Nerve growth factor localization in the nasal mucosa of patients with persistent allergic rhinitis.

BACKGROUND AND OBJECTIVES: Nerve growth factor (NGF) and NGF receptors have been shown to be expressed by structural and infiltrating inflammatory cells in the human allergic bronchial mucosa and conjunctiva. In the nose, a positive immunostaining for NGF was recently reported in biopsies of subjects undergoing surgery for refractory nasal obstruction. This study was aimed at studying by immunohistochemistry NGF expression and localization in the nasal mucosa from subjects with moderate/severe persistent allergic rhinitis and natural allergen exposure. METHODS: Immunostaining for NGF, tryptase and eosinophil cationic protein was performed in human nasal turbinate sections of 25 patients affected by persistent allergic rhinitis and sensitization to Dermatophagoides pteronyssinus. RESULTS: NGF was consistently expressed in the epithelium and in the submucosa of allergic rhinitic subjects, preferentially localized in eosinophils and mast cells. A strong NGF immunostaining was found in mucous cells of the epithelial lining and in the submucosal glands. CONCLUSIONS: As previously shown for allergic asthma and allergic conjunctivitis, NGF is also detectable in the nasal mucosa of patients with persistent allergic rhinitis. The preferential NGF localization in mucous cells of the epithelial lining and in the submucosal glands suggests a possible role for NGF in modulating secretion in allergic rhinitis and possibly other allergic diseases.

Angiotensin I converting enzyme in gastric mucosa of the rabbit: localization by autoradiography, immunofluorescence, and immunoelectron microscopy.

To localize angiotensin converting enzyme (ACE) in the fundic mucosa of the rabbit, we used autoradiography with the ACE inhibitor [3H]-trandolaprilate and post-embedding immunocytochemical techniques with a goat anti-rabbit ACE, using fluorescence and electron microscopy. Autoradiographic localization of [3H]-trandolaprilate in rabbit fundus sections shows that ACE is present in the fundic mucosa and mainly in the gland area. Fundic mucosa was fixed with 4% formaldehyde and embedded in Lowicryl K4M. Semi-thin (1 micron) or thin sections (800-900 A) were stained with anti-rabbit ACE followed by fluorescein isothyocyanate-labeled rabbit anti-goat IgG or protein A-gold reagent, respectively. Label was present on endothelium of all blood vessels running through the mucosa. Label was prominently localized in the granules of mucous surface and neck cells and on the granules of chief cells. The intracellular localization of ACE, and particularly its intragranular presence within chief and mucous cells, suggests that the enzyme, at the fundic level, is involved in the intragranular processing of a peptide, the nature of which remains to be determined.

Localization of angiotensin-converting enzyme (ACE) mRNA in rabbit gastric mucosa by in situ hybridization.

In a previous study we showed, by immunohistochemical analysis on rabbit fundic mucosa, that in addition to its usual presence on the luminal plasma membrane of endothelial cells, angiotensin converting-enzyme (ACE) was localized inside granules of surface and neck mucous cells and within granules of chief cells. The aim of the present study was to localize ACE mRNA in cells of the rabbit fundic mucosa by in situ hybridization with a 35S-labeled probe. This probe was a cDNA fragment (406 BP) encoding a portion of the rabbit ACE mRNA obtained by reverse transcription followed by polymerase chain reaction on total RNA extracted from fundic mucosa. ACE mRNAs were detected in mucous and chief cells and in endothelial cells of the mucosal vasculature. These results are in complete agreement with our prior studies which showed by immunohistochemical analysis that ACE is present in these cells. Our findings therefore suggest that ACE previously detected in epithelial cells of the rabbit gastric mucosa is actually synthesized within these cells.

A new experimental method for the dynamic study of the antibody transfer mechanism from mother to fetus in the rat.

The yolk-sac is known to be a route for the transport of passive immunity from mother to fetus in the rat. The main purpose of the present paper is to describe an experimental system for ultrastructurally studying the kinetics of the uptake and transport of immunoglobulin by rat yolk-sac. This system has the advantage of enabling the membrane to be externalized and then exposed to protein under controlled environmental conditions whilst at the same time maintaining the conceptus in connection with the in situ placenta. Preliminary investigations have utilized homologous anti-horseradish peroxidase (HRP) IgG (detected as antibody by application of HRP) or HRP alone. Comparison has been made with the localization of endogenous IgG transmitted in vivo after immunization of the female rat with HRP. The results show the rapid binding of IgG to membrane since only 30 sec were sufficient for this attachment to occur. Moreover, the endocytic process also appears to be very fast as localization of IgG in clusters or patches, caveolae or pits and even rare microvesicles is observed within 8 min. On the other hand, no binding of HRP to microvilli was observed and, unlike IgG, HRP became located in the apico-tubulocanalicular system.

