Biochemical interactions between methotrexate and 1-β-d-arabinofuranosylcytosine in hematopoietic cells of children: a Pediatric Oncology Group study

Summary Children with acute lymphocytic leukemia (ALL) in remission were treated with overlapping sequential infusions of methotrexate (MTX) and 1--d-arabinofuranosylcytosine (araC) as part of continuation therapy. The doses and the sequence were chosen to mimic conditions that produced greater than additive antineoplastic activity with these two drugs in preclinical studies. To assess the potential for the drug combination to exhibit greater than additive effect in vivo, we investigated several biochemical parameters that had been associated with synergism in vitro. Because the patients were in remission, the intracellular parameters could only be measured in cytologically normal hematopoietic cells. We observed that (1) the mean plasma concentrations of MTX and araC were above those required to obtain a greater than additive cytotoxicity with the two drugs in tissue culture; (2) MTX did not have a significant antipurine effect in bone marrow mononuclear cells; (3) the mean intracellular concentration of deoxycytidine triphosphate (dCTP) was significantly lower after treatment with the drug combination than after therapy with araC alone; and (4) the ratio of araC triphosphate (araCTP) to dCTP was 2.6 times higher after treatment with the combination than after araC alone. These results indicate that it is possible to achieve in patients the biochemical conditions associated with the greater than additive antineoplastic activity of MTX and araC in vitro.Abbreviations ALL acute lymphocytic leukemia - araC 1--d-arabinofuranosyluracil - araCTP araC triphosphate - araU 1--d-arabinofuranosyluracil - dNTPs deoxyribonucleoside triphosphates - MTX methotrexate - TCA trichloroacetic acid Supported in part by grants from the National Cancer Institute, National Institutes of Health (CA-38 053; CA-33572, CA-32278, CA-38 859, CA-29 691, and CA-30 969). Preliminary reports on the biochemical data were published by E. M. N., A. M. T., and D. P. inProc Am Assoc Cancer Res 24: 133 (1983) and those on the clinical data, by R. A K., E. M. N., D. P. R. B. R., M. B. H., Y. R., and A. I. F. inProc Am Soc Clin Oncol 3: 201 (1984)     

Antibody and memory B cell responses to the dengue virus NS1 antigen in individuals with varying severity of past infection

To further understand the role of NS1-specific antibodies (Abs) in disease pathogenesis, we compared neutralizing antibody levels (Nabs), NS1-Ab levels, IgG antibody subclass profiles and NS1-specific memory B-cell responses (Bmems) in individuals, with varying severity of past dengue. Nabs (Neut50 titres) were assessed using the Foci Reduction Neutralization Test (FRNT) and in-house ELISAs were used to assess NS1-Abs and NS1-Ab subclasses for all four DENV serotypes in individuals with past DF (n = 22), those with past DHF (n = 14) and seronegative (SN) individuals (n = 7). B-cell ELISpot assays were used to assess NS1-specific Bmem responses. 15/22 (68.18%) individuals with past DF and 9/14 (64.29%) individuals with past DHF had heterotypic infections. Neut50 titres were found to be significantly higher for DENV1 than DENV2 (p = 0.0006) and DENV4 (p = 0.0127), in those with past DHF, whereas there was no significant difference seen in titres for different DENV serotypes in those with past DF. Overall NS1-Ab to all serotypes and NS1-specific IgG1 responses for DENV1, 2 and 4 serotypes were significantly higher in those with past DHF than individuals with past DF. Those with past DHF also had higher IgG1 than IgG3 for DENV1 and DENV3, whereas no differences were seen in those with past DF. Over 50% of those with past DF or DHF had NS1-specific Bmem responses to >2 DENV serotypes. There was no difference in the frequency of Bmem responses to any of the DENV serotypes between individuals with past DF and DHF. Although the frequency of Bmem responses to DENV1 correlated with DENV1-specific NS1-Abs levels (Spearman r = 0.35, p = 0.02), there was no correlation with other DENV serotypes. We found that those with past DF had broadly cross-reactive Nabs, while those with past DHF had higher NS1-Ab responses possibly with a different functionality profile than those with past DF. Therefore, it would be important to further evaluate the functionality of NS1-specific antibody and Bmem responses to find out the type of antibody repertoire that is associated with protection against severe disease.     

