Immunohistochemical analysis of adhesive papillae of Clavelina lepadiformis (Müller, 1776) and Clavelina phlegraea (Salfi, 1929) (Tunicata, Ascidiacea).

Almost all ascidian larvae bear three mucus secreting and sensory organs, the adhesive papillae, at the anterior end of the trunk, which play an important role during the settlement phase. The morphology and the cellular composition of these organs varies greatly in the different species. The larvae of the Clavelina genus bear simple bulbous papillae, which are considered to have only a secretory function. We analysed the adhesive papillae of two species belonging to this genus, C. lepadiformis and C. phlegraea, by histological sections and by immunolocalisation of b-tubulin and serotonin, in order to better clarify the cellular composition of these organs. We demonstrated that they contain at least two types of neurons: central neurons, bearing microvilli, and peripheral ciliated neurons. Peripheral neurons of C. lepadiformis contain serotonin. We suggest that these two neurons play different roles during settlement: the central ones may be chemo- or mechanoreceptors that sense the substratum, and the peripheral ones may be involved in the mechanism that triggers metamorphosis.

Implementing strategies for data collection in chronic rhinosinusitis.

SUMMARY: Chronic rhinosinusitis (CRS) is a debated topic in the international rhinologic literature because of its high prevalence, heterogeneity of clinical manifestations and unpredictability of disease course. Recently, the focus in CRS research has moved to identify biological subtypes that might explain its aetiology and clinical variability. However, these analyses are still expensive and limited to scientific purposes, so that they cannot be used on a large scale in daily practice. For this reason, we wondered if it was possible to define a risk stratification for CRS patients based only on first level investigations. The heterogeneity of the disease has given us a large amount of data compelling to find an additional storage system. Herein, we present the results of our work, the RhinoBank, as we believe that it is an easy-to-use tool for those professionals dealing with CRS and an effective system to exploit in clinical research.

Septal flip flap for anterior skull base reconstruction after endoscopic resection of sinonasal cancers: preliminary outcomes.

Over the past decade surgery for sinonasal malignancies encroaching into the anterior skull base (ASB) has evolved from open craniofacial resection to the use of minimally invasive transnasal endoscopic approaches. Using these techniques, ASB reconstruction is most often performed in a multilayer fashion with autologous free grafts (fascia lata or iliotibial tract) which leads to the production of abundant nasal crusting in the postoperative months and discomfort for patients. In carefully selected cases, we propose harvesting a flap from the contralateral nasal septum based on the septal branches of the anterior and posterior ethmoidal arteries (Septal Flip Flap, SFF), which can be rotated to resurface the ASB defect. The exclusion criteria for using the SFF were as follows: cases where the tumour extended to both ethmoid complexes; cases where there was nasal septum or planum spheno-ethmoidalis involvement by the disease; cases of sinonasal malignant tumour with multifocal histology. In our tertiary care referral centre, skull base reconstruction using the SFF was performed in four patients; one was affected by ethmoidal teratocarcinosarcoma, one by persistence of sinonasal undifferentiated carcinoma after radio-chemotherapy, another by olfactory cleft esthesioneuroblastoma and the fourth by ethmoidal squamous cell carcinoma. Successful skull base reconstruction was obtained in all four cases without any intra- or post-operative complications. Post-operatively, nasal crusting was significantly reduced with faster healing of the surgical cavity. No recurrences of disease have been observed after a mean follow-up of 15 months. The SFF can be considered as a safe and effective technique for ASB reconstruction with high success rates similar to those obtained with other pedicled flaps. This flap also ensured a faster healing process with reduction of nasal crusting and improvement in the quality of life of patients in the postoperative period. This technique appears to be a safe and effective option for ASB reconstruction after endonasal resection of sinonasal malignancies in selected cases. Larger case series with a longer follow-up are needed to validate the preliminary results obtained with such an innovative and promising surgical technique.

[Rational treatment of nasal polyposis]. / Trattamento ragionato della poliposi nasale.

