Combination therapy for radiation-induced bone marrow aplasia in nonhuman primates using synthokine SC-55494 and recombinant human granulocyte colony-stimulating factor

Combination cytokine therapy continues to be evaluated in an effort to stimulate multilineage hematopoietic reconstitution after bone marrow myelosuppression. This study evaluated the efficacy of combination therapy with the synthetic interleukin-3 receptor agonist, Synthokine- SC55494, and recombinant methionyl human granulocyte colony-stimulating factor (rhG-CSF) on platelet and neutrophil recovery in nonhuman primates exposed to total body 700 cGy 60Co gamma radiation. After irradiation on day (d) 0, cohorts of animals subcutaneously received single-agent protocols of either human serum albumin (HSA; every day [QD], 15 micrograms/kg/d, n = 10), Synthokine (twice daily [BID], 100, micrograms/kg/d, n = 15), rhG-CSF (QD, 10 micrograms/kg/d, n = 5), or a combination of Synthokine and rhG-CSF (BID, 100 and 10 micrograms/kg/d, respectively, n = 5) for 23 days beginning on d1. Complete blood counts were monitored for 60 days postirradiation and the durations of neutropenia (absolute neutrophil count < 500/microL) and thrombocytopenia (platelet count < 20,000/microL) were assessed. Animals were provided clinical support in the form of antibiotics, fresh irradiated whole blood, and fluids. All cytokine protocols significantly (P < .05) reduced the duration thrombocytopenia versus the HSA-treated animals. Only the combination protocol of Synthokine + rhG-CSF and rhG-CSF alone significantly shortened the period neutropenia (P < .05). The combined Synthokine/rhG-CSF protocol significantly improved platelet nadir versus Synthokine alone and HSA controls and neutrophil nadir versus rhG-CSF alone and HSA controls. All cytokine protocols decreased the time to recovery to preirradiation neutrophil and platelet values. The Synthokine/rhG-CSF protocol also reduced the transfusion requirements per treatment group to 0 among 5 animals as compared with 2 among 5 animals for Synthokine alone, 8 among 5 animals for rhG-CSF, and 17 among 10 animals for HSA. These data showed that the combination of Synthokine, SC-55494, and rhG-CSF further decreased the cytopenic periods and nadirs for both platelets and neutrophils relative to Synthokine and rhG-CSF monotherapy and suggest that this combination therapy would be effective against both neutropenia and thrombocytopenia consequent to drug- or radiation- induced myelosuppression.     

p15ink4B and p16ink4 gene inactivation in acute lymphocytic leukemia

Malignant cells from 52 children with acute lymphocytic leukemia (ALL) were investigated for inactivation of the p15ink4B and p16ink4 genes and other genetic alterations on chromosome 9p21. Homozygous deletions of the p15ink4B and/or the p16ink4 genes were detected in 16 cases and a further 9 cases showed evidence of allelic loss either by hemizygous deletion or loss of heterozygosity (LOH) for 9p21 markers. Most cases had loss of both genes, but 5 patients had lost only p16ink4 and 2 cases had homozygous loss of p15ink4B only. Sequence analysis of all exons of p15ink4B and p16ink4 was performed in patients with hemizygous deletions or LOH for 9p21 markers. A frame shift mutation of p16ink4 exon 1 was shown in 1 case, whereas all other clones carried the wild- type sequence of p15ink4B and p16ink4 in the remaining allele. The data suggest that both the p15ink4B and p16ink4 genes can be inactivated in ALL. The existence of a hitherto undefined tumor-suppressor gene on chromosome 9p cannot be ruled out.     

Hemoglobin M equon beta 41 (C7) phenylalanine leads to tyrosine

A severe hemolytic crisis was observed in a 34-yr-old female of English- Irish extraction following a viral illness treated with acetaminophen. Heinz bodies and heat instability were present only during a transient hemolytic event. A challenge dose of acetaminophen caused no detectable hematologic abnormality. Structural studies of the hemoglobin during hemolysis and again after complete recovery localized the abnormality to tryptic peptide beta Tp-5, and automated sequencing of I 125-labeled beta chains indicated a replacement of phenylalanine (C7) beta 41 by tyrosine. Substitution of the next residue, phenylalanine (CD1) beta 42 by serine (Hb Hammersmith), has resulted in chronic severe Heinz body hemolytic anemia. The lack of chronic anemia in the present disorder may reflect the different relationships of beta41 and beta 42 and/or the similarities in volume and hydrophobicity of tyrosine and phenylalanine. It is suggested that substitution of tyrosine for phenylalanine in Hb Mequon may disturb the critical environment around the heme group and render it susceptible to oxidative denaturation in the presence of infections and/or drugs.     

