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BACKGROUND: Both individuals with intellectual disabilities (ID) and staff may be more inclined to use manual signs during formal than informal activities. In addition, the sign use of individuals with ID and staff is positively related. It is unclear if activity type and the sign use of staff interact as they shape the sign use of individuals with ID. METHODS: Through non-continuous partial interval coding, we observed frequency of manual sign usage in adults with ID during communicative activities, non-communicative activities and mealtimes in four special schools and 4-day centres. Using loglinear analysis and partial associations, we measured how sign use varied by activity between the people with ID and the staff. RESULTS: When staff used signs, clients and students did not vary their spontaneous signing rate between types of activities. When staff did not use signs, a differential influence appeared according to the type of activity: clients and students were significantly more likely to also refrain from using signs during mealtimes and leisure or work activities such as crafts (84% to 89% of the time) than during communicative activities such as signing sessions (65% of the time). CONCLUSIONS: Reluctance of staff to model sign use seemed to hinder signing implementation by the people with ID. Future studies should take into account various levels of sign prompting and increasing pragmatic functions of individuals' sign use.

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BACKGROUND: Staff may encourage individuals with intellectual disabilities to use manual signs by modelling its use, but implementing key word signing during daily activities can be demanding. METHOD: Staff's use of manual signs was observed in four special schools and four day centres for adults with intellectual disabilities during communicative group activities, non-communicative group activities and mealtimes. Using in situ partial interval coding, we measured staff's communication rate, semantic diversity of manual signs, sign reinforcement and sign imitation. With Chi-squared tests, associations were measured between these variables, the two settings and the three activity types. RESULTS: During communicative activities, staff used manual signs significantly more in adult services than special schools. During non-communicative activities and mealtimes, staff seldom used or reinforced signs. CONCLUSIONS: Staff communicated frequently but did not often model sign use during daily activities. To investigate influence from training background, further detailed measurements are warranted.

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BACKGROUND: One of the causes of unstable anticoagulant control in patients using vitamin K antagonists is a fluctuating intake of vitamin K. Research suggests that patients with a low dietary intake of vitamin K have a less stable anticoagulant control than patients with a higher intake. OBJECTIVES: To study whether supplementation with a low daily dose of vitamin K improves anticoagulant control. METHODS: We performed a double-blind, randomized, placebo-controlled trial. 200 patients of the Leiden anticoagulation clinic, who used the vitamin K antagonist phenprocoumon, were randomized to receive either adjusted-dose phenprocoumon and 100 mug vitamin K once daily or adjusted-dose phenprocoumon and a placebo. Treatment duration was 24 weeks. The primary outcome was the percentage of time the International Normalized Ratio was within the therapeutic range. RESULTS: The time in the therapeutic range was 85.5% in the placebo group and 89.5% in the vitamin K group (adjusted difference 3.6%; 95% CI -0.8% to 8.0%). The time below the therapeutic range was 3.1% in the placebo group and 2.1% in the vitamin K group (adjusted difference -0.7%; 95% CI -2.5% to 1.1%) and the time above the therapeutic range was 11.4% in the placebo group and 8.5% in the vitamin K group (adjusted difference -2.9%; 95% CI -6.9% to 1.1%). The relative risk (RR) of a maximal stability in the vitamin K group compared to the placebo group was 1.8 (95%, CI 1.1-2.7). CONCLUSION: Supplementation of vitamin K antagonists with 100 mug vitamin K improves stability of anticoagulant therapy. Because the risk of side effects is inversely related to anticoagulant stability, such an improvement is likely to reduce the number of bleeding and thrombotic events.

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BACKGROUND: Efforts to improve dosing quality in oral anticoagulant control include the use of computer algorithms. As current algorithms are simplistic and give dosage proposals in a small fraction of patients, we developed an algorithm based on principles of system and control engineering that gives proposals in nearly all patients. OBJECTIVE: To evaluate the new algorithm in clinical practice. PATIENTS AND METHODS: We conducted a double-blind randomized controlled trial among 712 patients with an indication for long-term anticoagulant treatment at the Leiden Anticoagulation Clinic. We compared oral anticoagulant dosing supported by the new algorithm (ICAD) with the standard algorithm (TRODIS). RESULTS: The percentage of time spent in the therapeutic range was similar for the new and standard algorithm groups, 79.8% vs. 80.2% (difference 0.4%, 95% CI: -1.7-2.6%). The new algorithm produced a dosage proposal in 97.5% of visits, and the standard algorithm in 60.8% (difference 36.7%, 95% CI: 35.4-38.0%). Of proposals of the new algorithm, 79.3% were accepted by the physician vs. 90.9% for the standard algorithm (difference 11.6%, 95% CI: 10.2-13.0%). This implies that the new algorithm gave an acceptable proposal in 77.4% of all patient visits vs. 55.3% for the standard algorithm (difference 22.1%, 95% CI 20.4-23.8%). CONCLUSIONS: Substantially more dosage proposals were generated and accepted with the new than with the standard algorithm, and the new algorithm will therefore improve the efficiency of anticoagulant monitoring without loss of quality.

