Chronic Pseudomonas aeruginosa endobronchitis in rhesus monkeys: II. A histopathologic analysis

We have recently established a rhesus monkey model of chronic Pseudomonas aeruginosa (PA) endobronchitis by bronchoscopic instillation of PA-embedded agar beads. All experimental animals developed chronic neutrophilic endobronchitis similar to chronic PA endobronchitis in cystic fibrosis (CF). Histopathologic studies further confirmed similarities to chronic PA endobronchitis in CF, including marked peribronchial inflammation, epithelial damage, presence of degraded cilia and ciliary abnormalities, appearance of PA bacterial clusters, mucosal hyperplasia, goblet cell hypertrophy/hypersecretion, airway obstruction, alveolar abnormalities, bronchiectasis, and fibrosis.     

Chronic Pseudomonas aeruginosa endobronchitis in rhesus monkeys: I. Effects of pentoxifylline on neutrophil influx.

Host defense abnormalities in cystic fibrosis (CF) against Pseudomonas aeruginosa (PA) lead to excessive neutrophil influx into the infected lungs, resulting in pulmonary complications. We have developed a rhesus monkey model of chronic PA endobronchitis by intrabronchial instillation of PA-embedded agar beads, utilizing flexible fiberoptic bronchoscopy. Treatment of infected monkeys with pentoxifylline suppressed neutrophil influx and ameliorated pulmonary damage. The results suggest a method by which neutrophil influx and pulmonary damage in CF patients can be managed or prevented.     

Rapid diagnosis of exacerbations of chronic bronchitis of pneumococcal etiology in children

The diagnostic possibilities of the microscopic examination of bronchial secretion smears, stained by Gram's method, from 56 children with chronic bronchitis at different stages of exacerbation have been studied. The following criteria have been proposed for confirming the etiological role of pneumococci in cases of endobronchitis in children: the number of paired diplococci in the visual field must exceed 10 with polynuclears covering the whole field. In 84% of cases the use of this method makes it possible to determine the pneumococcal etiology of the disease within two hours from the arrival of the pathological material to a bacteriological laboratory; this method can also be used for evaluating the effectiveness of antibacterial therapy at its different stages.     

Endobronchitis by Scedosporium apiospermum in a child with cystic fibrosis

A case of endobronchitis by Scedosporium apiospermum in a child with cystic fibrosis is presented. The bronchial aspirate's cytology showed the presence of a large amount of septated-dichotomized hyphae. The bronchial aspirate's culture showed the presence of Scedosporium apiospermum in a pure culture of three consecutive samples. The scanning electron microscopy study of the mucosal surface revealed scarce mycelia with the presence of abundant conidiae. The transmission electron microscopy of the mucosa revealed inflammatory infiltrates constituted by macrophages, polymorphonuclear leukocytes, a lot of dichotomized mycelia and macrophages with hyphae and conidiae within the phagosomes. The patient was treated with amphotericin B and itraconazole.     

Experience with maxipime in neurosurgical clinic]

Cefepime (Maxipime, Bristol-Myers Squibb), a 4th generation cephalosporin was used in the postoperative treatment of 121 patients of Anesthesiology and Intensive Care Unit of Neurosurgical Clinics. The patients were divided into groups by the risk factor of pyoseptic complications. The results were estimated by the number and nature of the complications such as increasing liquor neutrophilic cytosis, systemic inflammations and others. The findings (increasing liquor neutrophilic cytosis only in 2 patients and endobronchitis in 4 patients) and good tolerance of cefepime (Maxipime) were in favour of its use in a dose of 1 g administered intravenously dropwise during initial narcosis and in 12 hours as an efficient agent for perioperative prophylaxis in neurosurgical patients.     

Reduction of eicosanoid production by essential fatty acid depletion does not attenuate the inflammatory response induced by Pseudomonas aeruginosa pneumonia in rat lung.