Despite the therapeutical advances of the last decade, nasal polyposis represents still a problem for rhinology, practitioners. A number of hypotheses have been formulated about its etiopathogenesis, but no one is confirmed, so that nowadays therapy continues to be only symptomatic and does not cure definitively the underlying pathology. Recurrences are frequent and discourage both the practitioner and the patient. Purpose of this paper is to illustrate Authors' therapeutical rationale aimed to reestablish nasal flow, reduce rhinorrhea, improve olfaction, decrease rhinosinusinusal infection rate and maintain as long as possible such a symptomatic improvement. These targets are best achieved by a combination of medical and surgical treatments in order to optimize the results and reduce the side-effects of both the therapeutical options. Moreover the treatment should be tailored on each patient and follow up should be careful and performed at regular interval. Authors reviewed the clinical records of patients who underwent surgery for nasal polyposis between 2002 and 2004 at Ospedale di Circolo e Fondazione Macchi, University of Insubria, Varese, Italy, with a minimum follow-up of 12 months. All patients underwent the complete set of diagnostic work-up. The choice between surgical or medical options was based on both the kind of the polyposis and the staging of the pathology. The therapy was as more "personalized" as possible, but a homogeneity of treatment was maintained. The results show that a correct "staging" of the patient allows an appropriate therapy and reduces recurrence rate. In conclusion, authors report their experience and propose a scheme of diagnostic work-up in order to define grading/staging of the pathology and establish a "tailored" therapeutic protocol aimed to control a pathology which is rarely definitively treated.

Localization of tubulin mRNA during ascidian development.

In ascidians determination of the nervous system is known to occur at the gastrula stage, when the chorda-endoderm and ectoderm come in contact. In situ hybridization with beta-tubulin cDNA showed that tubulin transcripts were concentrated in presumptive neural cells at the early gastrula stage and continued to increase in these cells throughout neurulation. In the young larvae, the tubulin transcripts were also detectable in the adhesive papillae cells, in mesenchyme cells, muscle cells and button cells. The results suggest that expression of tubulin mRNA could be used as a marker for neural determination at early stages and, at later stages, as a marker for all cell types that elaborate many microtubules.

Behavior of endodermal "button cells" during metamorphosis of ascidian larvae.

Swimming larvae of Phallusia mamillata are known to have "button cells" of endodermal origin between the ventral surface of the pharynx and the epidermis, that are stainable by various techniques. By immunofluorescence with anti-tubulin antibody and confocal laser microscopy, we obtained a bright reaction at one pole of the cells, suggesting the presence of a cap of tubulin and of microtubules overlaying the nucleus. During metamorphosis the microtubule-rich pseudopods at their base reach the epidermis, especially in the area near the adhesive papillae. Then they emigrate through the epidermis and become roundish again.

Better red than dead: carotenoid-based mouth coloration reveals infection in barn swallow nestlings.

Nestling birds solicit food from their parents by displaying their open brightly coloured gapes. Carotenoids affect gape colour, but also play a central role in immunostimulation. Therefore, we hypothesize that, by differentially allocating resources to nestlings with more brightly coloured gapes, parents favour healthy offspring which are able to allocate carotenoids to gape coloration without compromising their immune defence. We demonstrated that, in the barn swallow Hirundo rustica, (i) parents differentially allocate food to nestlings with an experimentally brighter red gape, (ii) nestlings challenged with a novel antigen (sheep red blood cells, SRBCs) have less bright gape colour than their control siblings, (iii) nestlings challenged with SRBCs but also provided with the principal circulating carotenoid (lutein) have more brightly coloured red gapes than their challenged but unsupplemented siblings and (iv) the gape colour of nestlings challenged with SRBCs and provisioned with lutein exceeds that of siblings that were unchallenged. This suggests that parents may favour nestlings with superior health by preferentially feeding offspring with the brightest gapes.

Role of cine-fluoroscopy, transthoracic, and transesophageal echocardiography in patients with suspected prosthetic heart valve thrombosis.

