Prognostic importance of systemic clearance of methotrexate in childhood acute lymphoblastic leukemia

Summary Pharmacokinetic studies of methotrexate have been carried out in 21 children with acute lymphoblastic leukemia diagnosed in 1981. Children were treated with intermediate dose (500 mg/m²) methotrexate in keeping with the 1981 ALL treatment Protocol of the Hungarian Childhood Leukemia Working Group. Of the 21 children, 8 relapsed, and 13 are in continuous complete remission. In the relapsed patients significantly increased systemic clearance of methotrexate was observed at the time of the second methotrexate treatment cycle compared with the calculated value after the first administration of the drug. No such change in the clearance was found in patients who are still in remission. There was no difference between children who relapsed or who are in remission in the elimination half-time of the drug. Age, sex, WBC at diagnosis, and systemic clearance of methotrexate were found to be connected with the probability of relapse in the patients studied. The possible reasons for the prognostic role of systemic methotrexate clearance are discussed.     

Approaches to managing volunteer marrow donor registry HLA data. Algorithms for directing donor center-initiated HLA-DR typing of selected donors

The German bone marrow donor center (DKMS) hasrecruited over 732 500 donors during the first 9 years of its existence. Initially, donors were typed for HLA-A and B, and DR typing was only done on request for a patient-initiated search. In 1994, a project was started which led to the donor center-initiated DR typing (DCI-DRT) of >35,000 donors. These donors were selected by donor-specific criteria (age, sex, height and weight) and according to HLA-A and B phenotypes. The latter was done to avoid unnecessary DR typing of the most common A, B phenotypes With a follow up of >6 years, this strategy has led to a number of confirmatory typings (CT) (n=4588) and stem cell harvests (n=568), which is at least comparable to those ensuing after patient-initiated HLA-DR typing (126 000 DR typings, 8,213 CTs, 888 resulting in stem-cell donation). DCI-DRT seems to be a cost-effective strategy which may help to reduce search times and improve search outcome, and improve the overall efficiency of donor center operations     

Effect of exercise on coronary endothelial function in patients with coronary artery disease

BACKGROUND: Studies of the cardioprotective effects of exercise training in patients with coronary artery disease have yielded contradictory results. Exercise training has been associated with improvement in myocardial perfusion even in patients who have progression of coronary atherosclerosis. We therefore conducted a prospective study of the effect of exercise training on endothelial function in patients with coronary artery disease. METHODS: We randomly assigned 19 patients with coronary endothelial dysfunction, indicated by abnormal acetylcholine-induced vasoconstriction, to an exercise-training group (10 patients) or a control group (9 patients). To reduce confounding, patients with coronary risk factors that could be influenced by exercise training (such as diabetes, hypertension, hypercholesterolemia, and smoking) were excluded. In an initial study and after four weeks, the changes in vascular diameter in response to the intracoronary infusion of increasing doses of acetylcholine (0.072, 0.72, and 7.2 microg per minute) were assessed. The mean peak flow velocity was measured by Doppler velocimetry, and the diameter of epicardial coronary vessels was measured by quantitative coronary angiography. RESULTS: In the initial study, the two groups had similar vasoconstrictive responses to acetylcholine. After four weeks of exercise training, coronary-artery constriction in response to acetylcholine at a dose of 7.2 microg per minute was reduced by 54 percent (from a mean [+/-SE] decrease in the luminal diameter of 0.41+/-0.05 mm in the initial study to a decrease of 0.19+/-0.07 mm at four weeks; P<0.05 for the comparison with the change in the control group). In the exercise-training group, the increases in mean peak flow velocity in response to 0.072, 0.72, and 7.2 microg of acetylcholine per minute were 12+/-7, 36+/-11, and 78+/-16 percent, respectively, in the initial study. After four weeks of exercise, the increases in response to acetylcholine were 27+/-7, 73+/-19, and 142+/-28 percent (P<0.01 for the comparison with the control group). Coronary blood-flow reserve (the ratio of the mean peak flow velocity after adenosine infusion to the resting velocity) increased by 29 percent after four weeks of exercise (from 2.8+/-0.2 in the initial study to 3.6+/-0.2 after four weeks; P<0.01 for the comparison with the control group). CONCLUSIONS: Exercise training improves endothelium-dependent vasodilatation both in epicardial coronary vessels and in resistance vessels in patients with coronary artery disease.     

