Inability of oxymetholone to elicit an erythropoietin-hypersecretory state in orchidectomised male mice

The erythropoietin-hypersecretory state (EPO-HS) is a condition elicited by several inducers that is easily observed in hypertransfused-polycythaemic rats and mice, in which hypoxia-stimulated erythropoietin (EPO) secretion is higher than in polycythaemic controls. Steroids with different androgenic/anabolic ratios have proved to be potent inducers of EPO-HS. However, both steroid activities do not appear to have similar importance. The purpose of the present study was to assess the ability of oxymetholone, a synthetic derivate of testosterone that shows the highest anabolic/androgenic ratio, to elicit an EPO-HS. Mice were orchidectomised at 30 days of age. One month later, groups of animals were injected with graded doses of oxymetholone for 4 weeks. All orchidectomised mice were hypertransfused 4 days after the end of the injection period. On the next day, they were exposed to air maintained at 506.5 mbar for 6 h to stimulate EPO production. Plasma EPO titre was determined by immuno-analysis and taken as a reflection of the EPO production rate. Kidney, seminal vesicle and levator ani muscle weights were registered as index of renotrophic, androgenic and anabolic effects, respectively. The steroid brought out a significant anabolic, and a poor androgenic, response. Any of the doses of oxymetholone tested increased EPO production in orchidectomised polycythaemic mice and, therefore, an EPO-HS was not induced. This finding, coupled with previously reported data, suggests that only steroids showing a certain degree of androgenic action have the capacity to evoke an EPO-HS.     

Inability of tumour cells to elicit the respiratory burst in cytotoxic, activated macrophages.

Activated macrophages from Corynebacterium parvum-treated mice are cytotoxic to non-antibody-coated tumour cells and have an augmented respiratory burst potential when compared to resident macrophages. We have investigated the possible involvement of the respiratory burst as an effector mechanism in this type of tumour killing. Scavengers of toxic metabolites of oxygen such as catalase, superoxide dismutase, 2,3-dihydroxybenzoate, ethanol, and cytochrome c did not inhibit macrophage cytotoxicity in this system. To investigate whether or not neoplastic cells stimulate the macrophage respiratory burst, we exposed activated macrophages to viable tumour cells and monitored macrophage superoxide anion production, chemiluminescence, and hexose monophosphate shunt activity. None of these indicators of the macrophage respiratory burst was stimulated by the tumour cells towards which the macrophages were cytotoxic. The data suggest that the macrophages burst is not utilized as an effector mechanism in the non-antibody-mediated macrophage tumour cytotoxicity reaction.     

Differential bronchial and pulmonary vascular responses to vagal stimulation in the pig

The pulmonary and bronchial vascular responses and changes in bronchial tone upon vagal stimulation (240 impulses at 2 Hz or 10 Hz) were studied in anaesthetized pigs paralyzed with pancuronium. The acetylcholine-evoked vasodilatation in the tracheobronchial circulation had the same magnitude when using pancuronium or succinylcholine as skeletal muscle relaxants. Atropine-sensitive bradycardia, hypotension and bronchoconstriction were observed upon vagal stimulation. A vasoconstrictor response in the pulmonary vascular bed and clear-cut vasodilatation in the bronchial circulation supplied by the bronchial artery also occurred upon vagal stimulation. The vagallyevoked increase in pulmonary vascular resistance was markedly reduced after atropine while the bronchial vasodilatation was unchanged. This suggests that the vagallyinduced increase in bronchial blood flow was not secondary to changes in the pulmonary circulation. Furthermore, the pulmonary vasoconstrictor response caused by vagal stimulation under control conditions is probably explained by reflex sympathetic activation due to the fall in systemic blood pressure. These data indicate selective vagal non-cholinergic influence of blood flow in the bronchial vascular bed compared to the pulmonary circulation.     