Prosthetic heart valve thrombosis (PVT) is a rare but potentially life-threatening complication of heart valve replacement. An effective, quick, and easy diagnostic method is highly desirable. We evaluated the diagnostic efficacy of cine-fluoroscopy (CF), transthoracic (TTE), and transesophageal (TEE) echocardiography in 82 consecutive patients with mechanical valves and suspected PVT. Criteria for PVT were: leaflet(s) motion restriction at CF, increased Doppler gradients at TTE, and evidence of thrombi at TEE. Patients were divided in 4 groups (A, B, C, and D) according to results of CF and TTE. Group A was composed of 24 patients with positive CF and TTE. Thrombi were detected by TEE in all cases, suggesting that when both are positive, CF and TTE correctly identified PVT in all patients so that TEE may be deferred. Group B was composed of 12 patients with positive CF and negative TTE; TEE showed PVT in 4 patients (33%). These patients had very slight leaflet motion restriction as in the case of initial PVT. This suggests that CF compared with Doppler may identify patients with "hemodynamically significant" PVT. The remaining 8 patients in this group had monocuspid prostheses with negative TEE, suggesting that abnormal leaflet motion at CF may be due to functional changes. Therefore, TEE should always be performed in case of monocuspid prostheses with isolated CF abnormalities. Group C was composed of 18 asymptomatic patients with small-sized aortic prostheses and very high Doppler gradients on routine TTE. CF showed normal leaflet motion and TEE ruled out PVT in all cases outlining the diagnostic role of CF in this particular subset. Finally, group D was composed of 28 patients with negative CF and TTE. TEE did not show thrombi in 24 of 28 patients (86%), confirming that, when both yield negative results, CF and TTE are reliable methods to rule out valve thrombosis in most cases. However, in 4 of 28 patients (14%) TEE showed "nonobstructive" prosthetic thrombosis: these patients had mitral prostheses, chronic atrial fibrillation, and 3 of 4 had systemic embolisms. Thus, TEE should be performed in selected patients despite negative CF and TTE results. Sensitivity, specificity, and positive and negative predictive values were 87%, 78%, 80%, and 91% for CF and 75%, 64%, 57%, and 78% for TTE, respectively. CF and TTE correctly identified PVT in 70 of 82 patients (85%). TEE was actually required in 15% of the cases. Thus, CF and TTE are quick, effective, and complementary diagnostic tools to diagnose PVT in most patients. TEE still remains the gold standard technique in selected cases.

Adhesive Papillae of Phallusia mamillata Larvae: Morphology and Innervation.

The swimming larvae of most solitary ascidians belonging to the Ascidiidae family bear three anterior, simple conic adhesive papillae. They secrete adhesive substances that are used to effect transitory settlement at the beginning of the metamorphosis.The adhesive papillae of newly hatched Phallusia mamillata larvae examined by the SEM are covered by the tunic. When the larvae are about to settle, the tunic becomes fenestrated over the central part of the papilla and bulb-ended microvilli protrude through the holes. These papillae have two types of elongated cells: many peripheral cells and few larger central cells with microvilli and bundles of microtubules oriented along the major axis of the cells.We have done immunofluorescence experiments with an anti-beta-tubulin monoclonal antibody (clone 2-28-33) reacting with axonal microtubules. Only the central cells of the papillae were stained and the axons appeared to arise from the proximal ends of these cells. These axons form a long nerve that reaches the brain vesicle. Branches of the same nerve appear to connect to the basal ends of the peripheral cells. By confocal laser microscopy we were able to follow the course of the papillary nerve. The two nerves connecting the dorsal papillae fuse together into a single nerve that runs posteriorly. The nerve connecting the ventral papilla runs posteriorly for a long tract before fusing with the nerve of the dorsal papillae just near the brain.The reported observations raise the hypothesis that the central cells of the adhesive papillae might be primary sensory neurons and that they may have chemosensory function.

The postnodal piece (PNP) of the chick embryo as a model system for studying organ differentiation.

