Genomics approach to the analysis of bacterial communities dynamics in Hirschsprung's disease-associated enterocolitis: a pilot study.

INTRODUCTION: The most invalidating and life-threatening complication in Hirschsprung's disease patients (HSCR) is Hirschsprung's disease-associated enterocolitis (HAEC). The mechanisms underlying enterocolitis have not been identified. The limited knowledge of the role of intestinal microflora is in part due to the complexity of the intestinal microbiome and to the limitation of cultivation-based technologies, given that less than 25% of the intestinal bacterial species can be cultured. MATERIALS AND METHODS: We used amplified ribosomal DNA restriction analysis (ARDRA) with four different restriction enzymes to study variations of microflora composition of the stools of a selected HSCR patient in different clinical conditions (acute phase vs. remission). RESULTS: We assessed a total of 15 stool specimens belonging to the same 3-year-old male patient suffering from HSCR, which were harvested during 4 HAEC episodes and remission phases. Restriction analysis showed that HAEC episodes seem to cluster together at ARDRA analysis, thus suggesting a sort of predisposing bacterial community for HAEC development and the need for a microflora equilibrium to maintain wellness. CONCLUSIONS: This approach proved to be effective, useful and powerful in assessing microflora dynamics and indicated that the differences in microflora associated with acute HAEC or remission are likely to result from a combination of disease activity and different antibiotic therapies. ARDRA proved to be useful in discriminating disease versus remission. Our findings indicated that HAEC results from a change in the equilibrium between bacterial species or from altered discrimination of harmless from harmful microorganisms, challenging the definition of pathogenic and non-pathogenic species. Based on these results, we propose ARDRA as a rapid inexpensive tool to assess microflora dynamics during HAEC episodes.

[Action of substance P and one of its synthetic antagonists on the nucleus tractus solitarius]. / Azione della sostanza P e di un antagonista di sintesi sul nucleo del tratto solitario.

The study aims to establish the nature of the chemical mediator which produces the IP (presynaptic inhibition) of the mechanoreceptive afferents reaching the NTS (nucleus tractus solitarius) of the frog. To this end we have examined the effects of the administration of SP (substance P) and of one of its antagonists in the IV ventricle, in both normal and unilaterally axotomized preparations at the level of the glossopharyngeal nerve. In particular we have examined the size of the afferent discharge of the glossopharyngeal-hypoglossus reflex arc and the PAD (primary afferent depolarization) phenomena recorded from the dorsal root of the XII. While in normal preparations the SP reduces the size of the reflex discharge, on the contrary the antagonist increases it; the electrical activity of PAD appears to be enhanced by SP and reduced by the antagonist. Lastly SP normalises the enhanced response produced by axotomy. All the observed effects favour the hypothesis that the IP, which appears in the NTS with the activation of the mechanoreceptive afferents, is brought about by the release of S.P. from their central endings.

Fine structure of the fungiform papilla in a ranid frog (Rana esculenta).

The freetop of the fungiform papilla shows a sensorial area about 100 micron in diameter, surrounded by a ring of ciliated cells. Externally to the ciliated cells, i.e., in the lateral wall, numerous large goblet cells can be seen devoid of their mucous content. The sensorial area is composed by three types of cells: mucous, supporting, and neuroepithelial cells. Mucous cells form the most superficial layer, while the cell bodies of the other two are deep, and from them basal and apical processes arise. The above mentioned cells are connected by desmosomes preferentially located between the mucous and the supporting cells, rather than between the supporting and the neuroepithelial cells. The lateral wall of the papilla is made up of a multilayered epithelium that comprises two types of cells: the first type contains electron-dense granules and an abundant rough endoplasmic reticulum, the others are ciliated cells. In the connective axis of the papilla, numerous fenestrated capillaries with endothelial vesiculated cells and nerve fibers are found.

Electrical behaviour of the rat's tibia during growth and fracture healing.

The electrical behaviour of the exposed tibial surface of young rats has been studied in normal conditions, after fracture and during the repair process. Recordings of potentials were made after all the electrical artefacts (muscle injury potentials) had been neutralized. No significant potential difference was obtained on the exposed bony surface in all the specimens. The results suggest that the currents of the hypothetical "bone-generator" do not reach the tibial surface. The bioelectrical properties of bone cannot therefore be investigated by an external recording method.

Response of the hypothalamo-neurohypophyseal axis (AVP system) and the kidney to salt load in young propylthiouracil-treated rats.