Sipid mediators of inflammation have been implicated in the pathogenesis of Pseudomonas aeruginosa (PA) related pulmonary damage in patients with cystic fibrosis. We studied the role of these mediators in a rat model of PA endobronchitis using essential fatty acid deficient (EFAD) animals. Whole blood from EFAD animals produced significantly less leukotriene B4 (LTB4) and hydroxyheptadecatrienoic acid when stimulated ex vivo than did whole blood from control animals (p less than 0.005). Similarly, lung lavage fluid from EFAD animals infected with PA contained less LTB4 and thromboxane B2 (TXB2) than that from control animals. Despite these differences, cellular infiltration of airways in response to PA infection was virtually identical in animals from the regular diet and the EFAD groups. Both EFAD and control animals had a significant increase in white blood cells (WBC) in lung lavage fluid at 1, 3 and 6 days following infection with PA when compared to animals receiving sterile beads. Localized areas of consolidation and nodularity were grossly evident in the lungs of all PA infected animals irrespective of their ability to generate the lipid inflammatory mediators. Microscopic examination of lung sections demonstrated similar changes in all infected animals. We conclude that LTB4 and TXB2 production occurs early in the course of PA pulmonary infection in rats. This early rise in lipid mediators is temporally associated with an influx of WBC into the airways. However, attenuation of eicosanoid production by use of an EFAD diet does not lead to a reduction in the inflammatory response to PA infection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dopamine and the chemoregulation of ventilation in the rat

In pentobarbitalized rats, exogenous dopamine inhibits ventilation by acting on peripheral chemoreceptors. This inhibition is suppressed by haloperidol and by domperidone. Almitrine bismesilate, a potent glomic stimulant, potentiates dopaminergic inhibitory responses by depletion of dopamine endogenous stores. After blockade by dopamine antagonists, as well as after dopamine depletion, ventilatory responses to NaCN i.v. is increased. This potentiation can be explained by the suppression of a negative feed-back control dependent on endogenous dopamine release during the glomic stimulation itself.     

Pyruvate decarboxylation in astrocytes and in neurons in primary cultures in the presence and the absence of ammonia

Oxidative decarboxylation of [1-¹⁴C]pyruvate was studied in primary cultures of neurons and of astrocytes. The rate of this process, which is a measure of carbon flow into the tricarboxylic acid (TCA) cycle and which is inhibited by its end product, acetyl CoA, was determined under conditions which would either elevate or reduce the components of the malate-aspartate shuttle (MAS). Addition of aspartate (1 mM) was found to stimulate pyruvate decarboxylation in astrocytes whereas addition of glutamate (or glutamine) had no effect. Since aspartate is a precursor for extramitochondrial malate, and thus intramitochondrial oxaloacetate, whereas glutamate and glutamine are not, this suggests that an increase in oxaloacetate level stimulates TCA cycle activity. Conversely, a reduction of the glutamate content by 3 mM ammonia, which might reduce exchange between glutamate and aspartate across the mitochondrial membrane, suppressed pyruvate decarboxylation. This effect was abolished by addition of glutamate or glutamine or exposure to methionine sulfoximine (MSO). These findings suggest that impairment of MAS activity by removal of MAS constituents decreases TCA cycle activity whereas replenishment of these compounds restores the activity of the TCA cycle. No corresponding effects were observed in neurons.     

Localization of the gene-richest and the gene-poorest isochores in the interphase nuclei of mammals and birds

At a resolution of 850 bands, human chromosomes comprise two subsets of bands, the GC-richest H3⁺ and the GC-poorest L1⁺ bands, accounting for about 17 and 26%, respectively, of all bands. The former are a subset of the R bands and the latter are a subset of the G bands. These bands showed the highest and the lowest gene densities, respectively, as well as a number of other distinct features. Here we report that human and chicken interphase nuclei are characterized by the following features. (1) The gene-richest/GC-richest chromosomal regions are predominantly distributed in internal locations, whereas the gene-poorest/GC-poorest DNA regions are close to the nuclear envelope. (2) The interphase chromosomes seem to be characterized by a polar arrangement, because the gene-richest/GC-richest bands and the gene-poorest/GC-poorest bands are predominantly located in the distal and proximal regions, respectively, of chromosomes, and because interphase chromosomes are extremely long. While this polar arrangement is evident in the larger chromosomes, it is not displayed by the chicken microchromosomes and by some small human chromosomes, namely by chromosomes that are almost only composed by GC-rich or by GC-poor DNA. (3) The gene-richest chromosomal regions display a much more spread-out conformation compared to the gene-poorest regions in human nuclei. This finding has interesting implications for the formation of GC-rich isochores of warm-blooded vertebrates.     