The postnodal piece of the chick embryo is prepared by cutting the blastoderm 0.6 mm behind Hensen's node at the primitive streak stage. The expiants with their area opaca can be maintained in flat culture (New's method) for 24-30 hr. With this method the polarity and the connections between various sheets are maintained, but the expiants are stretched and difficult to handle for histology and immunostaining. Several PNPs from which the area opaca had been trimmed were cultured on one vitelline membrane (Niu and Deshpande's method) for up to 4 days without stretching effects. The polarity and connections between the embryonic sheets are hard to recognize, but expiants can be easily processed for histology and immunofluorescence. In both culture types the expiants can be easily treated, even with high molecular weight substances. Although the flat culture was useful for the induction of somites and of neural plates, we describe the advantages of culture without the area opaca of neural plates induced by tubulin mRNA or by TPA, which can differentiate into neural tubes. We also demonstrated that TPA is a powerful neural inducer in the chick embryo and stimulates cell proliferation in ectoderm and endoderm.

Immunohistochemical study of the nervous system of the tunicate Thalia democratica (Forsskal, 1775).

Thalia democratica is a cosmopolitan tunicate belonging to the Thaliacea class. To further investigate the anatomy of this species, immunohistochemical labelling was performed using anti-tubulin and anti-serotonin antibodies on specimens collected in the Mediterranean Sea. The anti-tubulin antibody stained the cilia of the endostyle, the pericoronal bands and of the gill bar, enabling a detailed description of these structures. Moreover, immunolabelling of the nervous system showed the presence of eight pairs of nerve fibres emerging from the neural ganglion. Serotonergic cells were observed in the distal tract of the intestine, along the pericoronal bands, and in the placenta of gravid blastozooids, as well as in the neural ganglion. The presence of serotonin in the central nervous system has also been reported in the larvae of ascidians and may be linked to the planktonic life of these animals, a condition shared by adult thaliaceans and ascidian larvae. This work improves our knowledge of the anatomy of T. democratica and demonstrates the presence of a complex serotonergic system.

Immunohistochemical analysis of adhesive papillae of Clavelina lepadiformis (Müller, 1776) and Clavelina phlegraea (Salfi, 1929) (Tunicata, Ascidiacea).

Almost all ascidian larvae bear three mucus secreting and sensory organs, the adhesive papillae, at the anterior end of the trunk, which play an important role during the settlement phase. The morphology and the cellular composition of these organs varies greatly in the different species. The larvae of the Clavelina genus bear simple bulbous papillae, which are considered to have only a secretory function. We analysed the adhesive papillae of two species belonging to this genus, C. lepadiformis and C. phlegraea, by histological sections and by immunolocalisation of ß-tubulin and serotonin, in order to better clarify the cellular composition of these organs. We demonstrated that they contain at least two types of neurons: central neurons, bearing microvilli, and peripheral ciliated neurons. Peripheral neurons of C. lepadiformis contain serotonin. We suggest that these two neurons play different roles during settlement: the central ones may be chemo- or mechanoreceptors that sense the substratum, and the peripheral ones may be involved in the mechanism that triggers metamorphosis.

Regional distribution of polyadenylated mRNA in Xenopus laevis embryos.

Xenopus laevis embryos were dissected into dorsal and ventral regions in post-gastrula stages. Polyadenylated and nonpolyadenylated ribonucleic acids were separated on oligo (dT) cellulose and translated in vitro. The radioactivity incorporated into the translation products directed by polyadenylated and nonpolyadenylated messenger ribonucleic acids shows that in the dorsal region most proteins are synthesized on polyadenylated messenger ribonucleic acid templates in all the stages, while in the ventral region the major templates seem to be, until the neural fold stage, nonpolyadenylated messenger ribonucleic acids. Later the polyadenylated messenger ribonucleic acid activity there too increases.

Polyadenylated mRNAs from various developmental stages of Xenopus laevis. Role of 26 S mRNA.

