Demonstration of erythroblastic differentiation after 14 days in blood in vitro during Vaquez's disease without addition of erythropoietin]

Leucocytes of normal individuals and patients with polycythemia vera were isolated from the peripheral blood by Ficoll-Hipaque density gradient centrifugation and cultured in vitro suing the bovine plasma clot culture technique with a minor modification: the addition of fresh normal serum. After 14 days in the presence of sheep erythropo?etin (3U/ml) erythropo?etic bursts containing between 3 and 10 subcolonies were observed in normal and polycythemia vera cultures. Blood leucocytes of patients with polycythemia vera rise to these erythropo?etic bursts without addition of erythropo?etin to the culture. This behavior was never observed in the blood of normal individuals. These results indicate that in polycythemia vera commited erythro?d stem cells of high proliferative capacity closely resembling the murine erythro?d burst forming unit have an abnormal sensitivity to erythropo?etin as well as the immediate precursors of the proerythroblasts. The culture of these cells from the peripheral blood offers some practical advantages.     

Differentiation of human megakaryocytes in culture starting from the primordial circulating cells in the newborn]

Human neonatal blood mononuclear cells were seeded in plasma clot containing high dose of a crude erythropietin. Pure megakaryocyte colonies were observed rarely and most of the colonies were mixed, megakaryocytes being located between subcolonies of erythrocytic bursts. The megakaryocytic nature of large cells could be clearly confirmed by the presence of platelet peroxidase, demarcation membranes, and alpha granules detected by electron microscopy; in addition mature micromegakaryocytes were recognized, shedding platelets.     

Granulocytosis induced in vivo by a mouse marrow stromal cell line,BMA1, which produces colony stimulating factor

Summary Nude mice were inoculated with BMA1 cells. These are cells which produce granulocyte-macrophage colony stimulating factor (GM-CSF); They are derived from mouse bone marrow stromal cells transfected with adenovirus 5 DNA. Progressive neutrophilia developed as the tumor grew, but disappeared quickly after local tumor excision. Media conditioned with tumor cells had GM-CSF but neither erythropoietin, nor burst-promoting activity. In all the tumors which developed, focal areas of bone formation were found among fibrosarcomatous tissues.     

Granulocytosis induced in vivo by a mouse marrow stromal cell line, BMA1, which produces colony stimulating factor

Nude mice were inoculated with BMA1 cells. These are cells which produce granulocyte-macrophage colony stimulating factor (GM-CSF); They are derived from mouse bone marrow stromal cells transfected with adenovirus 5 DNA. Progressive neutrophilia developed as the tumor grew, but disappeared quickly after local tumor excision. Media conditioned with tumor cells had GM-CSF but neither erythropoietin nor burst-promoting activity. In all the tumors which developed, focal areas of bone formation were found among fibrosarcomatous tissues.     

Unwarranted assumptions: Claude Bernard and the growth of the vera causa standard

The physiologist Claude Bernard was an important nineteenth-century methodologist of the life sciences. Here I place his thought in the context of the history of the vera causa standard, arguably the dominant epistemology of science in the eighteenth and early nineteenth centuries. Its proponents held that in order for a cause to be legitimately invoked in a scientific explanation, the cause must be shown by direct evidence to exist and to be competent to produce the effects ascribed to it. Historians of scientific method have argued that in the course of the nineteenth century the vera causa standard was superseded by a more powerful consequentialist epistemology, which also admitted indirect evidence for the existence and competence of causes. The prime example of this is the luminiferous ether, which was widely accepted, in the absence of direct evidence, because it entailed verified observational consequences and, in particular, successful novel predictions. According to the received view, the vera causa standard's demand for direct evidence of existence and competence came to be seen as an impracticable and needless restriction on the scope of legitimate inquiry into the fine structure of nature. The Mill-Whewell debate has been taken to exemplify this shift in scientific epistemology, with Whewell's consequentialism prevailing over Mill's defense of the older standard. However, Bernard's reflections on biological practice challenge the received view. His methodology marked a significant extension of the vera causa standard that made it both powerful and practicable. In particular, Bernard emphasized the importance of detection procedures in establishing the existence of unobservable entities. Moreover, his sophisticated notion of controlled experimentation permitted inferences about competence even in complex biological systems. In the life sciences, the vera causa standard began to flourish precisely around the time of its alleged abandonment.     

