Biologic and prognostic significance of the presence of more than two mu heavy-chain genes in childhood acute lymphoblastic leukemia of B precursor cell origin

We examined the arrangement of the mu heavy-chain immunoglobulin (Ig) genes in the leukemic blast cell DNA of 93 children with acute lymphoblastic leukemia (ALL). All cases met morphologic and cytochemical criteria for ALL, lacked detectable T cell surface antigens, and expressed HLA-DR (Ia) antigens. Eighty-three of the 93 patients (89%) were positive for the common acute lymphoblastic leukemia antigen (CALLA), and 20 of 91 (22%) tested had detectable cytoplasmic immunoglobulin. As expected, the heavy-chain lg gene was rearranged in all cases, and the pattern of rearrangements was variable; 23 had one allele rearranged and one in the germ line configuration; 15 had one rearranged and one deleted; and 37 had two rearranged. Unexpectedly, in 18 patients the presence of more than two mu gene-hybridizing bands was detected. Combinations of enzymes and heavy-chain gene probes were used to confirm that the extra bands were not the result of underdigestion of the DNA or DNA restriction site polymorphism. In eight of the 18 patients, we identified an extra chromosome 14 as a possible cause of the extra bands' hybridizing to the mu heavy-chain constant-region probe. In the remaining ten patients, the presence of three or four bands hybridizing with the mu probe suggests the presence of two populations of leukemic cells that may have arisen either by separate leukemic transformation events or by clonal evolution of one clone into two related lines. Although preliminary (2-year follow-up), our data suggest that childhood ALL of B lineage with more than two mu heavy-chain genes, but without extra copies of chromosome 14, may be more resistant to therapy.     

Factor VIII/von Willebrand factor protein: sensitivity of periodic acid Schiff stain to carbohydrate deficiency

We have investigated the periodic acid Schiff (PAS) Coomassie staining ratio of the human factor VIII/von Willebrand factor (fVIII/vWf) protein. The PAS-Coomassie staining ratio is consistent over 8 days. The PAS-Coomassie ratio of fVIII/vWf protein purified from different starting materials does not appear to be significantly different. The PAS stain can detect as little as 300 ng of carbohydrate in the fVIII/vWf protein. Desialation did not affect the PAS-Coomassie ratio, while removal of penultimate galactose resulted in a marked reduction in the PAS-Coomassie ratio. This reduction was further accentuated with the removal of N-acetylglucosamine. The smaller multimers of the fVIII/vWf protein have a reduced sialic acid and PAS-Coomassie staining ratio. This difference does not appear to be related to the sialic acid deficiency but may be related to the distribution or organization of the carbohydrate moieties on the smaller fVIII/vWf multimers.     

The mutagenic response at the ouabain resistance locus in T cells of mice exposed to N-ethyl-N-nitrosourea parallels the response at the Hprt locus and correlates with mutation target size

The lymphocyte Hprt gene has been used extensively as a reporter locus to monitor the mutational effects of the exposure of animals to genotoxicants. Implicit in this view of the function of a reporter gene is the assumption that its mutagenic response is representative of that of other genes in the organism. As a test of this hypothesis we compared the frequency of 6-thioguanine-resistant (TGr) mutants at the Hprt locus with the mutant frequency (MF) induced at another locus, the ouabain resistance (Oua) locus. The frequency of spontaneous OUA(R) mutants was estimated to be 1.1x10(-7) (MF between <0.3 and 1.1x10(- 7)), which was approximately 30-fold less than the spontaneous TGr MF. Following treatment with N-ethyl-N-nitrosourea (ENU), the induced OUA(R) MF at each of two dose levels (50 and 150 mg/kg ENU) and two time points (3 and 6 weeks post-exposure) was consistently 8- to 9-fold lower than the corresponding TGr MF. Thus the mutagenic response of the Oua locus closely paralleled that of the Hprt locus, indicating a similarity in their response to ENU. In addition, the Oua locus was 3-4 times more sensitive than the Hprt locus to the mutagenic effect of ENU, as measured by the fold increase in MF over the background level. The number of ENU-mutable sites capable of resulting in a TGr or OUA(R) phenotype, otherwise known as the mutation target size, was estimated to differ by an order of magnitude between the two loci. This difference in target size correlates with, and therefore may largely account for, the difference in induced MF between both loci.      

Hypoglycemic and anti-lipemic effects of the aqueous extract from Cissus sicyoides

Background

Psoriasis is a chronic inflammatory skin disease that can be successfully treated with a mixture of fumaric acid esters (FAE) formulated as enteric-coated tablets for oral use. These tablets consist of dimethylfumarate (DMF) and salts of monoethylfumarate (MEF) and its main bioactive metabolite is monomethylfumarate (MMF). Little is known about the pharmacokinetics of these FAE. The aim of the present study was to investigate the hydrolysis of DMF to MMF and the stability of MMF, DMF and MEF at in vitro conditions representing different body compartments.