Assessment of Photoinduced Frontal Polymerization Processes for a Stable 1K System

Frontal polymerization (FP) has attracted increasing interest in recent years in various applications. This polymerization method can be very promising for the polymerization of thick materials with high fillers content in the range of 50–80% (weight) by local application of a reasonable amount of energy. In this work, recent advances in controllable and predictive behavior for photoinduced frontal photopolymerization are reported. Here, tert-butyl peroxybenzoate (Luperox-P) is selected to initiate thermal polymerization at depth because its high polymerization ability and its decomposition temperature is in a promising range, i.e., neither extremely high (monomer decomposition) nor very low (storage stability issues). Thermal imaging experiments are used to follow the temperatures in the samples in real time. The number of cured layers and the depth of cure are also determined. This paper investigates various factors such as the contents of both photo and thermal initiators, the light intensity, the fiber contents, the irradiation time, etc., resulting in a statistical design of experiments with the factors: 1) content of Luperox P and 2) the irradiation time used to investigate the influence on photoinduced frontal polymerization. Markedly, FP appears to be fully controllable for a storage-stable, tunable 1K system.     

Adverse childhood experiences and the development of Down syndrome regression disorder

Down syndrome regression disorder (DSRD) is a clinical symptom cluster of acute or subacute neurocognitive regression in otherwise health persons with Down syndrome. The objective of this study was to evaluate if adverse childhood experiences (ACEs) were more prevalent in children with DSRD than those with DS alone. A survey-based, cohort-based study was performed. Caregivers of individuals with DSRD with onset of symptoms between age 10 and 30 years and DS alone were administered the ACEs questionnaire via an online REDCap survey. A total of 159 responses were collected after excluding incomplete surveys and those not meeting criteria for DSRD. Individuals with DSRD were not more likely to experience ACEs (p = 0.18, 95% confidence interval [CI]: 0.43–1.17). In those with ACEs prior to the onset of symptoms, the median time prior was 7 months (interquartile range: 5–10). Individuals with DSRD were more likely to report three or more ACEs (52, 33%) compared to those with DS alone (39, 22%) (p = 0.02, 95% CI: 1.08–2.87). Exposure to ACEs were not predictive of response to particular therapeutic interventions although those with multiple ACEs 3 months prior to the onset of symptoms was associated with lower response rates to benzodiazepines and immunotherapy (p = 0.02, 95% CI: −3.64–−1.13). This study provides preliminary data that individuals with DSRD experience ACEs at a similar rate to individuals with only DS alone, although three or more ACEs, often preceding the onset of symptoms, was more prevalent in individuals with DSRD.     

Enhancement of the response to purinergic agonists in P2Y1 transfected 1321N1 cells by antagonists suramin and PPADS

1. We have previously shown that both suramin and pyridoxal-phosphate-6-azophenyl-2′, 4′ disulphonic acid (PPADS) act as antagonists at transfected P2Y₁ receptors. Here we show that under certain experimental conditions these two P2 antagonists can enhance the response to agonists acting at these receptors.
2. The expression of either P2Y₁ or P2Y₂ receptors in 1321N1 human astrocytoma cells results, on a change of medium, in an elevation of basal (no added agonist) accumulation of [³H]-inositol(poly)phosphates([³H]-InsP_x) compared to cells not expressing these receptors. This elevation is much greater in P2Y₁ transfectants than in P2Y₂ transfectants.
3. Both PPADS and suramin reduced this basal level of [³H]-InsP_x accumulation in the P2Y₁ expressing cells.
4. When a protocol was used which required changing the culture medium, antagonists were added at a concentration which reduced the basal accumulation by about 50%, there was a significant stimulation in response to increasing concentrations of 2-methylthioadenosine 5′-triphosphate (2MeSATP), in the absence of antagonists there was no significant effect of the agonist.
5. However, when 2MeSATP was added in the absence of a change of medium and with no antagonist present, there was a several fold increase in [³H]-InsP_x accumulation. These results show that a release of endogenous agonist activity (possibly ATP/ADP) from the P2Y₁ expressing cells can create conditions in which a response to an agonist such as 2MeSATP can only be seen in the presence of a competitive antagonist.