Mechanism of strengthening of the contractile activity in the duodenum and in the jejunum in psychogenic stress in rabbits

In experiments on unanaesthetized rabbits myoelectric activity (contractile activity index) of proximal (postpyloric) and distal sites of duodenum, and proximal part of jejunum was studied under stress induced by fastening a rabbit to a table in supine position. In both sites of duodenum, the stress impact induced a short-time decrease of contractile activity which was followed by its increase that exceeded the initial level. In the proximal part ofjejunum, the increase of contractile activity took place only during the second part of stress response. The strengthening of the contractile activity of the proximal part of duodenum was preserved after muscarinic or nicotinic cholinoceptor blockage, and after beta-receptor blockage. It was concluded that the contractile response of the proximal part of duodenum did not result from the contribution of central or local neurogenic mechanism, including excitatory cholinergic one, but was humoral in origin. The strengthening of the contractile activity of the distal part of duodenum and proximal part ofjejunum was abolished by muscarinic cholinoceptor and beta-receptor blockage, and resulted from the action of circulating catecholamines on the excitatory beta-adrenoceptor, localized on the cholinergic neurones of the enteric nervous system.     

Relationship of the multiple forms of human alpha-D-galactosidase and alpha-D-fucosidase in the normal and in Fabry's disease

Activities and multiple forms of alpha-D-galactosidase of human kidney and liver in the normal and in Fabry's disease were comparatively studied using alpha-D-galactoside and alpha-D-fucoside as substrates. By isoelectric focusing alpha-D-galactosidase was shown to exist in multiple forms, one of which possesses both alpha-D-galactosidase and alpha-D-fucosidase activity. In Fabry's disease, caused by a deficiency of alpha-D-galactosidase A, we found only one form of alpha-D-galactosidase, which corresponded to form B (alpha-N-acetylgalactosaminidase) and was also able to split alpha-D-fucoside. Thus, in Fabry's disease the alpha-D-fucosidase profile was virtually unchanged, as compared with the normal. The results obtained indicate that the alpha-D-fucosidase activity is due to the action of alpha-D-galactosidase B, encoded for by an autosomal gene of chromosome 22. We suppose these data could be confirmed by revealing the significant reduction of the alpha-D-fucosidase activity in patients with alpha-N-acetylgalactosaminidase deficiency.     

Changes in LH and prolactin levels in diabetic male rats and the role of the opiate system in the control of their secretion

Diabetic male rat has low serum levels of luteinizing hormone (LH) and testosterone (T), which are accompanied by atrophy of the testes and accessory glands. The present study investigated changes in the serum levels of LH, prolactin (PRL) and glucose, following diabetes induction by streptozotocin. In addition, involvement of the opiate system in the control of LH and PRL secretion was evaluated. There was no difference in PRL levels between diabetic and control animals, except at 8 hours after streptozotocin injection. In contrast, the diabetic animals had consistently lower levels of LH, starting on the second day of diabetes. Blockade of the opiate system by naltrexone caused a sharp increase of LH levels in normoglycemic rats, while only a gradual decrease was observed in hyperglycemic animals. PRL secretion was inhibited by naltrexone, both in diabetic and control groups. It is concluded that, unlike normoglycemic rats, inhibition of LH secretion in diabetes is not under the control of the opiate system, probably as a result of T deficiency. In contrast, PRL secretion in diabetic rats, as in the control group, is under the influence of endogenous opiates.     

Interactions in the Metabolism of Glutamate and the Branched-Chain Amino Acids and Ketoacids in the CNS

Glutamatergic neurotransmission entails a tonic loss of glutamate from nerve endings into the synapse. Replacement of neuronal glutamate is essential in order to avoid depletion of the internal pool. In brain this occurs primarily via the glutamate-glutamine cycle, which invokes astrocytic synthesis of glutamine and hydrolysis of this amino acid via neuronal phosphate-dependent glutaminase. This cycle maintains constancy of internal pools, but it does not provide a mechanism for inevitable losses of glutamate N from brain. Import of glutamine or glutamate from blood does not occur to any appreciable extent. However, the branched-chain amino acids (BCAA) cross the blood–brain barrier swiftly. The brain possesses abundant branched-chain amino acid transaminase activity which replenishes brain glutamate and also generates branched-chain ketoacids. It seems probable that the branched-chain amino acids and ketoacids participate in a “glutamate-BCAA cycle” which involves shuttling of branched-chain amino acids and ketoacids between astrocytes and neurons. This mechanism not only supports the synthesis of glutamate, it also may constitute a mechanism by which high (and potentially toxic) concentrations of glutamate can be avoided by the re-amination of branched-chain ketoacids.     