Cardiac vagal and sympathetic nerve responses to baroreceptor stimulation in the dog

The effects of ascending stepwise pressure changes in the isolated carotid sinuses on cardiac vagal and sympathetic nerve activities were studied in anesthetized, open chest dogs. The steady state responses of the cardiac vagal and the sympathetic nerve activity and arterial blood pressure were plotted against the sinus pressure and the relations were approximated by the normal distribution function (response curve). The sinus pressure- vs. reflex gain relations (reflex gain curve) were approximated by the normal density function. The maximum gain and the range of change were found to be greater for the vagal than for the sympathetic and arterial pressure responses. The sinus pressure values derived from response curves and reflex gain curves for vagal and sympathetic nerve responses were close to each other, while these values and those obtained from arterial pressure responses were considerably apart. It was concluded that: (1) The cardiac vagal neurons are more sensitive to the baroreceptor input than the sympathetic neurons; (2) The similar type of baroreceptor afferent inputs reach the cardiac vagal and the sympathetic structures which are controlling the autonomic outflows.     

Characteristics of cardiac period responses to prolonged vagal stimulation in dogs

In 13 dogs anaesthetised with a-chloralose and urethane, and β-blocked with propranolol, the sensitivity of both the left and right cervical vagi was tested with constant-current electrical stimulation at 10 Hz. All 17 active vagi were stimulated for 60 s with supramaximal constant-current pulses at frequencies between 2·5 and 12·5 Hz. In all 17 vagi tested, statistically significant (p<0·0001) linear relationships were found between the vagal stimulus frequency and (i) the steady-state bradycardia, (ii) postvagal tachycardia and (iii) the slope of the response at the onset and termination of stimulation. Postvagal tachycardia was also correlated to the preceding bradycardia and to the slope of the response at the end of stimulation. In all 13 experiments and over 70 responses the steady-state bradycardia was highly correlated (r=0·9382) to the slope at the onset of stimulation. A simple model describing the release, hydrolysis and effect of ACh at the SA node is presented, which predicts the experimental results obtained and gives the average time constant for the hydrolysis of ACh at the cardiac pacemaker as 1·76±0·14 s (p<0·01).     

Influence of adenosine on responses to vagal nerve stimulation in the anesthetized rabbit

The influence of local adenosine infusion into the celiac artery on the gastric contractile responses to centrifugal vagal nerve stimulation was studied in anesthetized rabbits, and was compared with the effects of systemic administration of equivalent amounts of adenosine. Close arterial infusion of adenosine caused a marked reduction of gastric contractions induced by nerve stimulation, whereas corresponding responses to close arterial infusions of acetylcholine were enhanced during adenosine. The comparison with systemic adenosine administration revealed that the influence on gastric neurotransmission was not related to the hypotensive effect of the compound. No effects of adenosine were seen on bronchial activity as measured by insufflation pressure. Variable effects were obtained on cardiac responses to vagal stimulation. Gastric smooth muscle contractions elicited in vitro by transmural nerve stimulation were affected by adenosine in a biphasic manner, initial inhibition followed by potentiation of the apparently cholinergic responses. It is suggested that adenosine may modulate cholinergic neurotransmission in vivo by a dual effect, prejunctional inhibition and postjunctional enhancement.     

Neuronal responses to transient hypoglycaemia in the dorsal vagal complex of the rat brainstem

Several regions of the mammalian brain contain glucosensing neurones. In vivo studies have suggested that those located in the hypothalamus and lower brainstem are involved in glucoprivic feeding and homeostatic control of blood glucose. We have identified and characterized hypoglycaemia-sensitive neurones in the dorsal vagal complex of the brainstem using in situ hybridization, single-cell RT-PCR and whole-cell patch-clamp recordings from rat brainstem slices. Approximately 80% of neurones did not respond to hypoglycaemia (changing artificial cerebrospinal fluid (ACSF) glucose from 10 m m to 0 m m ) within 5 min (non-responsive: NR). Another 10% depolarized within 155 ± 31 s (mean ± s.e.m. ) of glucose removal (glucose-inhibited: GI), and the remaining neurones hyperpolarized within 53 ± 7 s (glucose-excited: GE). The hyperpolarization was reversed by the K_ATP channel blocker tolbutamide. Single-cell RT-PCR revealed that GI and GE, but not NR, cells expressed glucokinase (GLK). In contrast, SUR1, a K_ATP channel subunit, was expressed in GE and some NR cells. In situ hybridization with biotin-labelled riboprobes in the dorsal vagal complex revealed ubiquitous expression of SUR1, and widespread, but sparse, expression of GLK. Identification of astrocytes using a GFAP (glial fibrillary acidic protein) antibody showed that GLK and GFAP were not colocalized. In summary, we have demonstrated that GI and GE neurones exist in the brainstem and that GLK is essential for their function. It seems likely that GE neurones work in a way analogous to pancreatic β-cells in that they require both GLK and K_ATP channels.     