The development of the ability of the kidney to concentrate urine was studied in normal and propylthiouracil-treated rats by measuring urinary sodium concentration and osmolarity at different ages. It was shown that the normal animals, but not the PTU-treated ones were able to concentrate urine at 35 days of age. The response of the hypothalamo-neurohypophyseal axis (AVP system) and that of the kidney were investigated in the two categories of animals at 35 days of age by measuring concomitantly the AVP content of the hypothalamus and neurohypophysis, the AVP plasma concentration and the natremia and plasma osmolarity at various intervals after intraperitoneal injection of a 5% NaCl solution. In normal, as well as in PTU-treated rats, salt load did not lead to significant modifications of the hypothalamic AVP content. In comparison with normal rats, the neurohypophysis of the PTU-treated ones released AVP more slowly, but with a similar amplitude. In normal rats, the plasma AVP concentration was already maximal 30 min after salt load and then decreased and returned to the normal value at 2 hrs; at the same time, the natremia and plasma osmolarity also recovered their normal value. In contrast, the plasma AVP concentration as well as the natremia remained high 1 h 30 after salt load in PTU-treated rats. It is concluded that PTU-treated young rat is unable to compensate the salt load in normal delays. Beside the slowing in AVP release by neurohypophysis and decreased AVP plasma level, the responsiveness of the kidney seems to be altered.

[The ciliary apparatus of the lingual mucosa in the frog]. / Gli apparati ciliari della mucosa linguale di rana.

By means of scanning and transmission microscopy it has been examined the ciliary system of the tongue mucosa. The scanning electronmicrographs of the fungiform papillae have revealed three ciliary apparatuses allocated respectively: at the papillary summit (corona ciliata and a narrow but separated paracoronal ciliary system) and on the peduncolar papillary stem. The cilia of both paracoronal and peduncolar groups have not been yet described. Also the filiform papillae are supplied with cilia but as irregularly distributed groups. The border of the tongue is a continuous and normal ciliary epithelium and finally groups of cilia are scattered also on the whole sublingual epithelium. At the transmission microscopy the cells of all the examined mucosal ciliary groups are showing a normal ultrastructural aspect.

[Capillary networks and osmotic uptake in fungiform papillae]. / Rete capillare e richiamo osmotico delle papille fungiformi.

In the research it has been tested the ability of convoluted capillary network of the fungiform papillae to act as countercurrent ionic exchanger and therefore to give rise a water uptake from the external environment. Manometric determinations of the water inflow during immersion of the tongue in solutions of different osmolality have shown a conspicuous water uptake, osmotic in nature and largely supported by the Na+ absorbed from the external solutions. The Na+ inhibits also the receptor electrical response to the stimulating Ca++ solutions. These observations show that the papillae can be considered as osmotic devices and emphasise also that the Na+ is directly involved in the papillary functional activity.

[Effect of metavanadate on papillary water uptake]. / Influenza del metavanadato sul richiamo idrico papillare.

It has been examined the influence of vanadium on the papillary osmotic water-salts uptake in order to differentiate it from water flow of other biological substrates as amphibia epithelia. Between the different vanadium compounds only the metavanadate is active and only after administration in the abdominal vein. The general influence of metavanadate is a facilitating one and concerns: electrical receptor afferent discharge, osmotic water uptake and ciliary motility. At the papillary level therefore vanadium is not at all an inhibitory agent as observed in many biological substrates. This observation rule out any analogy in the processes of water inflow operating respectively at the fungiform papillae and amphibia epithelia.

[Paracoronal cavity system and papillary water uptake]. / Sistema cavitario paracoronale e richiamo idrico papillare.

In the research it has been carried out a morphological investigation of the paracoronal area of the fungiform papillae. By means of scanning, transmission and light microscopy it has been observed in this area a series of superficial openings around and external to the ciliary crown; and in the epithelium corresponding cavitary system. Each cavity on the other hand appears surrounded by extremely narrow epithelial cells and thus appears able to facilitate the papillary exchange activity. This paracoronal cavitary system is proposed as morphological candidate for the conspicuous water entry in the papillae during osmotic phenomena.

[Quantitative aspects of ciliary apparatus motility of fungiform papillae]. / Aspetti quantitativi della motilità dell'apparato ciliare delle papille fungiformi.

The present research is intended to evaluate the frequency of the ciliary beat in different formations of the frog's oral cavity. The phenomenon has been quantitatively evaluated on the television images of mucus-ciliary waves by means of a photometric method, in order to obtain an electrical indication of the beat frequency. The results show that frequency of the ciliary beat is not uniform in all formations examinated. Differences in the beat frequency were evidenced in the single coronae ciliatae, further the frequencies of the beat decreases sistematically from the base to the tip of the tongue. The cilia on the lingual edge and on the palat exhibit a similar behaviour. The uniformity of the ciliary behaviour in the whole oral cavity is stressed and the factors contributing to a particular ciliary frequency in the fungiform papillae are discussed.

