Sequence and schedule-dependent synergy of trimetrexate in combination with 5-fluorouracil in vitro and in mice

The purpose of this study was to determine the conditions for optimum synergistic efficacy of the two-drug combination of trimetrexate and 5-fluorouracil. Synergistic cell killing of Chinese hamster ovary cells in these clonogenic survival assays was observed only when the cells had been exposed to trimetrexate (25 microM) for 2 to 4 h prior to 5-fluorouracil exposure (either 125 or 250 microM). The schedule dependence of the observed synergy in vitro was closely linked to trimetrexate-induced changes in cellular 5-phosphoribosyl 1-pyrophosphate (PRPP) pools. Exposure to 25 microM trimetrexate induced increases in PRPP pools to 398% and 761% of control values at 2 and 4 h, respectively. Methotrexate (20 microM) also increased Chinese hamster ovary cell PRPP content in a time-dependent fashion to values of 280 and 511% of control after 2 and 4 h of drug exposure. Previous in vivo studies demonstrated a modest degree of therapeutic synergy between trimetrexate and 5-fluorouracil against P388 leukemia. Our in vitro results suggested that the degree of synergy seen in vivo could be increased with appropriate schedule changes. Mice were implanted i.p. with 10(6) P388 leukemia cells on Day 0 and were treated with trimetrexate (every 3 h for eight injections; Days 1, 5, and 9) and 5-fluorouracil (Days 1, 5, and 9) as single agents or in combination on one of two schedules; 5-fluorouracil was administered with either the first or the last of the eight trimetrexate doses on Days 1, 5, and 9. Both treatment regimens demonstrated therapeutic synergy but, as predicted from the in vitro data, the "5-fluorouracil last" was superior to the "5-fluorouracil first" sequence. Treatment with the optimal doses on the "5-fluorouracil last" sequence (trimetrexate, 31; 5-fluorouracil, 33 mg/kg/injection) produced an increased life span of 183% and a net reduction in tumor cell burden of 6.7 logs compared with a 111% increased life span (net reduction in tumor burden of 2.6 logs) produced by the most active of the single agents, 5-fluorouracil. Thus the efficacy of the combination of trimetrexate with 5-fluorouracil was sequence and time dependent both in vitro and in vivo. The synergy, observed in vitro and probably in vivo, was linked to a trimetrexate-induced elevation of intracellular PRPP, thus facilitating the production of 5-fluoropyrimidine nucleotides. These data are similar to the sequence and schedule dependency of the methotrexate/5-fluorouracil combination with important differences.(ABSTRACT TRUNCATED AT 400 WORDS)     

Oxidative response of human neutrophils, monocytes, and alveolar macrophages induced by unopsonized surface-adherent Staphylococcus aureus.

In contrast to results with bacterial suspensions, phagocytosis of unopsonized bacteria readily occurs when bacteria are adhered to glass or plastic surfaces. However, in contrast to neutrophils, alveolar macrophages produced much less DNA denaturation as measured by acridine orange metachromasia of phagocytized Staphylococcus aureus. We have studied the phagocytosis of unopsonized surface-adherent S. aureus and the subsequent production of reactive oxygen species by peripheral blood neutrophils, monocytes, and alveolar macrophages. Phagocyte-free systems were then used to show the relationship of the reactive oxygen species produced by neutrophils and alveolar macrophages and the denaturation of unopsonized S. aureus DNA with acridine orange. Peripheral blood neutrophils, monocytes, and alveolar macrophages from normal human volunteers were added to vials with adherent S. aureus without opsonin. Bacterial uptake and luminol- and lucigenin-dependent chemiluminescence were measured. Neutrophils developed much greater luminol-dependent chemiluminescence than monocytes or alveolar macrophages. Compared with neutrophils and monocytes, alveolar macrophages developed significantly greater concentrations of superoxide, as measured by lucigenin-dependent chemiluminescence and ferricytochrome c reduction. These findings suggested that products of the myeloperoxidase-hydrogen peroxide-halide pathway were generated when peripheral blood neutrophils were stimulated and that alveolar macrophages primarily produced superoxide. When these reactive oxygen species were generated in phagocyte-free systems containing S. aureus, products of the myeloperoxidase-hydrogen peroxide-halide pathway produced denaturation of S. aureus DNA, whereas superoxide did not. Thus, differences in reactive oxygen species produced during phagocytosis may be related to the different capacities of neutrophils and alveolar macrophages to denature unopsonized adherent S. aureus DNA.     

