Evidence for direct action of human biosynthetic (recombinant) GM-CSF on erythroid progenitors in serum-free culture

The biologic activity of human biosynthetic granulocyte-monocyte colony stimulating factor (GM-CSF) was investigated in serum-free culture of erythroid progenitors derived from adult peripheral blood. The morphology of erythroid bursts and the cloning efficiency of BFU-E under serum-free conditions were similar to those observed in dishes with fetal bovine serum (FBS). For these experiments, progenitor cells were partially purified by Ficoll-Paque density centrifugation, adherence to a plastic surface, and complement-mediated cytotoxicity of Leu-1+ elements. For some studies, blastlike cells were harvested directly from 6-day-old semisolid cultures. In serum-free culture of the light-density cell fraction, biosynthetic erythropoietin (Ep) was sufficient for formation of pure and mixed erythroid colonies whereas GM-CSF was required for granulocyte-monocytic colonies. When adherent and Leu-1+ cells were removed, or when in vitro differentiated blast cells were used as a source of progenitors, neither Ep or GM-CSF alone induced colony formation. In dishes supplemented with both growth factors, erythroid bursts were detected. Although the presence of GM- CSF alone did not induce formation of any colony or clusters, BFU-E were recorded when Ep was added 8 days later, suggesting that BFU-E could be maintained. Terminal maturation of the resulting erythroid bursts was delayed by 8 days. These results provide evidence that GM- CSF acts directly on early erythroid progenitors. Furthermore, they suggest that both Ep and GM-CSF are necessary to start the differentiation process.     

Enhancement of chemotactic factor-stimulated neutrophil oxidative metabolism by leukotriene B4

Leukotriene B4 (LTB4) is a potent primary stimulator of neutrophil chemotaxis, aggregation, and degranulation and induces superoxide production at higher concentrations. In order to determine whether LTB4 modulates neutrophil responses to oxidative stimuli, human neutrophils (PMNs) were incubated with LTB4 prior to stimulation with f-Met-Leu-Phe (fMLP, 10(-7) mol/L), opsonized zymosan (OZ, 250 micrograms/mL), or phorbol myristate acetate (PMA, 32 nmol/L). Superoxide (O2-) production by stimulated PMNs was assessed by the superoxide dismutase-inhibitable reduction of cytochrome c. LTB4 alone did not stimulate O2- production in concentrations below 10(-7) mol/L and had no effect on the O2- assay. In the concentration range of 10(-12) to 10(-8) mol/L, LTB4 did not alter O2- release induced by OZ or PMA. In contrast, LTB4-treated cells demonstrated enhanced O2- production following exposure to fMLP, and in the presence of 10 nmol/LLTB4, generated 180% +/- 41% of O-2 quantities produced by control cells (n = 23). Enhancement was LTB4 dose-dependent, was maximal in the range of 1 to 10 nmol/L LTB4, was not reversed by removal of the lipid from the medium prior to fMLP stimulation, and was not dependent on the presence of Ca++ or Mg++ in the suspending medium. Chemiluminescence of fMLP-stimulated neutrophils was increased to 323% of controls in neutrophils preincubated with 10 nmol/L LTB4. Unlike augmentation of oxidative responses to fMLP seen with other degranulating stimuli, enhancement by LTB4 was not correlated with an increase in 3H-fMLP receptor binding. These results indicate that, in addition to its primary effects on neutrophil function, LTB4 modulates PMN oxidative responses to the chemotactic peptide and, thus, may amplify the release of oxygen metabolites at inflammatory foci.     