     

Endothelin-like immunoreactivity in aqueous humor of patients with primary open-angle glaucoma and cataract

Background: Experimental evidence suggests a role of endothelin-1 (ET) in the regulation of intraocular pressure (IOP). Method: Therefore, inpatients undergoing cataract surgery, ET-like immunoreactivity (STIR) was measured by radioimmunoassay in pooled samples of aqueous humor of eyes with primary open-angle glaucoma (POAG) and normotensive eyes with cataract only. Results: ETIR was significantly (P < 0.05) higher in patients with cataract and POAG (20.5 ± 1.8 pg/ml,n = 12; preoperative IOP 21.4 ± I.1 mmHg,n = 33) than in patients with cataract only (15.8 ± 1.6 pg/m1,n = 15; preoperative IOP 16.0 ± 0.6 mmHg,n = 77). Conclusion: This finding may indicate a role of ET in POAG or ocular antihypertensive treatment, and its relevance should be further investigated.     

Variability of contour line alignment on sequential images with the Heidelberg Retina Tomograph

Background: The influence of the contour line alignment software algorithm on the variability of the Heidelberg Retina Tomograph (HRT) parameters remains unclear. Methods: Nine discrete topographic images were acquired with the HRT from the right eye in six healthy, emmetropic subjects. The variability of topometric data obtained from the same topographic image, analyzed within different samples of images, was evaluated. A total of four mean topographic images was computed for each subject from: all nine discrete images (A), the first six of those images (B), the last six of those nine images (C), and the first three combined with the last three images (D). A contour line was computed on the mean topographic image generated from the nine discrete topographic images (A). This contour line was then applied to the three other mean topographic images (B, C, and D), using the contour line alignment in the HRT software. Subsequently, the contour line on the mean topographic images was applied to each of the discrete members of the particular images subsets used to compute the mean topographic image, and the topometric data for these discrete topographic images was computed successively for each subset. Prior to processing each subset, the contour line on the discrete topographic images was deleted. This strategy provided a total of three analyses on each discrete topographic image: as a member of the nine images (mean topographic image A), and as a member of two subsets of images (mean topographic image B, C, and/or D). The coefficient of variation (100×SD/mean) of the topographic parameters within those three analyses was calculated for each discrete topographic image in each subject (intraimage coefficient of variation). In addition, a coefficient of variation between the nine discrete topographic images (interimage coefficient of variation) was calculated. Results: The intraimage and interimage variability for the various topographic parameters ranged between 0.03% and 3.10% and between 0.03% and 24.07% respectively. The intraimage coefficients of variation and interimage coefficients of variation correlated significant (r ²=0.77;P<0.0001). Conclusion: A high intraimage variability, i.e. a high variability in contour line alignment between sequential images, might be an important source of test re-test variability between sequential images.     

Hypoglycaemic effects of the novel antidiabetic agent repaglinide in rats and dogs

1. Repaglinide, a novel compound with a nonsulphonylurea structure, is currently being clinically tested as a therapeutic agent. In the present study, the hypoglycaemic effects of repaglinide in rats and dogs were investigated.
2. Whereas the R-enantiomer, AG-EE 624 ZW, showed only weak hypoglycaemic activity, the S-enantiomer, repaglinide, turned out to be a potent hypoglycaemic compound in rats after oral as well as after intravenous administration. Only 50% of the dose of repaglinide was needed to be equieffective with the racemic mixture AG-EE 388 ZW. The corresponding ED₅₀ values calculated for the effects after 120 min p.a. (intravenous administration) were 3.4 μg kg⁻¹ (repaglinide) and 6 μg kg⁻¹ (AG-EE 388 ZW).
3. When compared to glimepiride or glibenclamide, repaglinide displayed a 18 to 25 times higher potency in fasted rats. The ED₅₀ values calculated for the effects after 120 min p.a. (oral administration) were 10 μg kg⁻¹ (repaglinide), 182 μg kg⁻¹ (glimepiride) and 255 μg kg⁻¹ (glibenclamide).
4. In glucose loaded rats (0.5, 1.0, 2.0 and 3.0 g kg⁻¹ glucose, p.o.) repaglinide exerted a very strong antihyperglycaemic activity which was even more pronounced than under normoglycaemic conditions. So for a reduction in blood glucose of 1 mmol l⁻¹, 10.3, 9.3, 7.0 8.4 and 7.2 μg kg⁻¹ repaglinide were needed after glucose loads of 0.0, 0.5, 1.0, 2.0 and 3.0 g kg⁻¹, respectively.
5. In beagle dogs repaglinide again showed a pronounced hypoglycaemic effect (ED₅₀ 28.3 μg kg⁻¹) which lasted for up to 24 h. However, insulin levels were only transiently increased.
6. The in vivo data presented are well supported by recently published in vitro findings. From its activity profile, repaglinide appears to be a promising new therapeutic agent.