Recombinant human interleukin-3 and granulocyte-macrophage colony- stimulating factor show common biological effects and binding characteristics on human monocytes

Two human hemopoietic growth factors involved in monocytopoiesis, interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were studied for their ability to stimulate blood monocytes and to bind to the monocyte membrane. Both cytokines maintained monocyte/macrophage numbers during long-term culture and increased cell size as compared with controls. Effects on cell numbers were present at low cytokine concentrations (6 to 20 pmol/L), whereas enhanced 3H-thymidine incorporation was observed only at higher concentrations (greater than or equal to 60 pmol/L). Autoradiographic studies showed only 1% to 3% of stimulated monocytes with nuclear grains. These results suggest that the primary mechanism for IL-3 and GM-CSF-induced maintenance of monocyte/macrophage numbers in humans is through an effect on cell survival. Surface receptors for both IL-3 and GM-CSF were studied by using 125I-labeled recombinant human (rh) cytokines and performing Scatchard analyses. Both cytokines showed curvilinear Scatchard plots, and computer analyses favored a two-site binding model. High-affinity binding data for 125I rhIL-3 (Kd 7.7 to 38.2 pmol/L; receptor number/cell 95 to 580) and for 125I rhGM-CSF (Kd 4.7 to 38.9 pmol/L; receptor number/cell 8 to 67) show similar binding affinities for the two cytokines but a lower receptor number/cell for 125I rhGM-CSF. Low-affinity binding characteristics for 125I rhIL-3 (Kd 513 to 939 pmol/L; receptor number/cell 179 to 5,274) and for 125I rhGM- CSF (Kd 576 to 1,120 pmol/L; receptor number/cell 130 to 657) show a similar pattern for the two cytokines. Specificity of 125I rhIL-3 and 125I rhGM-CSF binding to monocytes was established by the ability of the homologous cytokine to inhibit binding and the inability of a range of other cytokines to compete at 100-fold excess molar concentration. It is important, however, that binding of 125I rhIL-3 was partially inhibited by rhGM-CSF and that rhIL-3 partially inhibited binding of 125I rhGM-CSF to the monocyte membrane under conditions shown to prevent receptor internalization. The degree of inhibition varied between 25% and 80% in different experiments, and quantitative inhibition experiments showed that 1,000-fold excess concentrations of competitor failed to inhibit binding of the heterologous ligand completely. These results demonstrate that human IL-3 and GM-CSF have similar effects on growth and survival of human monocytes in vitro and suggest that these and other common biological effects may be mediated either through a common receptor or through distinct receptors associated on the monocyte membrane.     

Growth signaling promotes chronological aging in budding yeast by inducing superoxide anions that inhibit quiescence

Inhibition of growth signaling pathways protects against aging and age-related diseases in parallel with reduced oxidative stress. The relationships between growth signaling, oxidative stress and aging remain unclear. Here we report that in Saccharomyces cerevisiae, alterations in growth signaling pathways impact levels of superoxide anions that promote chronological aging and inhibit growth arrest of stationary phase cells in G0/G1. Factors that decrease intracellular superoxide anions in parallel with enhanced longevity and more efficient G0/G1 arrest include genetic inactivation of growth signaling pathways that inhibit Rim15p, which activates oxidative stress responses, and downregulation of these pathways by caloric restriction. Caloric restriction also reduces superoxide anions independently of Rim15p by elevating levels of H?O?, which activates superoxide dismutases. In contrast, high glucose or mutations that activate growth signaling accelerate chronological aging in parallel with increased superoxide anions and reduced efficiency of stationary phase G0/G1 arrest. High glucose also activates DNA damage responses and preferentially kills stationary phase cells that fail to arrest growth in G0/G1. These findings suggest that growth signaling promotes chronological aging in budding yeast by elevating superoxide anions that inhibit quiescence and induce DNA replication stress. A similar mechanism likely contributes to aging and age-related diseases in complex eukaryotes.     

Silibinin prevents amyloid β peptide-induced memory impairment and oxidative stress in mice

Background and purpose:

Accumulated evidence suggests that oxidative stress is involved in amyloid β (Aβ)-induced cognitive dysfunction. Silibinin (silybin), a flavonoid derived from the herb milk thistle (Silybum marianum), has been shown to have antioxidative properties; however, it remains unclear whether silibinin improves Aβ-induced neurotoxicity. In the present study, we examined the effect of silibinin on the memory impairment and accumulation of oxidative stress induced by Aβ_25–35 in mice.

Experimental approach:

Aggregated Aβ_25–35 (3 nmol) was intracerebroventricularly administered to mice. Treatment with silibinin (2, 20 and 200 mg·kg⁻¹, once a day, p.o.) was started immediately after the injection of Aβ_25–35. Locomotor activity was evaluated 6 days after the Aβ_25–35 treatment, and cognitive function was evaluated in a Y-maze and novel object recognition tests 6–11 days after the Aβ_25–35 treatment. The levels of lipid peroxidation (malondialdehyde) and antioxidant (glutathione) in the hippocampus were measured 7 days after the Aβ_25–35 injection.