Expression of angiotensin I converting enzyme mRNA in rabbit gastric epithelial cells.

Angiotensin I converting enzyme (ACE) is a dipeptidyl carboxypeptidase synthesized by endothelial cells from many vascular beds as well as by extravascular tissues. Two forms of ACE have been characterized, a pulmonary form and a testicular form. Previously, in the gastrointestinal tract, we localized ACE in the rabbit gastric fundic tissue. In the present study, Northern blot analysis demonstrated the expression of a 5 kb ACE mRNA in fundic mucosa, identical in size to pulmonary ACE mRNA. In order to confirm the epithelial origin of this ACE, we have purified fundic epithelial cells by a flow cytometry technique by which endothelial cells were excluded and the population was enriched in intermediate and chief cells. Using reverse transcription and polymerase chain reaction with specific oligonucleotides, we have amplified from the enriched fundic epithelial cell RNA a 874 bp fragment, the restriction map of which is identical to that of rabbit lung. These findings demonstrate that in gastric mucosa ACE is expressed in fundic epithelial cells and seems to be similar to the pulmonary form.

Evidence for autocrine growth stimulation by a gastrin/CCK-like peptide of the gastric cancer HGT-1 cell line.

Gastrin has been shown to promote the growth of some colonic tumor cell lines. To evaluate the involvement of this hormone in the proliferation of gastric tumors, we studied the effects of gastrin/CCK-receptor antagonists (L365,260 and L364,718), proglumide and C terminal-specific gastrin antibodies on the human gastric adenocarcinoma cell line HGT-1. L365,260, but not L364,718, dose-dependently inhibited cell proliferation (72% after 4 days at 10 nM) and [3H]thymidine incorporation (68% after 2 days at 10 nM) in serum-free medium. No cytotoxic effects of proglumide or L365,260 on this cell line were detected. Proglumide inhibited cell proliferation in serum-free medium (40% and 66.5% after 2 and 4 days of treatment; IC50 = 1.4 mM) and in 5% fetal calf serum (FCS)-supplemented medium (30% and 22% after 2 and 4 days of treatment; IC50 = 3.25 mM). [3H]Thymidine incorporation was also inhibited by proglumide in serum-free medium (IC50 = 2.3 mM) and 5% FCS-supplemented medium (IC50 = 3.35 mM). Gastrin did not induce cell proliferation or increase [3H]thymidine incorporation and no high-affinity gastrin binding sites were observed. However, C terminal-specific gastrin antibodies, even at low concentration, caused a dramatic decrease in both cell number (IC50 = 1:4000 antiserum dilution) and [3H]thymidine incorporation (IC50 = 1:400 antiserum dilution) in the HGT-1 cell line. In addition, immunofluorescence analysis revealed that these antibodies specifically bind HGT-1 cells and radioimmunoassay analysis confirms the presence of gastrin/CCK-like peptide in cell extracts.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of angiotensin I-converting enzyme in the human gastric HGT-1 cell line.

We have previously shown that angiotensin I-converting enzyme (ACE) was expressed by epithelial cells of the rabbit gastric mucosa. In a search to obtain a cell model to study the regulation of ACE expression of gastric origin and its relationship with gastrin-cholecystokinin peptides, which have been proposed as ACE substrates, we investigated whether the HGT-1 human gastric cell line, which expresses gastrin, could also express ACE, using enzymatic and immunodetection methods as well as Northern-blot analysis and polymerase chain reaction. Results show that HGT-1 cells expressed a protein with a molecular weight of 130-140 kDa whose enzymatic and immunological properties were identical to those of ACE. More than 80% of ACE activity was found to be ectoenzymatic. However, immunocytochemical localization has mainly shown an intracellular localization, suggesting that most of intracytoplasmic ACE was not enzymatically active. In addition, Northern-blot analysis and polymerase chain reaction showed that the mRNA encoding that protein displayed a size and a sequence identical to those of somatic ACE. It therefore appears that the HGT-1 cell line could be a useful model to study both the regulation of gastric ACE and its interactions with gastrin-cholecystokinin peptides.

Endothelin expression in human megakaryoblastic leukemia cell lines and normal platelet precursors.

The aim of the present investigation was to determine whether endothelin (ET) could be expressed in and released from the human leukemia megakaryoblastic cell lines HEL, MEG-01, DAMI and the normal human platelet progenitors. Using the reverse transcriptase-polymerase chain reaction (RT-PCR) on total RNA isolated from the cells, we amplified a cDNA of the expected size (453 bp). Southern-blotting hybridization revealed that RT-PCR products from the cell lines were specific of ET-1 mRNA. Immunocytochemical analyses highlighted immunoreactive ET-1 in the cytoplasm of these cells which also released the mature peptide. ET-1 release from the three cell lines was increased by thrombin exposure. Although MEG-01 cells express ET receptors, ET-1, the selective ETB agonist sarafotoxin 6C and the non-selective ET-receptor antagonist PD 142893 showed no proliferative or antiproliferative action in basal or stimulating medium. This indicated a lack of autocrine ET-mediated effect on growth. These results demonstrate for the first time that human megakaryoblastic leukemia cell lines and normal bone marrow platelet precursors express ET-1 mRNA and release the mature peptide.