Effect of aminoglutethimide on murine fetal hepatic erythroid colony formation

Pretreatment of pregnant mice with aminoglutethimide phosphate, an inhibitor of glucocorticoid synthesis, increases the content of fetal liver erythroid colony-forming cells (CFU-E), as assessed by the formation of erythroid colonies in vitro by fetal liver cells in plasma clots containing exogenous erythropoietin. In addition, the inability of aminoglutethimide to influence erythroid colony formation in vitro suggests that endogenous glucocorticoids exert a suppressive effect on the number of functional CFU-E in the fetal liver.     

Effects of midline cerebellar splitting and of lesions in cerebral cortex on cerebellum cholinesterase activity,in the albino rat

Riassunto La divisione sagittale mediana del cervelletto è seguita, nel ratto, da una eguale diminuzione della attività colinesterasica vera nelle due metà del cervelletto, che raggiunge, nei casi di divisione completa, valori che si avvicinano a quelli osservati nella metà controlaterale per sezione totale, unilaterale, dei peduncoli.Lesioni della corteccia cerebrale, anche se estese, non modificano l'attività colinesterasica vera del cervelletto.In nessun caso vennero osservate modificazioni dell'attività pseudocolinesterasica.     

Erythropoietin formation during hypoxia in mice with impaired responsiveness to erythropoietin induced by irradiation or 5-fluorouracil injection

Summary Plasma erythropoietin levels during continuous exposure to hypobaric hypoxia in mice with marrow aplasia induced by whole body X-irradiation or 5-fluorouracil injection were higher than in control mice similarly exposed. These findings give support to the hypothesis that a relationship exists between erythropoietin production rate and erythroid responsiveness to the hormone.Supported by Conicet and Subcyt grants. Request for reprints should be addressed to C. E. B.     

Systemic oxygen transport and erythropoiesis in the mouse

Summary Removal of 15% of blood volume in the mouse increases erythropoiesis by a factor of 2.2 when measured 12 h after bleeding. Exposure of normal mice to 40% reduced barometric pressure for the same period of time increases erythropoiesis only by a factor of 1.6. The response to hypoxia takes place in the presence of a 40% reduction of oxygen consumption and tissue-venous P_{o 2}, changes which are concomitant with a 5-fold increase in plasma erythropoietin activity. The larger response in anemic animals on the other hand occurs without any detectable change in these parameters. These results cast serious doubts about the interpretation of the quantitative homeostatic control of erythropoiesis based solely on the action of erythropoietin.Acknowledgments. This work was supported by a grant from the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina. We thank Isabel Zingariello for excellent technical assistance.     

Effects of chronic lesions of the peduncles on cerebellum cholinesterase activity,in the albino rat

Riassunto Lesioni unilaterali nell'ambito dei peduncoli cerebellari sono seguite, nel ratto, da una caduta dell'attività colinesterasica vera del cervelletto, che raggiunge i valori massimi del 60% nella metà ipsilaterale e del 40% in quella controlaterale nel caso di sezione totale dei tre peduncoli. In nessun caso l'attività della pseudo-colinesterasi è risultata significativamente modificata.     

