitro radiation survival of peripheral blood T lymphocytes was studied in 15 clinically normal adults and 4 patients with Fanconi's anemia. Tritiated thymidine incorporation in a whole blood lymphocyte stimulation test (LST) and a newly developed whole blood T-lymphocyte colony assay were used to measure lymphocyte blastogenesis and colony formation in response to phytohemagglutinin (PHA) or concanavalin-A (Con-A) stimulation. Lymphocyte colony formation was found to be consistently more sensitive than the LST for detection of low-level radiation effects using both normal cells and lymphocytes from Fanconi's anemia patients. Lymphocytes from patients with Fanconi's anemia were significantly more sensitive to in vitro x-irradiation than lymphocytes from clinically normal individuals as measured by their ability to divide when stimulated by PHA in the LST (patients, D37 = 198 R; normals, D37 = 309 R, p = 0.057) and colony formation assay (patients, D37 = 53 R; normals, D37 = 109 R, p = 0.016). No significant difference in the radiosensitivity of the Con-A response was observed between the two groups. The PHA-responsive T-lymphocyte subpopulation in Fanconi's anemia patients appears to be intrinsically defective. The nature of this defect, significance in the disease process, and relevancy of these findings to the establishment of radiation protection standards are discussed.     

Participation of haemato‐oncological patients in medical decision making and their confidence in decisions

ERNST J., WEISSFLOG, G., BRÄHLER E., NIEDERWIESER D., KÖRNER A. & SCHRÖDER C. (2010) European Journal of Cancer Care
Participation of haemato‐oncological patients in medical decision making and their confidence in decisions Increasingly more clinical care and research acknowledge the patients' interest in participating in medical decision making. However, for haematological patients, there are as yet only modest findings. The current study explores patients' perceptions of their role in the medical decision‐making process in a sample of 117 haematological patients. The majority of patients surveyed (63.9%) took a passive role in the medical decision‐making process, which is a significantly greater proportion compared with individuals suffering from solid cancers. Despite passive majority, most of the participants reported a positive evaluation of the decision‐making process. Importantly, patients' evaluations were significantly more negative either if patients were treated as inpatients (vs. outpatients), or if they experienced no control over the decision (vs. collaboration with the doctor, or deciding autonomously). The results and limitations of the study are discussed.     

Contemporary open partial nephrectomy is associated with diminished procedure-specific morbidity despite increasing technical challenges: a single institutional experience

Objective  

To review trends in open partial nephrectomy (OPN) at our center, concentrating on patient selection, technique and perioperative complications.     

Interobserver Agreement for the Detection of Barrett’s Esophagus with Optical Frequency Domain Imaging

Background

Optical frequency domain imaging (OFDI) is a second-generation form of optical coherence tomography (OCT) providing comprehensive cross-sectional views of the distal esophagus at a resolution of ~7 μm.

Aim

Using validated OCT criteria for squamous mucosa, gastric cardia mucosa, and Barrett’s esophagus (BE), the objective of this study was to determine the inter- and intra-observer agreements by a large number of OFDI readers for differentiating these tissues.

Methods

OFDI images were obtained from nine subjects undergoing screening and surveillance for BE. Sixty-four OFDI image regions of interest were randomly selected for review. A training set of 19 images was compiled distinguishing squamous mucosa from gastric cardia and BE using previously validated OCT criteria. The ten readers then interpreted images in a test set of 45 different images of squamous mucosa (n = 15), gastric cardia (n = 15), or BE (n = 15). Interobserver agreement differentiating the three tissue types and BE versus non-BE mucosa was determined using multi-rater Fleiss’s κ value. The images were later randomized again and four readers repeated the test 3 weeks later to assess intraobserver reliability.

Results

All ten readers showed excellent agreement for the differentiation of BE versus non-BE mucosa (κ = 0.811 p < 0.0001) and for differentiating BE versus gastric cardia versus squamous mucosa (κ = 0.866, p < 0.0001). For the four readers who repeated the test, the median intraobserver agreement (BE vs. non-BE) was high (κ = 0.975, IQR: 0.94, 1.0).

Conclusions

Trained readers have a high interobserver agreement for differentiating BE, squamous, and gastric cardia mucosa using OFDI.