Nutridynamics--studying the dynamics of food components in products and in the consumer

The concentrations and biological effects of nutrients, antinutrients and bioactive compounds, including microbes and their constituents, are affected by production and processing steps, the food matrix in which they reside, the way they are digested and metabolized in the human body, and whether or not and in what form they subsequently reach their target site. A new scientific concept, denoted here as 'nutridynamics', aims to unravel the dynamics of these processes by using a systematic approach to study how a food component is affected by the food matrix itself and what it does in the body. This holistic concept has potential synergy with the areas of food technology and nutrigenomics, and provides a link between food production and the mechanistic effects of bioactive ingredients.     

Activities and regulation of the enzymes involved in the first and the third steps of the aspartate biosynthetic pathway in Enterococcus faecium

The enzymes aspartokinase and homoserine dehydrogenase catalyze the reaction at key branching points in the aspartate pathway of amino acid biosynthesis. Enterococcus faecium has been found to contain two distinct aspartokinases and a single homoserine dehydrogenase. Aspartokinase isozymes eluted on gel filtration chromatography at molecular weights greater than 250,000 and about 125,000. The molecular weight of homoserine dehydrogenase was determined to be 220,000. One aspartokinase isozyme was slightly inhibited by meso-diaminopimelic acid. Another aspartokinase was repressed and inhibited by lysine. Although the level of diaminopimelate-sensitive (DAP^s) enzyme was not much affected by growth conditions, the activity of lysine-sensitive (Lys^s) aspartokinase disappeared rapidly during the stationary phase and was depressed in rich media. The synthesis of homoserine dehydrogenase was controlled by threonine and methionine. Threonine also inhibited the specific activity of this enzyme. The regulatory properties of aspartokinase isozymes and homoserine dehydrogenase from E. faecium are discussed and compared with those from Bacillus subtilis.     

Estrogens and cell multiplication in the adenohypophysis of the male rat: in vivo and in vitro studies

A single dose (1 microgram) of oestradiol sub-cutaneously injected to an immature male rat promotes a transitory increase of the pituitary mitotic activity, the maximum of which is reached between 32 and 48 hours ; the observed fluctuations are similar to those previously described for the thymidine kinase activity. In these conditions, the concentration of blood prolactin remains unaltered, as were those of LH and FSH. It follows that hyperplasy of the pituitary can be quickly induced by doses of oestrogen that do not affect significantly the hormone release. Using Moxestrol, a synthetic oestrogen not bound by the oestradiol plasma binding protein, we show that in the very young rat, the in vivo responsiveness of the pituitary increases and reaches its maximum by day 17. This results can be tentatively related to the ontogeny of the oestradiol receptors in the pituitary described by others ; all our attempts to induce the thymidine kinase in cultured glands remained unsuccessful.     

Processes of excitation in the dendrites and in the soma of single isolated sensory nerve cells of the lobster and crayfish

