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.

Autoimmune alterations in the neurohypophysis of rabbits immunized against vasopressin.

Rabbits immunized against vasopressin developed clinical signs of diabetes insipidus persisting for up to two months after the last boost. Ultrastructural and immunocytochemical studies of the neurohypophysis demonstrated autoimmune alterations: infiltration by immune cells and extracellular deposits of immunoglobulins. No alterations were observed in the hypothalamic nuclei synthesizing vasopressin nor in hormone-target cells of the kidney. Immunization against vasopressin may provide the first example of an autoimmune disease in the hypothalamo-neurohypophysial system.

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.

[Radioimmunoassay for human plasma 8-arginine-vasopressin (author's transl)]. / Dosages plasmatiques radioimmunologiques de la 8-arginine vasopressine. Réponse à la stimulation par la nicotine

The authors have developed a radioimmunoassay for human plasma vasopressin (AVP) which permits the estimation of antidiuretic hormon (ADH) levels as low as 0,8 pg/ml. The average plasma level of AVP after overnight water restriction was found to be 14,3 pg/ml (sd = 4,4 pg/ml) in normal subjects. They provoked a hypersecretion of ADH by the intravenous injection of 1-2 mg of nicotine. In 11 volunteer normal subjects this stimulation by nicotine provoked ADH hypersecretion which reached a maximum between 2nd and 15th minutes after injection. In 3 cases of diabetes insipidus, nicotine injection did not induce ADH hypersecretion; in 1 case of potomania this response was weak; in 2 cases of syndrome of inappropriate ADH secretion, AVP plasma levels were elevated and the response after nicotine stimulation was exaggerated.

[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.

[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.

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.

[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.

[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.

[Integration of oral mechanoreception of amphibia]. / L'integrazione della sensibilita' meccanocettiva orale degli anfibi.

The study concerns the effects obtained at the medulla by electrical stimulation of mechanoreceptive afferences associated to the hyomandibularis nerve and branches of V and VII cranial nerves of the frog. Each of the afferences examined activates both nuclei of XI and XII in spite of the different medullary input; this action is very similar to that carried out by the mechanoreceptor afferences coming from papillae fungiformes. These result show very well the common ability of all the afferences coming from the mouth to obtain a similar action at the medulla. These properties suggest a convergence of the oral mechanoreceptor information on a mechanism of integration in order to obtain a coordination of the tongue and oesophagus motor reflex.

[Extrapapillary mechanoceptive afferent system of the frog tongue]. / Il sistema afferente meccanocettivo extrapapillare della lingua di Rana.

Tactile stimuli applied to the interpapillary spaces of the frog's tongue surface, activated mechanoreceptor afferences. The neurons of these afferences are associated only to the hypoglossal nerve and are able to make a complex central influence. A first effect is a hypo-hypoglossal reflex concerning the muscles of the tongue and of its floor. A second effect is a antidromic discharge of impulses travelling in the dorsal root of hypoglossal nerve and thus representing a DRR. Possibly this effect is related to the PAD phenomena of central terminals of the mechanoreceptors extrapapillary afferences.

[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.

[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.

[Activation of afferent pathways and of control frog olfactory bulb and extracellular field potentials]. / Attivazione delle vie afferenti e di controllo del bulbo olfattivo di rana e potenziali di campo extracellulari.

It has been recorded the extracellular field potentials of the frog's olfactory bulb produced by electrical stimulation of the commissura anterior and small branches of the olfactory nerve. The activation of the commissural system obtain a positive, long-lasting field potential generated at the soma of the granular cells. The olfactory nerve stimulation produce also a positive surface wave bath at the end of bulbar reply: this effect can be attributed to the activation of the bulbar component of commissural system at the NOA. The recognizable electrical effects of the commissural system allow to make clear the sources of the extracellular field potentials in the bulbar neuronal circuits.