In vitro interactions of PGE and cAMP with murine and human erythroid precursors

Addition of prostaglandins of the E series (PGE1, PGE2) in methylcellulose cultures of murine marrow results in a dose-dependent inhibition of the cloning efficiency of both BFU-E and CFU-C. However, CFU-E growth is unaffected. The inhibitory action of PGE is progressively overcome by increasing amounts of colony-stimulating factor (CSF), and with some limitations, also of erythropoietin (Ep). Addition of PGF2 alpha' associated or not with indomethacin, does not exert any significant effect on these hemopoietic precursors. In an attempt to unvail the mechanism(s) underlying these phenomena, dibutyryl-cyclic AMP (db-cAMP), theophylline (an inhibitor of phosphodiesterase), or theophylline + PGE were plated at various concentrations. Both db-cAMP and theophylline induce an inhibitory influence on both BFU-E and CFU-C growth, which mimicks that by PGEs; additionally, theophylline potentiates the inhibitory action of PGE1. In all these studies, the CFU-E number was not significantly modified. PGE action on BFU-E proliferation is clearly species-dependent, since PGE1 addition to human marrow methylcellulose cultures induces a significant enhancement of the number of both BFU-E and CFU-E derived colonies. This action was abolished upon removal of adherent cells, thus suggesting that PGE1 evokes a release of factor(s) enhancing human erythroid colony growth by adherent cells.     

Effects of dipyridamole, soluflazine and related molecules on adenosine uptake and metabolism by isolated human red blood cells

Summary— The suggestion that adenosine may have beneficial effects on post reperfusion survival following cardiac ischaemia has led to the search for agents which increase the concentration of this substance in the ischemic region as a possible therapeutic approach to the treatment of angina and myocardial infarction. In the present study, dipyridamole, soluflazine and lidoflazine, known inhibitors of the nucleotide exchange system, have been shown using an HPLC method to prevent the decrease in the concentration od added adenosine outside human red blood cells in vitro. However, the results suggest that this effect was due to inhibition of adenosine deaminase rather than inhibition of nucleotide exchange as had previously been suggested. The selective inhibitor of adenosine deaminase erythro-9-(2 hydroxy-3-nonyl adenosine) exhibited the same profile of activity in the human red blood cell assay. pIC₅₀ values for the four compounds named above were found to be 6.80 ± 0.09, 6.95 ± .03, 6.10 ± 0.14 and 7.39 ± 0.05 vs adenosine disapearance observed in the extracellular incubation medium respectively. Thus, as the disappearance of adenosine outside the cells was not due to its uptake but to its catabolism, this in vitro method does not appear to be predictive for the ability of compounds to act on adenosine uptake into cardiac myocytes. Any antiischemic action of these agents is more readily explained by an inhibition of the catabolism of adenosine and not by the inhibition of its transport across the membrane of cardiac myocytes.     

Cerebral microgyria, thalamic cell size and auditory temporal processing in male and female rats

Induction of microgyria by freezing injury to the developing somatosensory cortex of neonatal rats causes a defect in fast auditory processing in males, but not in females. It was speculated that early damage to the cortex has sexually dimorphic cascading effects on other brain regions mediating auditory processing, which can lead to the observed behavioral deficits. In the current series of experiments, bilateral microgyri were induced by placement of a freezing probe on the skulls of newborn male and female rats, and these animals were tested in adulthood for auditory temporal processing. Control animals received sham surgery. The brains from these animals were embedded in celloidin, cut in the coronal plane and the following morphometric measures assessed: microgyric volume, medial geniculate nucleus (MGN) volume, cell number, and cell size, and, as a control, dorsal lateral geniculate nucleus (dLGN) volume, cell number and cell size. There were no sex differences in the cortical pathology of lesioned animals. However, microgyric males had more small and fewer large neurons in the MGN than their sham-operated counterparts, whereas there was no difference between lesioned and sham-operated females. There was no effect on dLGN cell size distribution in either sex. Microgyric males were significantly impaired in fast auditory temporal processing when compared to control males, whereas lesioned females exhibited no behavioral deficits. These results suggest that early injury to the cerebral cortex may have different effects on specific thalamic nuclei in males and females, with corresponding differences in behavioral effects.      

Platelet glycine, glutamate and aspartate in primary headache

Platelet levels of glutamic and aspartic acid and glycine were measured in patients with migraine with aura, migraine without aura, tension headache and cluster headache. High levels of these amino acids were found in patients with migraine with aura compared to normal subjects and other headache groups. During headache, glutamate levels further increased in migraine with aura patients. These findings may have relevance to the neurological symptoms of migraine with aura.