The stretch receptor organs of Alexandrowicz in lobster and crayfish possess sensory neurons which have their cell bodies in the periphery. The cell bodies send dendrites into a fine nearby muscle strand and at the opposite pole they give rise to an axon running to the central nervous system. Mechanisms of excitation between dendrites, cell soma, and axon have been studied in completely isolated receptor structures with the cell components under visual observation. Two sensory neuron types were investigated, those which adapt rapidly to stretch, the fast cells, and those which adapt slowly, the slow cells. 1. Potentials recorded from the cell body of the neurons with intracellular leads gave resting potentials of 70 to 80 mv. and action potentials which in fresh preparations exceeded the resting potentials by about 10 to 20 mv. In some experiments chymotrypsin or trypsin was used to make cell impalement easier. They did not appreciably alter resting or action potentials. 2. It has been shown that normally excitation starts in the distal portion of dendrites which are depolarized by stretch deformation. The changed potential within the dendritic terminals can persist for the duration of stretch and is called the generator potential. Secondarily, by electrotonic spread, the generator potential reduces the resting potential of the nearby cell soma. This excitation spread between dendrites and soma is seen best during subthreshold excitation by relatively small stretches of normal cells. It is also seen during the whole range of receptor stretch in neurons in which nerve conduction has been blocked by an anesthetic. The electrotonic changes in the cells are graded, reflecting the magnitude and rate of rise of stretch, and presumably the changing levels of the generator potential. Thus in the present neurons the resting potential and the excitability level of the cell soma can be set and controlled over a wide range by local events within the dendrites. 3. Whenever stretch reduces the resting membrane potential, measured in the relaxed state in the cell body, by 8 to 12 mv. in slow cells and by 17 to 22 mv. in fast cells, conducted impulses are initiated. It is thought that in slow cells conducted impulses are initiated in the dendrites while in fast cells they arise in the cell body or near to it. In fresh preparations the speed of stretch does not appreciably influence the membrane threshold for discharges, while during developing fatigue the firing level is higher when extension is gradual. 4. Some of the specific neuron characteristics are: Fast receptor cells have a relatively high threshold to stretch. During prolonged stretch the depolarization of the cell soma is not well maintained, presumably due to a decline in the generator potential, resulting in cessation of discharges in less than a minute. This appears to be the basis of the relatively rapid adaptation. A residual subthreshold depolarization can persist for many minutes of stretch. Slow cells which resemble the sensory fibers of vertebrate spindles are excited by weak stretch. Their discharge rate remains remarkably constant for long periods. It is concluded that, once threshold excitation is reached, the generator potential within slow cell dendrites is well maintained for the duration of stretch. Possible reasons for differences in discharge properties between fast and slow cells are discussed. 5. If stretch of receptor cells is gradually continued above threshold, the discharge frequency first increases over a considerable range without an appreciable change in the firing level for discharges. Beyond that range the membrane threshold for conducted responses of the cell soma rises, the impulses become smaller, and partial conduction in the soma-axon boundary region occurs. At a critical depolarization level which may be maintained for many minutes, all conduction ceases. These overstretch phenomena are reversible and resemble cathodal block. 6. The following general scheme of excitation is proposed: stretch deformation of dendritic terminals → generator potential → electrotonic spread toward the cell soma (prepotential) → dendrite-soma impulse → axon impulse. 7. Following release of stretch a transient hyperpolarization of slow receptor cells was seen. This off effect is influenced by the speed of relaxation. 8. Membrane potential changes recorded in the cell bodies serve as very sensitive detectors of activity within the receptor muscle bundles, indicating the extent and time course of contractile events.     

Heterochromatin and late replication in the chromosomes of the rat

The distribution of heterochromatin in the chromosomes of the rat was determined by analysing two of its properties: late replication and differential stain with the DNA d-r method. The presence of late and non-late replicating c-heterochromatin in the genome of Rattus norvegicus indicates that this chromatin is an heterogeneous substance exhibiting different properties. Furthermore, the existence of heterochromatin formed by nonrepeated sequences or by sequences with a low degree of repetitiveness is suggested by the presence of late replicating areas which do not react with the DNA d-r method.Supported by grants from the Conicet and Cic.     

Identification of oxytocin and vasopressin in the testis and in adrenal tissue

Oxytocin, vasopressin and neurophysin-like immunoreactivity have been identified and measured by radioimmunoassay in extracts of human and rat testis and human fetal adrenal tissue. The authenticity of these polypeptides has been confirmed by their behaviour on high performance liquid chromatography. The concentrations of the hormone were too great to be explained by known circulating levels of the polypeptides, and their presence in steroid secreting organs suggests a possible role for them in steroidogenesis. The peptides may be taken up and concentrated by the tissues but the co-localisation of neurophysins with the hormones points towards local synthesis.     

Neurosecretion in the external and internal zone of the median eminence of the cat and dog

Summary The immunoglobulin-enzyme bridge technique, in association with rabbit antiporcine neurophysin-II has been applied to the median eminence of the dog and cat in order to study the distribution of neurophysin-like antigens throughout this area of the brain and correlate the findings with the corresponding distribution of neurosecretory material (NSM) as revealed by the crotonaldehyde fuchsin stain.Neurophysin and NSM were both present in the hypothalamo-supraoptico-neurohypophysial system—the pathway taken by oxytocin, vasopressin and neurophysin from the hypothalamus to the posterior pituitary lobe.Whereas the tuberoinfundibular tract of the median eminence was almost devoid of NSM, the presence of neurophysin-like material was clearly evident using immunoperoxidase histochemistry. The significance of a protein in the external median eminence possessing determinants cross-reactive against anti-neurophysin serum is discussed.This work was financed by a grant from the Medical Research Council of New Zealand.