[The electrical activity of olfactory bulb II in the frog]. / Le manifestazioni elettriche del bulbo olfattivo II di rana.

The paper refers to the extracellular field potentials of frog's olfactory secondary bulb elicited by means of electrical stimulation of vomero-nasal nerve. These potentials become evident as compound waveform both in situ and in vitro and their components are easily reliable to the activity of intrinsic neuronal circuitry of the bulb. The similarity of the field potentials in the olfactory primary and secondary bulbs is discussed.

[Facilitating effects of axotomy of the glossopharyngeal nerve on the motor reflex response of the frog medulla oblongata]. / Effetti facilitanti dell'assotomia del glossofaringeo sulla risposta riflessa motoria del midollo allungato di rana.

In the previous papers we have reported that the innervation of the frog lingual mucosa exercises a presynaptic inhibition at the bulbar region in order to integrate the mechanoreceptive information coming from the tongue. The present investigation was undertaken to establish if also the complementary tactile innervation of fungiform papillae has a similar ability. First of all it has been assayed the evocation of PAD tests by papillary innervation; then the facilitating effects, induced by the glossopharyngeal axotomy, on the afferents mass discharges recorded from hypoglossal nerve have been examined. Electrical stimulation of mechanical neurons at the root of IX produced DRP and DRR both at glossopharyngeal and hypoglossal nerves. After 5 and 10 days from glossopharyngeal axotomy becomes evident a considerable enhancement of efferent responses in many diastaltic arches of bulb. These effects strongly suggest a very evident inhibitory activity at the bulbar projection of papillary innervation. From all these observations it follows that presynaptic inhibition is joint to all the lingual mechanoreceptive afferences.

[Inhibitory effect on crossed bands in the tongue mechanoreceptor innervation]. / Influenza inibitoria dei fasci crociati cell'innervazione meccanocettiva linguale.

The inhibitory influence exercised at the bulbar level by crossed afferent innervation of the lingual mechanoreceptors has been examined in the frog. To reach this aim it has been electrically stimulated the central stump of both glossopharyngeal and hypoglossal unilaterally axotomized nerves and recorded the crossed reflex effects in the controlateral hypoglossal motoneurones. By comparison a similar reflex has been evoked by means of stimulation of intact glossopharyngeal and hypoglossal nerves in the same preparation. As regards the glossopharyngeal crossed afferences it has been observed that the stimulation of axotomized nerves intensifies controlateral reflex response, while stimulated intact afference produces an increased reflex only after controlateral hypoglossal section. This last kind of effect has been observed also in the case of a crossed innervation of the hypoglossal lingual afference. These results show a notable extension of lingual crossed mechanoreceptors innervation and indicate its ability to obtain complex contralateral inhibitory effects. This ability however is more evident as regards the glossopharyngeal nerve. These observations are useful in order to explain the properties of the same inhibitory influences exercised homolaterally by the mechanoreceptors afference.

[Direct observation of the inhibitory action of lingual mechanoreceptor afferents in the frog]. / Osservazione diretta dell'azione inibitoria delle afferenze meccanocettive linguali di rana.

In previous researches it has been demonstrated that the frog's tongue mechanoreceptors afferences exercise a presynaptic inhibition on the motor reflex activity evoked by themselves at the bulbar level. Since the indication on this effect were only indirectly obtained this present investigation is now devoted to obtain on the same subject also more direct observations. Therefore it has been examined the behavior of glossopharyngeal-hypoglossal reflex response after conditioning stimulation in different experimental conditions. In intact preparation it has been tested that the crossed inhibition between the mucosal and papillary tongue afferences. This conditioning activation of mucosal afference obtains persistent inhibition of the small reflex response evoked by electrical stimulation of single fungiform papillae. As expected this effect is no evident when the hypoglossal nerve was axotomized. In these preparations however the papillary test response is enhanced and becomes maximal after conditioning stimulation. Also the glossopharyngeal-hypoglossal reflex is depressed after the conditioning stimulation, but the inhibitory influence remains present as much after axotomization of glossopharyngeal nerve. The results show directly the inhibitory influence of frog lingual mechanoreceptors innervation on the bulbar reflex activity and also its correlation with presynaptic properties of the afferences themselves. In fact the facilitating effects observed after axotomy in the reflex response clearly indicate the ability of presynaptic inhibition to control the spread of excitation in the bulbar neuron circuitry. In this context the presynaptic inhibition appears an essential property of lingual mechanoreceptors innervation in the frog.