Erythropoietin but not VEGF has a protective effect on auditory hair cells in the inner ear

It has recently been shown that the oxygen-regulated factors erythropoietin (Epo) and vascular endothelial growth factor (VEGF) confer protection on different cells, including neuronal-derived ones. The receptors for Epo and VEGF are widely expressed in different organs. Since mammalian auditory hair cells can irreversibly be damaged by different agents, we aimed to identify otoprotective compounds. We focused on the role of Epo and VEGF in the inner ear and review the recent studies. Epo and its receptor are expressed in the inner ear. In vitro experiments on auditory hair cells showed a protective effect of Epo in ischemia- and gentamicin-induced hair cell damage. In contrast, an in vivo study using an animal model of noise-induced hearing loss showed a negative effect of Epo. Also VEGF and its receptors are expressed in the inner ear. Changes in the expression of VEGF or its receptors have been found in the cochlea after noise exposure, transcranial vibration and diabetic or aged animals. Until now, there are no studies about a direct effect of VEGF on auditory hair cells in vitro or in vivo. We could exclude a protective effect of VEGF on gentamicin-induced auditory hair cell damage in vitro. Thus, we conclude that Epo but not VEGF has a protective effect on auditory hair cell damage at least in vitro. (Part of a multi-author review.)     

Shear-dependence of endothelial functions

Endothelial cells are subjected to shear forces which influence important cell functions. Shear stress induces cell elongation and formation of stress fibers, increases permeability, pinocytosis and lipoprotein internalization, is involved in the formation of atherosclerotic lesions, increases the production of tissue plasminogen activator, and enhances von Willebrand factor release and hence platelet aggregation. It decreases adherence of erythrocytes and leukocytes, and increases the release of prostacyclin, endothelium derived relaxing factor, histamine and other compounds, but decreases erythropoietin secretion. The mechanism of signal transduction to the endothelial cell is not known exactly; shear-sensitive ion channels seem to be involved. It is concluded that a better understanding of shear-dependent endothelial functions will influence pathophysiologic concepts and therapeutic interventions.     

Oncogenic mechanisms in myeloproliferative disorders

Myeloproliferative disorders (MPDs) are clonal haematopoietic malignancies involving the abnormal proliferation of myeloid lineages. The World Health Organisation (WHO) classification of haematopoietic malignancies distinguishes MPDs from myelodysplastic/ myeloproliferative disorders and systemic mastocytosis. These malignancies frequently involve constitutive tyrosine kinase activity, resulting from either oncogenic fusion protein production or from point mutations. Chronic myelogenous leukaemia is the model used for studies of the consequences of such molecular defects. However, the heterogeneity of the clinical course of MPDs should be seen in a more rationale conceptual framework, including the many molecular events associated with these diseases. This review focuses on the various tyrosine kinase-related molecular mechanisms underlying both MPDs and rare diseases with myeloproliferative features. We pay particular attention to the newly identified JAK2 V617F mutation in polycythaemia vera, essential thrombocythaemia and idiopathic myelofibrosis and deal with disease heterogeneity and putative additional molecular mechanisms. Received 9 June 2006; received after revision 28 July 2006; accepted 11 September 2006     

Behaviour of glycogen and related enzymes in the sertoli cell syndrome

Riassunto Nella «sindrome da sole cellule di Sertoli congenita o idiopatica» (aplasia germinale vera) la 1–4 AP, ricercata con metodi istochimici, risulta assente in corrispondenza dell'epitelio del tubulo seminifero e si presenta in quantità apprezzabile in sede peritubulare (cellule muscolari). Nella «sindrome da sole cellule di Sertoli acquisita», conseguente a trattamento radiante, il comportamento della 1–4 AP è del tutto simile a quello del testicolo normale. Il metodo appare pertanto utile per la diagnosi differenziale fra le due forme, su base istochimica.     

Transfection of primary brain capillary endothelial cells for protein synthesis and secretion of recombinant erythropoietin: a strategy to enable protein delivery to the brain