Lipopolysaccharides from different bacterial sources elicit disparate cytokine responses in whole blood assays

The stimulatory effects of different purified lipopolysaccharide (LPS) preparations from E. coli, S. typhosa, P. aeruginosa, and K. pneumoniae on cytokine and chemokine production were measured in whole blood assays by ELISA. Incubation of 0.5 ml whole blood with 10 ng/ml E. coli and S. typhosa resulted in a time-dependent production of TNF-alpha, IL-1beta, IFN-gamma, IL-10 and MCP-1. K. pneumoniae, however, showed preferential effects on IL-1beta, IL-10 and MCP-1 production with less potent effects on TNF-alpha and IFN-gamma. LPS derived from P. aeruginosa showed a similar potency to other LPS preparations on MCP-1 production, yet completely failed to elicit the production of other cytokines. To further investigate potencies of the different LPS preparations, mediator production was determined following stimulation with agonist concentrations of 0.1 ng and 1000 ng per ml over a 24 h time period. Dose-response curves were obtained with LPS derived from E. coli, S. typhosa and K. pneumoniae on all mediators apart from IL-1beta and MCP-1. Most strikingly though, was the ability of LPS derived from P. aeruginosa to selectively elicit a significant dose-response effect on MCP-1 production, despite its very weak stimulatory effects on all other cytokines. These data imply that the bacterial origin of different LPS preparations can exhibit disparate effects on inflammatory mediator production. Furthermore, the potent, selective dose-response effect of P. aeruginosa LPS on MCP-1 production could help to explain the preponderance of a relentless inflammatory cellular infiltrate in diseases such as cystic fibrosis (CF).     

Differential bronchial and pulmonary vascular responses to vagal stimulation in the pig.

The pulmonary and bronchial vascular responses and changes in bronchial tone upon vagal stimulation (240 impulses at 2 Hz or 10 Hz) were studied in anaesthetized pigs paralyzed with pancuronium. The acetylcholine-evoked vasodilatation in the tracheobronchial circulation had the same magnitude when using pancuronium or succinylcholine as skeletal muscle relaxants. Atropine-sensitive bradycardia, hypotension and bronchoconstriction were observed upon vagal stimulation. A vasoconstrictor response in the pulmonary vascular bed and clear-cut vasodilatation in the bronchial circulation supplied by the bronchial artery also occurred upon vagal stimulation. The vagally-evoked increase in pulmonary vascular resistance was markedly reduced after atropine while the bronchial vasodilatation was unchanged. This suggests that the vagally-induced increase in bronchial blood flow was not secondary to changes in the pulmonary circulation. Furthermore, the pulmonary vasoconstrictor response caused by vagal stimulation under control conditions is probably explained by reflex sympathetic activation due to the fall in systemic blood pressure. These data indicate selective vagal non-cholinergic influence of blood flow in the bronchial vascular bed compared to the pulmonary circulation.     

Characterization of an antigen from Leishmania amazonensis amastigotes able to elicit protective responses in a murine model.

Lymphoproliferative responses to an antigen from Leishmania amazonensis amastigotes with an apparent molecular mass of 30 kDa, termed p30, were evaluated with BALB/c mice. The p30 antigen was purified after separation of parasite extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electroelution. Lymphoproliferative responses to p30 were obtained by subcutaneous immunization of animals with L. amazonensis amastigote extracts, and maximal stimulation indices were observed at an antigen concentration of 5 microg/ml. Induction of lymphoproliferation by p30 is stage specific, and no differences in the responses to this antigen between mice susceptible and resistant to L. amazonensis were detected. The predominant T cells characterized in the lymphocyte cultures were CD4+. Lymphokine analysis of the supernatants from these cultures indicated that Th1 is the subset involved in the lymphoproliferative responses to the antigen. BALB/c mice immunized with p30 and challenged with L. amazonensis amastigotes showed a very low level of infection, indicating a protective role for p30 and a correlation between Th1 and protection. Further biochemical characterization studies showed that this antigen presents cysteine proteinase activity.     