Results

DMF is hydrolyzed to MMF in an alkaline environment (pH 8), but not in an acidic environment (pH 1). In these conditions MMF and MEF remained intact during the period of analysis (6 h). Interestingly, DMF was hardly hydrolyzed to MMF in a buffer of pH 7.4, but was rapidly hydrolyzed in human serum having the same pH. Moreover, in whole blood the half-life of DMF was dramatically reduced as compared to serum. The concentrations of MMF and MEF in serum and whole blood decreased with increasing time. These data indicate that the majority of the FAE in the circulation are metabolized by one or more types of blood cells. Additional experiments with purified blood cell fractions resuspended in phosphate buffered saline (pH 7.4) revealed that at concentrations present in whole blood monocytes/lymphocytes, but not granulocytes and erythrocytes, effectively hydrolyzed DMF to MMF. Furthermore, in agreement with the data obtained with the pure components of the tablet, the enteric-coated tablet remained intact at pH 1, but rapidly dissolved at pH 8.

Conclusion

Together, these in vitro data indicate that hydrolysis of DMF to MMF rapidly occurs at pH 8, resembling that within the small intestines, but not at pH 1 resembling the pH in the stomach. At both pHs MMF and MEF remained intact. These data explain the observation that after oral FAE intake MMF and MEF, but not DMF, can be readily detected in the circulation of human healthy volunteers and psoriasis patients.     

Specificity of mutagenesis by 4-aminobiphenyl: mutations at G residues in bacteriophage M13 DNA and G-->C transversions at a unique dG(8-ABP) lesion in single-stranded DNA

Mutagenesis by the human bladder carcinogen 4-aminobiphenyl (ABP) was studied in single-stranded DNA from a bacteriophage M13 cloning vector. In comparison to ABP lesions in double-stranded DNA, lesions in single- stranded DNA were approximately 70-fold more mutagenic and 50-fold more genotoxic. Sequencing analysis of ABP-induced mutations in the lacZ gene revealed exclusively base-pair substitutions, with over 80% of the mutations occurring at G sites; the G at position 6310 accounted for 25% of the observed mutations. Among the sequence changes at G sites, G- ->T transversions predominated, followed by G-->C transversions and G-- >A transitions. In order to further elucidate the mutagenic mechanism of ABP, an oligonucleotide containing the major DNA adduct, N- (deoxyguanosin-8-yl)-4-aminobiphenyl (dG(8-ABP)), was situated within the PstI site of a single-stranded M13 genome. After in vivo replication of the adduct containing ABP-modified and control (unadducted) genomes, the mutational frequency and mutational specificity of the dG(8-ABP) lesion were determined. The targeted mutational efficiency was approximately 0.01%, and the primary mutation observed was the G-->C transversion. Thus dG(8-ABP), albeit weakly mutagenic at the PstI site, can contribute to the mutational spectrum of ABP lesions.      

Epileptic spasms without hypsarrhythmia in infancy and childhood: tonic spasms as a seizure type

Epileptic spasms were defined by the International League Against Epilepsy Task Force on Classification and Terminology in 2001 as a specific seizure type. Epileptic spasms without hypsarrhythmia have been described in some series of patients, occurring either in infancy or childhood. More prolonged epileptic spasms without hypsarrhythmia were previously defined as a different seizure type, and referred to as “tonic spasm seizures”. Here, we present a 5‐year‐old boy who started having epileptic spasms without hypsarrhythmia at 8 months of age, effectively treated with oxcarbazepine. With the withdrawal of medication, epileptic spasms returned. Video‐EEG monitoring revealed high‐voltage slow waves superimposed by low‐voltage fast activity, followed by an electrodecremental phase and a burst of asymmetric fast activity, time‐locked to clinical tonic spasm seizures. Brain MRI showed left temporal atrophy with temporal pole grey/white matter junction blurring and ictal PET‐CT showed left basal frontal hypermetabolism. Seizures were refractory to several AEDs and vigabatrin was introduced with seizure cessation. Despite efforts to classify epileptic spasms, these are still considered as part of the group of unknown seizure types. In some cases, a focal origin has been suggested, leading to the term “periodic spasms” and “focal spasms”. In this case, epileptic spasms without hypsarrhythmia, associated with tonic spasms, may be a variant of focal spasms and might be considered as an epileptic syndrome. [Published with video sequence]     

First Trimester Aneuploidy Screening Using Nuchal Iranslucency, Free Beta Human Chorionic Gonadotrophin and Maternal Age

Summary: Screening for aneuploidy using maternal age has a low detection rate and high false positive rate. Second trimester maternal serum screening increases trisomy 21 detection and decreases die false positive rate. First trimester screening would enable definitive diagnosis with chorionic villus sampling, and simple surgical termination of affected pregnancies would still be an option. Nuchal translucency (NT), free beta human chorionic gonadotrophin (fβHCG) and maternal age were assessed in 302 patients before chorionic villus sampling. NT positively and fβHCG negatively correlated with gestation, but neither correlated with maternal age nor with each other. Both NT and fβHCG were increased in trisomy 21. NT was increased and fβHCG was decreased in trisomy 18. Multivariate discriminant analysis enabled 87.5% detection of trisomy 21 in this high-risk population, for a 14% false positive rate. In a simulated normal population, using a risk cut-off of 1 in 250, 71% detection was achieved for a 7% false positive rate. The combination of NT, fβHCG and maternal age is a simple, readily available and viable first trimester screening strategy.