Treatment of chronic disorders affecting the central nervous system (CNS) is complicated by the inability of drugs to cross the blood–brain barrier (BBB). Non-viral gene therapy applied to brain capillary endothelial cells (BCECs) denotes a novel approach to overcome the restraints in this passage, as turning BCECs into recombinant protein factories by transfection could result in protein secretion further into the brain. The present study aims to investigate the possibility of transfecting primary rat brain endothelial cells (RBECs) for recombinant protein synthesis and secretion of the neuroprotective protein erythropoietin (EPO). We previously showed that 4% of RBECs with BBB properties can be transfected without disrupting the BBB integrity in vitro, but it can be questioned whether this is sufficient to enable protein secretion at therapeutic levels. The present study examined various transfection vectors, with regard to increasing the transfection efficiency without disrupting the BBB integrity. Lipofectamine 3000? was the most potent vector compared to polyethylenimine (PEI) and Turbofect. When co-cultured with astrocytes, the genetically modified RBECs secreted recombinant EPO into the cell culture medium both luminally and abluminally, and despite lower levels of EPO reaching the abluminal chamber, the amount of recombinant EPO was sufficient to evolve a biological effect on astrocytes cultured at the abluminal side in terms of upregulated gene expression of brain-derived neurotropic factor (BDNF). In conclusion, non-viral gene therapy to RBECs leads to protein secretion and signifies a method for therapeutic proteins to target cells inside the CNS otherwise omitted due to the BBB.     

The use of 5-fluorocytosine and ketoconazole in the culture of the erythrocytic stages of Plasmodium falciparum and some tumor cell lines

In vitro culture systems are often contaminated by bacteria and fungi. It is therefore often necessary to supplement culture media with agents such as penicillin/streptomycin, gentamycin or amphotericin B. The latter cannot be used in the in vitro culture of erythrocytic stages of P. falciparum, and thus anti-fungal agents have not been regularly used in this system. We describe the prophylactic use of 5-fluorocytosine (5-FC) and ketoconazole (KTZ) in tissue cultures at concentrations up to 300 and 10 micrograms/ml respectively which have no effect on the growth of P. falciparum (FCR-3 strain). A melanoma cell line (C32) and a line of uterine carcinoma (C41) were also unaffected by similar concentrations of 5-FC and KTZ. When dissolved in complete culture medium (RPMI 1640) with 10% human plasma, the minimum inhibitory concentration of 5-FC for a susceptible strain of Candida remained below 2 micrograms/ml. These experiments suggest that 5-FC (at 50 micrograms/ml) alone or in combination with KTZ (at 1 microgram/ml) is a useful addition to the armamentarium of antimicrobials available to the tissue culture biologist for a variety of cell culture systems.     

Inhibition of invasion and intraerythrocytic development ofPlasmodium falciparum by kinase inhibitors

We have examined the effects of seven protein kinase inhibitors (staurosporine, genistein, methyl 2,5-dihydroxycinnamate, tyrphostins B44 and B46, lavendustin A and R03) on the erythrocytic cycle of the malaria parasite,Plasmodium falciparum. One (staurosporine) strongly inhibits serine/threonine kinases, but the remainder all exhibit a strong preference for tyrosine kinases. We have been able to discriminate between effects on invasion and on intraerythrocytic development. All reagents impeded development of intraerythrocytic parasites, though at widely differing concentrations, from the sub-micromolar to the millimolar. Several inhibitors, including staurosporine, also reduced invasion. The phosphatase inhibitor, okadaic acid, had a strong inhibitory effect both on invasion and development. The regulation of malaria development by phosphorylation or dephosphorylation reactions at several points in the blood-stage cycle is implied.     

Erythropoietin modulates the neural control of hypoxic ventilation

Numerous factors involved in general homeostasis are able to modulate ventilation. Classically, this comprises several kind of molecules, including neurotransmitters and steroids that are necessary for fine tuning ventilation under different conditions such as sleep, exercise, and acclimatization to high altitude. Recently, however, we have found that erythropoietin (Epo), the main regulator of red blood cell production, influences both central (brainstem) and peripheral (carotid bodies) respiratory centers when the organism is exposed to hypoxic conditions. Here, we summarize the effect of Epo on the respiratory control in mammals and highlight the potential implication of Epo in the ventilatory acclimatization to high altitude, as well as in the several respiratory sickness and syndromes occurring at low and high altitude. (Part of a multi-author review.)