An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow

As dendritic cells (DC) are rare populations in all organs, their generation from hematopoietic precursors in large quantities has proven critical to study their biology. From murine bone marrow about 5 x 10(6) cells at 70% purity are obtained per mouse after 8 days of culture with GM-CSF. We have improved this standard method and routinely achieve a 50-fold higher yield, i.e., 1-3 x 10(8) immature and mature DC per mouse at 90-95% purity. The major modifications were: (i) the avoidance of any active depletion of bone marrow cell subpopulations to circumvent loss of precursors, (ii) a lower plating density of bone marrow cells, (iii) a prolonged culture period of 10-12 days, (iv) the reduction of the GM-CSF dose from day 8 or 10 onwards to reduce granulocyte contaminations. The final non-adherent population at day 10-12 constitutes a mixture of immature and mature DC. Further maturation of DC could be induced by high doses of LPS or TNF-alpha for the last 24 h, where 50-70% of the non-adherent fraction represented mature DC with high levels of NLDC-145, CD86 and CD40. This method allows by simple means the generation of high numbers of murine DC with very low B cell or granulocyte contaminations. It will be valuable to study DC biology notably at the molecular level.     

Gemins modulate the expression and activity of the SMN complex

Reduction in the expression of the survival of motor neurons (SMN) protein results in spinal muscular atrophy (SMA), a common motor neuron degenerative disease. SMN is part of a large macromolecular complex (the SMN complex) that includes at least six additional proteins called Gemins (Gemin2-7). The SMN complex is expressed in all cells and is present throughout the cytoplasm and in the nucleus where it is concentrated in Gems. The SMN complex plays an essential role in the production of spliceosomal small nuclear ribonucleoproteins (snRNPs) and likely other RNPs. To study the roles of the individual proteins, we systematically reduced the expression of SMN and each of the Gemins (2-6) by RNA interference. We show that the reduction of SMN leads to a decrease in snRNP assembly, the disappearance of Gems, and to a drastic reduction in the amounts of several Gemins. Moreover, reduction of Gemin2 or Gemin6 strongly decreases the activity of the SMN complex. These findings demonstrate that other components of the SMN complex, in addition to SMN, are critical for the activity of the complex and suggest that Gemin2 and Gemin6 are potentially important modifiers of SMA as well as potential disease genes for non-SMN motor neuron diseases.     

Immune response against the T-independent antigen alpha (1 leads to 3) dextran. I. Demonstration of an unexpected IgG response of athymic and germ-free-raised euthymic BALB/c mice

The primary antibody response in BALB/c mice to the T-independent bacterial antigen dextran B1355S [alpha(1 leads to 3)dextran] (Dex) was studied by means of isoelectric focusing, hemagglutination and immunodiffusion techniques. In response to a single immunization with 10 micrograms Dex all mice produce specific IgM antibodies. In addition, about 30% of conventionally raised BALB/c and BALB/c nu/ + mice, but 95% of germ-free (GF)-raised normal BALB/c and 100% of athymic BALB/c nu/nu mice produce specific IgG class anti-Dex antibodies. These antibodies include all IgG subclasses, carry predominantly the lambda light chain and the cross-reactive J558 idiotype and are specific for the alpha(1 leads to 3)glucosidic linkage. As compared to athymic and GF-raised mice, conventionally raised mice exhibit only a weak IgG response. The pronounced IgG production of GF-raised mice was not altered when adult mice were removed from their GF environment and housed under conventional conditions for several weeks prior to immunization with Dex. Reconstitution with isolated splenic T cells from conventionally raised, unprimed BALB/c mice reduces the remarkable capacity of BALB/c nu/nu mice to produce IgG anti-Dex antibodies. These findings suggest that the reduced capacity of conventionally raised BALB/c mice to mount an IgG response to the T-independent antigen Dex is due to a T cell-mediated suppressive mechanism which is neonatally induced by contact with environmental, i.e. bacterial, antigens.     

Molecular genetics of PKU in Eastern Europe: A nonsense mutation associated with haplotype 4 of the phenylalanine hydroxylase gene

Phenylketonuria (PKU) is a genetic disorder secondary to a deficiency of hepatic phenyalanine hydroxylase (PAH). Several mutations in thePAH gene have recently been reported, and linkage disequilibrium was observed between RFLP haplotypes and specific mutations. A new molecular lesion has been identified in exon 7 of thePAH gene in a Hungarian PKU patient by direct sequencing of PCR-amplified DNA. The C-to-T transition causes the substitution of Arg²⁴³ to a termination codon, and the mutant allele is associated with haplotype 4 of thePAH gene. The mutation is present in two of nine mutant haplotype 4 alleles among Eastern Europeans and is not present among Western Europeans and Asians. The rarity of this mutant allele and its restricted geographic distribution suggest that the mutational event occurred recently on a normal haplotype 4 background in Eastern Europe.