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We investigated the effects of 2,4-dinitrophenol (DNP), the uncoupler of mitochondrial oxidative phosphorylation, on the Ca2+ -sensitive Cl- current component of the transient outward current (I(TO2)). Amphotericin B perforated-patch, whole-cell patch-clamp technique was employed (35 degrees C) using enzymatically isolated single rabbit atrial myocytes. We defined I(TO2) as the amplitude of the 2 mM 4-aminopyridine resistant transient outward current sensitive to anthracene-9-carboxylic acid (A9C). Between +5 and +45 mV, 0.2 mM A9C inhibited I(TO2) by approximately 70% (n = 13). Within 30 s after application of 0.2 mM DNP, both normal I(TO2) transients (n = 8) and the I(TO2) transients that remained after A9C treatment (n = 8) were inhibited completely. In cells expressing I(TO2) (70% of total), DNP also suppressed an A9C-insensitive slow outward current by approximately 40%, but the holding current at -80 mV was unaffected. There was a approximately 2 min latency between inhibitory effects of DNP and subsequent membrane current increase, presumably caused by activation of the ATP-sensitive K+ channels (n = 16). We conclude that DNP acutely inhibits I(TO2) via a mechanism presumably separate from metabolic inhibition.

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Gap junctions (GJs) provide for a unique system of intercellular communication (IC) allowing rapid transport of small molecules from cell to cell. GJs are formed by a large family of proteins named connexins (Cxs). Cx43 has been considered as the predominantly expressed Cx by hematopoietic-supporting stroma. To investigate the role of the Cx family in hemopoiesis, we analyzed the expression of 11 different Cx species in different stromal cell lines derived from murine bone marrow (BM) or fetal liver (FL). We found that up to 5 Cxs are expressed in FL stromal cells (Cx43, Cx45, Cx30.3, Cx31, and Cx31.1), whereas only Cx43, Cx45, and Cx31 were clearly detectable in BM stromal cells. In vivo, the Cx43-deficient 14.5- to 15-day FL cobblestone area-forming cells (CAFC)-week 1-4 and colony-forming unit contents were 26%-38% and 39%-47% lower than in their wild-type counterparts, respectively. The reintroduction of the Cx43 gene into Cx43-deficient FL stromal cells was able to restore their diminished IC to the level of the wild-type FL stromal cells. In addition, these Cx43-reintroduced stromal cells showed an increased support ability (3.7-fold) for CAFC-week 1 in normal mouse BM and 5-fold higher supportive ability for CAFC-week 4 in 5-fluorouracil-treated BM cells as compared with Cx43-deficient FL stromal cells. These findings suggest that stromal Cx43-mediated IC, although not responsible for all GJ-mediated IC of stromal cells, plays a role in the supportive ability for hemopoietic progenitors and stem cells. (Blood. 2000;96:498-505)

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The severe combined immunodeficient (SCID) mouse model may be used to evaluate new approaches for the treatment of acute myeloid leukemia (AML). We have previously demonstrated the killing of SCID mouse leukemia initiating cells by in vitro incubation with human GM-CSF fused to Diphtheria toxin (DT-huGM-CSF). In this report, we show that in vivo treatment with DT-huGM-CSF eliminates AML growth in SCID mice. Seven cases of AML were studied. SCID mice were treated intraperitoneally with the maximally tolerated dose of 75 microg/kg/day for 7 days. Antileukemic efficacy was determined at days 40 and 80 after transplantation, by enumerating the percentages of human cells in SCID bone marrow using flow cytometry and short tandem repeat polymerase chain reaction (STR-PCR) analysis. Four out of seven AML cases were sensitive to in vivo treatment with DT-huGM-CSF at both evaluation time points. In three of these cases, elimination of human cells was demonstrated by flow cytometry and STR-PCR. One AML case showed moderate sensitivity for DT-huGM-CSF, and growth of the two remaining AML cases was not influenced by DT-huGM-CSF. Sensitivity was correlated with GM-CSFR expression. Our data show that DT-huGM-CSF can be used in vivo to reduce growth of AML and warrant further development of DT-huGM-CSF for the treatment of human AML.