Identification by RT-PCR and immunolocalization of arginine vasopressin in rat pancreas.

Arginine vasopressin (AVP), a hormone of the hypothalamic pituitary axis, has been described in several peripheral tissues, including pancreas. To demonstrate the ectopic synthesis of AVP at the pancreatic level, we explored the expression of the AVP-neurophysin-II (AVP-NP-II) precursor gene by reverse-transcriptase polymerase chain reaction (RT-PCR) and sequencing and attempted to localise the peptide by immunocytochemistry in normal rat pancreas. Primers designed at the 3' and 5' ends of the AVP-NP-II gene, RT-PCR, and automatic sequencing of PCR products from rat pancreas revealed transcripts of the predicted size with an identical sequence to those from the hypothalamus. In addition, AVP antiserum revealed immunoreactive material of perivascular localisation. These data provide the first direct evidence for the presence of AVP transcripts in rat pancreatic tissue, whereas concurrent immunodetection of this hormone offers further support for the potential role of ectopic AVP in local regulation of the secretory activity of the pancreas.

Local safety of intranasal triamcinolone acetonide: clinical and histological aspects of nasal mucosa in the long-term treatment of perennial allergic rhinitis.

Intranasal corticosteroids are increasingly used to treat allergic rhinitis and their long-term use is generally safe. However, the long-term safety of each molecule should be assessed. The main aim of this multicenter, prospective, randomized, open-label study was to evaluate the effect of triamcinolone acetonide aqueous intranasal spray on nasal mucosal thickness, macroscopic appearance, and mucociliary function. Patients with perennial allergic rhinitis were treated with triamcinolone acetonide 220 micrograms/day for six months. Nasal biopsies taken before and after treatment were compared with biopsies from patients who had been randomized to oral cetirizine 10 mg day or intranasal beclomethasone dipropionate 400 micrograms/day. In the evaluable population (n = 70), there were no significant differences between groups in terms of histologically evaluated thickness and endoscopically evaluated macroscopic appearance of the nasal mucosa, or indigocarmine saccharine test mucociliary function. In the intent-to-treat population (n = 92), there was no difference between treatment groups in the incidence of overall adverse events. This study indicates that sustained treatment with intranasal triamcinolone acetonide does not lead to atrophy of the nasal mucosa or impairment of mucociliary function.

Subjective and behavioral effects associated with repeated exposure to narcosis.

Below 30 m, nitrogen narcosis can severely degrade the performance of air breathing divers. Within the diving community it is generally thought that this effect can be reduced by repeating deep air dives on successive days but laboratory studies have found no strong evidence to support the notion of adaptation to narcosis. One possible explanation for this discrepancy is that one's subjective impression or perception of narcosis may decrease during repeated exposure to hyperbaric air without parallel improvement on task performance. To examine this possibility, symptoms and performance were examined over the course of 5 days of repeated exposure to 30% nitrous oxide at 1 ATA. While the results revealed no clear cut changes in global perceptions of narcosis across days, several symptoms from an adjective checklist showed unequivocal signs of adaptation. With respect to performance effects, reaction time yielded no indications of improvement over days relative to the control. These findings suggest that subjective adaptation can occur without parallel performance improvement, an effect which could compromise safety and which may be of concern in other operational settings that involve repeated exposure to stimulus conditions which impact on performance and symptoms.

Dissociation of the behavioral and subjective components of nitrogen narcosis and diver adaptation.

We investigated adaptation to nitrogen narcosis by compressing 11 highly experienced divers in a hyperbaric chamber to the equivalent of 54.6 meters of seawater once a day for 5 consecutive days. The behavioral component of narcosis was assessed with a serial choice-reaction time (RT) task, and the subjective component with a global magnitude estimate. Supplementary magnitude estimates were obtained with adjectives describing work effectiveness and body sensations. The results showed that there was no adaptation on the RT task, although learning was evident. In contrast, the global estimate dissociated from RT and showed clear adaptation by Day 3. The work effectiveness adjectives followed RT and did not show adaptation. Some body sensation adjectives showed clear adaptation, but others did not. These results lead to the conclusion that the anecdotal reports of adaptation by divers can probably be attributed to the subjective rather than the behavioral component of narcosis. Dissociation of these components suggests mediation by different brain mechanisms, and it is speculated that the gamma-aminobutyric acidA/benzodiazepine receptor complex, which has been implicated in both the anesthetic and anxiolytic properties of agents such as nitrous oxide, may be involved.