     

Effects of [3H]-BIDN,a novel bicyclic dinitrile radioligand for GABA-gated chloride channels of insects and vertebrates

1. The radiolabelled bicyclic dinitrile, [³H]-3,3-bis-trifluoromethyl-bicyclo[2.2.1]heptane-2,2-dicarbonitrile ([³H]-BIDN), exhibited, specific binding of high affinity to membranes of the southern corn rootworm (Diabrotica undecimpunctata howardi) and other insects. A variety of γ-aminobutyric acid (GABA) receptor convulsants, including the insecticides heptachlor (IC₅₀, 35±3 nM) and dieldrin (IC₅₀, 93±7 nM), displaced [³H]-BIDN from rootworm membranes. When tested at 100 μM, 1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane(EBOB), 4-t-butyl-2,6,7-trioxa-1-phosphabicyclo[2.2.2]octane-1-thione (TBPS), 1-phenyl-4-t-butyl-2,6,7-trioxabicyclo[2.2.2]octane (TBOB) and picrotoxin failed to displace 50% of [³H]-BIDN binding to rootworm membranes indicating that the bicyclic dinitrile radioligand probes a site distinct from those identified by other convulsant radioligands.
2. Dissociation studies showed that dieldrin, ketoendrin, toxaphene, heptachlor epoxide and α and β endosulphan displace bound [³H]-BIDN from rootworm membranes by a competitive mechanism.
3. Rat brain membranes were also shown to possess a population of saturable, specific [³H]-BIDN binding sites, though of lower affinity than in rootworm and with a different pharmacological profile. Of the insecticidal GABAergic convulsants that displaced [³H]-BIDN from rootworm, cockroach (Periplaneta americana) and rat brain membranes, many were more effective in rootworm.
4. Functional GABA-gated chloride channels of rootworm nervous system and of cockroach nerve and muscle were blocked by BIDN, whereas cockroach neuronal GABA_B receptors were unaffected.
5. Expression in Xenopus oocytes of either rat brain mRNA, or cDNA-derived RNA encoding a GABA receptor subunit (Rdl) that is expressed widely in the nervous system of Drosophila melanogaster resulted in functional, homo-oligomeric GABA receptors that were blocked by BIDN. Thus, BIDN probes a novel site on GABA-gated Cl⁻ channels to which a number of insecticidally-active molecules bind.

     

Activation of benzylic alcohols to mutagens by human hepatic sulphotransferases

Four primary and five secondary benzylic alcohols derived frompolycyclic aromatic hydrocarbons were tested for mutagenicityin Salmonella typhimurium TA98 in the presence of 3'-phosphoadenosine-5'-phosphosulphate,the cofactor for sulphotransferases, and varying amounts ofhepatic cytosol from three or four different human subjects,a 3-year-old child, an adult female, an adult male and one unknown.All compounds except one, 4H-cyclopenta[def] phenanthren-4-ol,were activated to mutagens. The interindividual variation inthe activities was at most 3-fold and the individual activitiestowards the different substrates were correlated with each other.The same compounds had previously been tested in the presenceof hepatic cytosol from rats and all compounds activated inone species were also activated in the other species. However,there were marked quantitative differences, which were furthercomplicated by the observation of a substantial sex differencein the rat. Male and female rat liver cytosol showed highersulphotransferase activities towards 1-hydroxymethylpyrene,9-hydroxymethylanthracene, 7-hydroxymethyl-12-methylbenz[a]anthraceneand 4H-cyclopenta[def]chrysen-4-ol than human liver cytosol.The largest difference in activity was seen with 7-hydroxymethyl-12-methylbenz[a]anthracene,reaching a factor of