In Xenopus laevis embryos, the 26 S polyadenylated mRNA, coding for the myosin heavy chain, shows a maximum relative concentration as measured by the 3H-poly(U)-hybridization technique, at the neural-plate stage (st. 12.5 of Nieuwkoop and Faber, [NF]) only in the dorsal (ectodermal and mesodermal) region, i.e. shortly before the morphological appearance of the somites. On the contrary, myosin-heavy-chain mRNA cannot be observed in the ventral region with the same technique. This 26 S mRNA begins to be hybridizable at the gastrula stage (st. 10 NF). Polyadenylated mRNAs from postgastrula stages were translated in a cell-free system of rabbit reticulocytes, and the translation products were analyzed by polyacrylamide-gel electrophoresis and immunoprecipitation. It was found that protein recognizable as the heavy chain of the muscle myosin had been translated. Its possible role on the induction of somites is discussed.

Transition from non-muscle to muscle myosin light chains in chick embryo development.

In chick embryo blastoderm the electrophoretic pattern of myosin light chains changes between the 4-somite and the 19-somite stages (stages 8-13 of Hamburger and Hamilton) from that of non-muscle to muscle myosin. This transition seems to follow the differentiation of the myotomes and to be developmentally regulated.

Action of valproic acid on Xenopus laevis development: teratogenic effects on eyes.

Valproic acid (VPA) is an anticonvulsive drug used in the treatment of epilepsy. Teratogenic effects of VPA have been described in different animal species. In this study, we investigate the effects of VPA on the development of Xenopus laevis embryos, by short pulse treatments (4 h) with relation to the dose and the stage of exposure to the drug. We exposed Xenopus embryos from blastula to stage 32 to three different doses of VPA (0.25, 5, and 10 mM) and we allowed these to develop until the controls reached stage 47. The embryos became more sensitive during the stages of neurulation, as observed in mouse and differently from Amblystoma, in which the more severe effects were produced by treatments at blastula stage. The malformations observed were similar to those described in mammals and other amphibians and consisted in developmental delay, perturbation of neural crest migration, and somite segmentation. We also observed abnormal development of the retina, which had never been described for VPA treatments. Therefore we analyzed the relation between VPA-induced eye malformations and the expression of Pax-6. We examined VPA-treated Xenopus embryos by whole mount in situ hybridization for mis-expression of Pax-6 in correlation with eye anomalies. Our results are consistent with the hypothesis that different members of Pax gene family are candidate target of VPA teratogenic action and in particular the decreased level of Pax-6 expression, shown by Northern blot analysis, is responsible for the retinal malformations we observed in VPA-treated Xenopus embryos. Teratogenesis Carcinog. Mutagen. 21:121-133, 2001.

Regulation of ectodermal differentiation in Xenopus laevis animal caps treated with TPA and ammonium chloride.

Animal caps isolated from Xenopus laevis embryos at the blastula stage were treated sequentially with NH4Cl, a known cement gland inducer, and with 12-O-tetradecanoyl phorbol-13-acetate (TPA), a known neural inducer. The two artificial inducers were also used in reverse order to see if they can mimic the natural inducers acting during the progressive determination of the ectodermal organ. Immunofluorescence and whole-mount in situ hybridization were used to study the expression of tubulin, taken to indicate an early step on the pathway of cell elongation, and neural cell adhesion molecule (N-CAM) taken to indicate an early step in the determination of the nervous system. The expression of XCG-1, a marker of early specification of the cement gland, was also studied. The results showed that the two artificial inducers can mimic the effects of the natural inducers in animal cap explants. The TPA behaves like a neural inducer, reducing the number and the extension of the cement gland when added to the medium in addition to NH4Cl, before or after NH4Cl treatment. In the process of cement gland/neural induction, it is possible to redirect the ectoderm already specified as cement gland to neural tissue, but it does not seem possible to respecify the neural tissue as cement gland. Moreover, the animal caps were also cut into dorsal and ventral parts and the two halves were treated separately. The results were similar to those obtained with treatment of the entire animal cap, suggesting that a dorsal-ventral pattern is not yet established before the gastrula stage, and that in normal embryos there are boundaries between the effects of different inducers.