A human myeloma immunoglobulin G binding four moles of calcium associated with asymptomatic hypercalcemia

A calcium-binding immunoglobulin G (IgG_I^RUP) was identified in the serum of a patient with multiple myeloma, asymptomatic hypercalcemia, and a normal ionized serum calcium. Calcium binding by IgG^RUP was confirmed by two-dimensional electrophoresis with calcium-45 and equilibrium dialysis. Amino acid analyses indicated an unusually high number of glutamic (or glutamine) residues in the L chain and Fab fragment but no detectable -carboxyglutamic acid. As determined by equilibrium dialysis with⁴⁵Ca, the intact IgG^RUP and its Fab fragments bound calcium at an optimum pH of 7.4. There was minimal binding of calcium to H chains and no binding by L chains or the Fc fragment. Recombination of H and L chains partially restored the binding activity. By Scatchard analysis, the binding affinity (K _d) of IgG^RUP was 1.7×10^–3 M and the binding capacity was 4 mol of calcium/mol of IgG. The binding of 4 mol of calcium/mol of IgG is twice that reported previously for two other calcium-binding myeloma proteins and suggests unique properties of IgG^RUP.     

Inhibition of Dugbe nairovirus replication by human MxA protein

Sensitivity to the interferon-induced protein, MxA, has previously been demonstrated for viruses belonging to the Orthobunyavirus, Hantavirus and Phlebovirus genera of the Bunyaviridae family. We have extended these findings to a member of the fourth and remaining genus containing viruses that infect man and other animals, the nairovirus Dugbe virus (DUGV). Indirect immunofluorescence experiments using VA9 cells (Vero cells permanently transfected with MxA cDNA) revealed strongly reduced DUGV antigen expression, suggesting that MxA inhibited DUGV replication. Western and Northern blot analyses showed significantly lower DUGV nucleocapsid (N) protein expression and DUGV genomic RNA, respectively, in the presence of MxA. Viral titres were also reduced by more than two orders of magnitude in VA9 cells compared with control VN36 cells. This finding may have application to nairovirus therapeutics.     

Which treatment for whom for ADHD? Moderators of treatment response in the MTA

Using receiver operating characteristics, the authors examined outcome predictors (variables associated with outcome regardless of treatment) and moderators (variables identifying subgroups with differential treatment effectiveness) in the Multimodal Treatment Study of Children with Attention-Deficit/Hyperactivity Disorder (ADHD; MTA). Treatment response was determined using parent- and teacher-reported ADHD and oppositional defiant symptoms, with levels near or within the normal range indicating excellent response. Among 9 baseline child and family characteristics, none predicted but 3 moderated treatment response. In medication management and combined treatments, parental depressive symptoms and severity of child ADHD were associated with decreased rates of excellent response; when these 2 characteristics were present, below-average child IQ was an additional moderator. No predictors or moderators emerged for behavioral and community comparison treatments. The authors discuss conceptual and clinical implications of research on treatment moderators.     

Progression of self-reported symptoms in laboratory animal allergy

BACKGROUND: Laboratory animal allergy is a common illness among workers exposed to laboratory animals and can progress to symptoms of asthma. OBJECTIVES: This study evaluates the continuum of disease from allergy symptoms to asthma symptoms in a dynamic cohort of workers exposed to animals in a pharmaceutical company. METHODS: Data arose from annual questionnaires administered to workers in a surveillance program established to monitor exposure to animals and the development of allergy. The life-table method was used to compare asthma-free survival between workers with and without symptoms of allergy. A Cox proportional hazards model was used to examine the effects of covariates on the development of asthma. RESULTS: A total of 603 workers contributed 2527.4 person-years to the study over the 12.3-year period. The probabilities of experiencing asthma symptoms by the 11th year of follow-up were 0.367 for workers with allergy symptoms and 0.052 for those without allergy symptoms. The hazard ratio for asthma symptoms when comparing workers with and without allergy symptoms was 7.39 (95% CI, 3.29-16.60) after adjustment for sex and family history of allergy. Female subjects developed asthma at a rate 3.4 times that of male subjects. CONCLUSIONS: This study supports the hypothesis that laboratory animal allergy symptoms are a major risk factor for the development of asthma. It also suggests a heightened risk of asthma for women who work with laboratory animals, a finding that has not been previously reported.     

Effect of errors in baseline optical properties on accuracy of transabdominal near-infrared spectroscopy in fetal sheep brain during hypoxic stress

A continuous-wave (cw) near-infrared spectroscopy (NIRS) instrument has been developed to noninvasively quantify fetal cerebral blood oxygen saturation (StO2). A linear Green's function formulism was used to analytically solve the photon diffusion equation and extract the time-varying fetal tissue oxy- and deoxy-hemoglobin concentrations from the NIR measurements. Here we explored the accuracy with which this instrument can be expected to perform over a range of fetal hypoxic states. We investigated the dependence of this accuracy on the accuracy of the reference optical properties chosen based on the literature. The fetal oxygenation of a pregnant ewe model was altered via maternal aortic occlusion. The NIR cw instrument was placed on the maternal abdomen directly above the fetal head, continuously acquiring diffuse optical measurements. Blood was sampled periodically from the fetus to obtain fetal arterial saturation (SaO2) measurements from blood gas analysis. The NIR StO2 values were compared with the fetal SaO2 measurements. Variations in the NIR results due to uncertainty in the reference optical properties were relatively small within the fetal SaO2 range of 30 to 80%. Under hypoxic conditions, however, the variability of the NIR StO2 calculations with changes in the assumed reference properties became more significant.