Key results:

Silibinin prevented the memory impairment induced by Aβ_25–35 in the Y-maze and novel object recognition tests. Repeated treatment with silibinin attenuated the Aβ_25–35-induced accumulation of malondialdehyde and depletion of glutathione in the hippocampus.

Conclusions and implications:

Silibinin prevents memory impairment and oxidative damage induced by Aβ_25–35 and may be a potential therapeutic agent for Alzheimer''s disease.     

Moderate iron deficiency in infancy: biology and behavior in young rats

Iron deficiency anemia in early childhood is associated with developmental delays and perhaps, irreversible alterations in neurological functioning. The goals were to determine if dietary induced gestational and lactational iron deficiency alters brain monoamine metabolism and behaviors dependent on that neurotransmitter system. Young pregnant rats were provided iron deficient or control diets from early in gestation through to weaning of pups and brain iron concentration, regional monoamine variables and achievement of specific developmental milestones were determined throughout lactation. Despite anemia during lactation, most brain iron concentrations did not fall significantly until P25, and well after significant changes in monoamine levels, transporter levels, and D₂R density changed in terminal fields. The changes in D₂R density were far smaller than previously observed models that utilized severe dietary restriction during lactation or after weaning. Iron deficient pups had normal birth weight, but were delayed in the attainment of a number of milestones (bar holding, vibrissae-evoked forelimb placing). This approach of iron deficiency in utero and during lactation sufficient to cause moderate anemia but not stunt growth demonstrates that monaminergic metabolism changes occur prior to profound declines in brain iron concentration and is associated with developmental delays. Similar developmental delays in iron deficient human infants suggest to us that alterations in iron status during this developmental period likely affects developing brain monaminergic systems in these infants.     

The consequences of uncontrollable stress are sensitive to duration of prior wheel running

The behavioral consequences of uncontrollable stress, or learned helplessness (LH) behaviors, are thought to involve hyperactivity of serotonergic (5-HT) neurons in the dorsal raphe nucleus (DRN). Other brain regions implicated in LH and capable of affecting 5-HT systems, such as the bed nucleus of the stria terminalis (BNST), amygdala, and habenula, could contribute to DRN 5-HT hyperactivity during uncontrollable stress. Six weeks of wheel running prevents LH and attenuates uncontrollable stress-induced c-Fos expression in DRN 5-HT neurons, although the duration of wheel running necessary for these effects is unknown. In the current study, 6 but not 3, weeks of wheel running blocked the shuttle box escape deficit and exaggerated fear produced by uncontrollable tail shock in sedentary rats. Corresponding to the duration-dependent effects of wheel running on LH behaviors, 6 weeks of wheel running was required to attenuate uncontrollable stress-induced 5-HT neural activity, indexed by c-Fos protein expression, in the DRN and c-Fos expression in the lateral ventral region of the BNST. Wheel running, regardless of duration, did not affect c-Fos expression anywhere in the amygdala or habenula. These data indicate that the behavioral effects of uncontrollable stress are sensitive to the duration of prior physical activity and are consistent with the hypothesis that attenuation of DRN 5-HT activity contributes to the prevention of LH by wheel running. The potential role of the BNST in the prevention of LH by wheel running is discussed.     

The embryo versus endometrium controversy revisited as it relates to predicting pregnancy outcome in in-vitro fertilization-embryo transfer cycles

To evaluate embryonic and endometrial factors for their value in predicting pregnancy outcome in in-vitro fertilization (IVF) and embryo transfer, a retrospective data collection and prospective uterine artery colour Doppler imaging study was performed in a university-based IVF-embryo transfer programme setting. A total of 210 patients were included and grouped as follows: (I) IVF with controlled ovarian stimulation (214 cycles); (II) frozen-thaw cycle of autologous embryos (30 cycles); (III) oocyte donation, no cryopreservation (12 cycles); (IV) frozen-thaw cycle with embryos from donated oocytes (10 cycles). Embryo quality was significantly better in pregnant than non-pregnant cycles (group I, P = 0.0104; groups II-IV, P = 0.0418). The endometrial echo was significantly thicker in pregnant versus non-pregnant patients in group I (P = 0.0059), but not in groups II-IV (P = 0.741). Past uterine surgery or abnormalities had no effect on pregnancy outcome. There were no significant differences in mean uterine artery resistance index or peak systolic velocity in pregnant versus non-pregnant patients in groups II-IV. Thus, embryo quality is the most reliable predictor of pregnancy outcome. Endometrial measurements were significantly thicker in subsequently pregnant patients only in group I, where the endometrium reflects the hormonal environment. Doppler parameters were not useful in predicting pregnancy outcome.