Serotonin localization in Phallusia mammillata larvae and effects of 5-HT antagonists during larval development.

The neurotransmitter 5-hydroxytryptamine (5-HT, serotonin) plays an important role in a wide range of non-neural processes. Using immunofluorescence with an antiserotonin antibody, 5-HT was localized in the brain and in some neurons of the larval tail of Phallusia mammillata. To test the effect of 5-HT on development, we treated embryos with two different 5-HT receptor subtype antagonists. Treatment at the gastrula stage with 10 microM ondansetron, an antagonist of the 5-HT(3) receptor, induced anterior truncation and a short tail. At 10 microM, ritanserin, a 5-HT(2B) receptor antagonist, induced larval phenotypes characterized by a roundish trunk region with flat papillae. The juveniles developed from these larvae had an abnormal cardiocirculatory system: their heart contractions were ineffective and their blood cells accumulated in the heart cavity. We conclude that an appropriate level of 5-HT is necessary for correct development and morphogenesis. Moreover, a different key role for multiple receptors in modulating the morphogenetic effects of 5-HT is suggested.

[A noninvasive assessment of the gain in baroceptor control of the heart rate in man]. / Valutazione noninvasiva del guadagno del controllo barocettivo della frequenza cardiaca nell'uomo.

In this study we addressed the problem of the noninvasive evaluation of the overall gain of baroreceptor control of heart period. We studied a population of healthy controls (n = 49, age 30 +/- 2 years, systolic arterial pressure [by Finapres] 114 +/- 1 mmHg) and a group of mild hypertensive subjects (n = 14, age 51 +/- 2 years, systolic arterial pressure 151 +/- 5 mmHg). Subjects were studied at rest both in absence and in presence of chronic beta-adrenergic receptor blockade (atenolol: controls 50 mg po oid x 4 days; hypertensives 100 mg po oid x 2 weeks). Spectral analysis of RR interval and of systolic arterial pressure variabilities provided noninvasive markers of autonomic control of the SA node and of the vasculature. The index alpha, obtained from bivariate cross spectral and spectral analysis provided a quantitative assessment of the closed loop gain of baroreceptor control of the heart period. The index alpha resulted more elevated in the normotensive than in the hypertensive group. Additionally it appeared significantly increased at the end of the treatment with the beta-adrenergic blocking drug atenolol. Furthermore, alpha appeared significantly and negatively correlated with age, systolic arterial pressure and, although weakly, with low frequency; it was positively correlated with the average RR interval. In conclusion, this study suggested an important link between the average level of sympathetic activity and baroreceptor control of heart rate in normotensive and mild hypertensive subjects. The clinical importance of this sympathetic modulation of the gain of the heart period/arterial pressure relationship can now be assessed with this noninvasive approach.

Induction of somites by myosin mRNA.

The effects of the messenger for myosin heavy chain (26S mRNA) on postnodal explants of chick embryo blastoderm were studied. Somites do not differentiate in the postnodal explants of chick embryo cultivated by New's method. They are induced when postnodal pieces are cultivated in the presence of a 26S mRNA extracted from chick leg muscles or in the presence of myosin. 26S mRNA plus actinomycin D induces small somites. 26S mRNA of duck, rabbit, or trout induce somite structures often built up of cells separated by large spaces and joined around a large myocele. Crayfish 26S mRNA or chick myosin light chain induce columnar cells connected around a cavity. Liver or kidney mRNA do not induce. The induction process can be summarized as follows: 26S mRNA codes for myosin (heavy chain) and the myosin (heavy chain) induces the somites. Induction of somites by mRNA can occur in the presence of actinomycin D but not when there is mRNA plus puromycin. It does occur when myosin acts in the presence of puromycin. Myosin and its heavy chain are present in chick blastoderm before the appearance of somites. Induced somites are able to induce neural plates. We conclude that in normal development somites are induced by myosin.