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We have previously demonstrated that diphtheria toxin (DT) fused to human GM-CSF effectively eliminates human long-term leukemia initiating cells in SCID mice. However, because huGM-CSF does not react with the murine GM-CSF receptor possible side-effects to nonleukemic tissues could not be analyzed in the AML/SCID model. To overcome this problem, we used murine GM-CSF fused to DT and studied the therapeutic index in the rat leukemia model BNML/LT12. In DT-mGM-CSF dose escalation experiments, severe dose-dependent toxicity to organs such as liver, kidney and lung was observed. Therefore, the antileukemic effects were evaluated with the lower doses. Daily intraperitoneal bolus injections of 75 microg/kg/day for 7 days induced a 3 log leukemic cell kill. The dose of 75 microg/kg/day had no effect on the hemopoietic progenitor cell subsets. These in vivo studies show that the DT-GM-CSF fusion protein can be used for specifically targeting leukemic cells and thus has potential as a therapeutic agent in the treatment of AML.

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We studied the cell kill induced by granulocyte-macrophage colony-stimulating factor (GM-CSF ) fused to Diphtheria Toxin (DT-GM-CSF ) in acute myeloid leukemia (AML) samples and in populations of normal primitive hemopoietic progenitor cells. AML samples from three patients were incubated in vitro with 100 ng/mL DT-GM-CSF for 48 hours, and AML cell kill was determined in a proliferation assay, a clonogenic assay colony-forming unit-AML (CFU-AML) and a quantitative long-term bone marrow (BM) culture ie, the leukemic-cobblestone area forming cell assay (L-CAFC). To measure an effect on cells with in vivo leukemia initiating potential DT-GM-CSF exposed AML cells were transplanted into immunodeficient mice. In two out of three samples it was shown that all AML subsets, including those with long-term abilities in vivo (severe combined immunodeficient mice) and in vitro (L-CAFC assay) were reduced in number by DT-GM-CSF. Cell kill induced by DT-GM-CSF could be prevented by coincubation with an excess of GM-CSF, demonstrating that sensitivity to DT-GM-CSF is specifically mediated by the GM-CSF receptor. Therefore, binding and internalization of GM-CSF probably occur in immature AML precursors of these two cases of AML. The third AML sample was not responsive to either GM-CSF or DT-GM-CSF. The number of committed progenitors of normal bone marrow (burst-forming unit-erythroid, colony-forming unit granulocyte- macrophage, and cobble stone area forming cell [CAFC] week 2) and also the number of cells with long-term repopulating ability, assayed as week 6 CAFC, were unchanged after exposure to DT-GM-CSF (100 ng/mL, 48 hours). These studies show that DT-GM-CSF may be used to eliminate myeloid leukemic cells with long-term potential in vitro and in immunodeficient mice, whereas normal hemopoietic stem cells are spared.

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The potential to selectively eliminate acute myeloid leukaemia (AML) cells with the GM-CSF-diphtheria toxin fusion protein (DT-GM-CSF) was studied under conditions of autonomous proliferation in vitro with no growth factors (GFs) added and after growth stimulation with a mixture of human (hu)G-CSF, huIL-3 and huSCF. DNA synthesis was maximally inhibited after 48 h exposure to DT-GM-CSF. Cell viability and AML colony forming ability in vitro were reduced. 18/22 samples were found to be sensitive to DT-GM-CSF, with 50% inhibition of DNA synthesis (ID50) at concentrations ranging from 0.1 to 16 ng/ml, and four samples were minimally or not sensitive to DT-GM-CSF (ID50 > or = 99 ng/ml). From the 15 samples which showed autonomous proliferation, 13 were sensitive to inhibition of proliferation by DT-GM-CSF. The level of GM-CSF receptor (GM-CSFR) expression was determined by flow cytometry after labelling with specific antibodies for the alpha and beta subunits. Although the toxicity to DT-GM-CSF was specifically mediated by the GM-CSFR, no correlation was found between the level of expression of the GM-CSFR alpha or beta subunit and the sensitivity for DT-GM-CSF. These in vitro studies show that the DT-GM-CSF fusion protein can be used for specifically targeting and eliminating leukaemic cells in the majority of AML cases.

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