Nervous control of pancreatic endocrine secretion in pigs IV. The effect of somatostatin on the insulin and glucagon responses to electrical vagal stimulation and to intraarterial acetylcholine

We studied the effect of a primed i.v. infusion of somatostatin (0.5 μg×min^–1×kg^–1 on the glucose dependent insulin and glucagon responses to electrical stimulation of the vagus nerves or to i.a. acetylcholine in anesthetized pigs. Somatostatin completely abolished the insulin and glucagon responses to ongoing vagal stimulation; after 70 min somatostatin infusion the response to reiterated stimulation was profoundly inhibited. After termination of the somatostatin infusion, a considerable rebound secretion of insulin and glucagon was noted. By contrast, the endocrine response to acetylcholine persisted in spite of the somatostatin administration. Blood glucose increased slightly during somatostatin infusion. The results suggest that somatostatin inhibits the responses to vagal stimulation by interference with the neural transmission to the pancreatic islets rather than by inhibition of the islet cells themselves; acetylcholine may be involved in this neural transmission (acting on nicotinic receptors.     

Influence of microgravity on astronauts' sympathetic and vagal responses to Valsalva's manoeuvre

When astronauts return to Earth and stand, their heart rates may speed inordinately, their blood pressures may fall, and some may experience frank syncope. We studied brief autonomic and haemodynamic transients provoked by graded Valsalva manoeuvres in astronauts on Earth and in space, and tested the hypothesis that exposure to microgravity impairs sympathetic as well as vagal baroreflex responses. We recorded the electrocardiogram, finger photoplethysmographic arterial pressure, respiration and peroneal nerve muscle sympathetic activity in four healthy male astronauts (aged 38–44 years) before, during and after the 16 day Neurolab space shuttle mission. Astronauts performed two 15 s Valsalva manoeuvres at each pressure, 15 and 30 mmHg, in random order. Although no astronaut experienced presyncope after the mission, microgravity provoked major changes. For example, the average systolic pressure reduction during 30 mmHg straining was 27 mmHg pre-flight and 49 mmHg in flight. Increases in muscle sympathetic nerve activity during straining were also much greater in space than on Earth. For example, mean normalized sympathetic activity increased 445 % during 30 mmHg straining on earth and 792 % in space. However, sympathetic baroreflex gain, taken as the integrated sympathetic response divided by the maximum diastolic pressure reduction during straining, was the same in space and on Earth. In contrast, vagal baroreflex gain, particularly during arterial pressure reductions, was diminished in space. This and earlier research suggest that exposure of healthy humans to microgravity augments arterial pressure and sympathetic responses to Valsalva straining and differentially reduces vagal, but not sympathetic baroreflex gain.     

Humoral and cardiovascular responses to feeding in pigs

In young pigs, allowed to feed after various periods of food deprivation, the changes in plasma levels of insulin, glucose, and corticosteroids were measured. Food was presented in bowls or obtained by operant response. In pigs deprived of food for 17 h there was a marked rise in insulin from 15 +/- 1 to 91 +/- 10 microU/ml and glucose from 94 +/- 2 to 112 +/- 4 mg/dl when feeding began, while the corticosteroid levels were reduced from 48 +/- 8 to 29 +/- 9 ng/ml. Evidence for a cephalic phase of insulin secretion was seen in 17-h fasted pigs. In pigs fed ad libitum there were no significant changes in the levels of insulin, glucose, or corticosteroids. The changes observed in the 5-h fasted pigs were intermediate. No differences were seen between bowl-fed and operant pigs. Pressure transducers were surgically implanted in the carotid arteries of three pigs. When 17-h fasted pigs started to eat there was a marked rise of blood pressure from 114 +/- 3 to 147 +/- 5 mmHg and a similar increase was seen when satiated pigs drank milk.     

How dendritic cells and microbes interact to elicit or subvert protective immune responses

B and T lymphocytes recognize antigens with high specificity, but neither initiate immune responses, nor decide their types. These functions rest upon dendritic cells (DCs), which can determine and maintain Th1/Th2 polarization. Immune responses are thus dependent on the DC subset, the receptors that recognize each pathogen and the microenvironment. Microbes employ an array of mechanisms to evade and disrupt DC functions; some even hijack DCs for transport around the body. Our progress in the understanding of DC physiology will hopefully help us create the necessary vaccines to counteract the infectious agents that still plague mankind.