Visual/vestibular effects of inert gas narcosis.

Divers breathing compressed air at depths beyond 30 m experience a type of behavioural impairment known as inert gas narcosis. This condition degrades performance on a wide range of tasks and has the potential to compromise safety. Symptoms associated with narcosis include slowed response time, amnesia, and euphoria. Studies have also found disturbances to mechanisms regulating ocular control in response to vestibular stimulation; however, these experiments have been limited to very low frequency head movement (0.2 Hz). Thus, to further examine the effects of narcosis on visual/vestibular mechanisms, the vestibular ocular reflex (VOR) was assessed across a range of higher frequencies more representative of natural head movement (2.0-4.7 Hz). Seven subjects were tested prior to, during and after exposure to narcosis which was induced using 30% nitrous oxide. Standard room air was breathed as a control. The results indicated that narcosis decreased the velocity of compensatory eye movements in response to head rotation (decrease in VOR-gain), with more pronounced decreases occurring at the higher frequencies. The lag between eye and head position (phase lag) was also decreased by nitrous oxide; an effect that was again more pronounced at higher frequencies. These results indicate that narcosis disrupts ocular regulatory mechanisms which help to stabilize images on the retina during head movement.

Dynamics of antibody transfer from mother to fetus through the yolk-sac cells in the rat.

In rodents, maternal immunoglobulins are transported intact by the yolk-sac visceral epithelium from mother to fetus. The main purpose of the present paper is to study the dynamics of the uptake and transport of immunoglobulins by the rat yolk-sac using a new experimental design. The results show the rapid binding of IgG to the cell membrane microvilli since only 30 sec were sufficient for this attachment to occur. The endocytic process also appears to be very fast as localization of IgG in clusters, pits and microvesicles were observed after 5 min of contact between the yolk-sac and the IgG solution. Moreover, the antibodies were detected in the intracellular spaces within 15 min of incubation.

Clinical and pathologic methods to assess the long-term safety of nasal corticosteroids. French Triamcinolone Acetonide Study Group.

BACKGROUND: The main objective of this long-term prospective local safety study was to evaluate endoscopic and histologic changes in nasal epithelium after 6-month treatment with triamcinolone acetonide (TAA). We describe here a method to measure quantitatively epithelium thickness. Results were compared with those seen with the use of cetirizine (an antihistamine) and another oral intranasal corticosteroid, beclomethasone dipropionate (BDP). METHODS: Patients were examined by an ENT specialist who first performed an endoscopic evaluation of the nasal cavities, assessing any morphologic abnormalities and the aspect of the mucosa. Biopsies were taken from the inferior turbinate before and after 24 weeks of treatment. Biopsies were immediately fixed in cold acetone (-20 degrees C) and embedded in glycolmethacrylate; sections of 2 microm were cut on an ultramicrotome. Morphometric evaluations were done in a blinded fashion by computerized image analysis to measure an epithelial area over a minimum length of 50 microm. The thickness was ascertained by the ratio of area to length. RESULTS: 1) For all three treatment groups, the nasal epithelium thickness decreased slightly from pretreatment to the end of treatment. 2) No statistically significant differences between the three treatment groups were found in epithelium thickness. 3) Macroscopically, nasal tissues in all treated groups were normal. CONCLUSIONS: These results clearly indicate that long-term treatment with TAA has no atrophic effect on nasal mucosa.

Immunohistochemistry of angiotensin I-converting enzyme in rat eye structures involved in aqueous humor regulation.

Angiotensin-converting enzyme (ACE) was localized by immunohistochemical methods in rat eye structures involved in aqueous humor secretion and reabsorption, i.e., the ciliary processes, the trabeculum, and Schlemm's canal. The three-layer peroxidase-antiperoxidase method was used with light and electron microscopy. In the ciliary processes, ACE was found on the basal plasma membrane of the internal epithelium facing the aqueous humor and on vesicles in its vicinity, whereas neither the external epithelium nor the blood vessels running through the ciliary processes were immunoreactive. A faint ACE immunoreactivity was found on some endothelial cell plasma membranes of both the internal collector channels and Schlemm's canal. These results suggest that the ciliary process internal epithelium could be an important site for the production of angiotensin II and/or the metabolism of bradykinin. In addition, the present investigation provides evidence for ACE involvement in aqueous humor secretion, whereas its involvement in aqueous humor reabsorption may be minor.

Cellular and subcellular immunohistochemical localization of angiotensin-converting enzyme in the rat adrenal gland.

Angiotensin-converting enzyme (ACE) was localized by immunocytochemical methods in the rat adrenal gland. The three-layer peroxidase-antiperoxidase method was used with light and electron microscopy. At the cortical level, ACE immunoreactivity occurred only on the luminal side of vascular endothelial cell membranes in the adrenal capsule, whereas no ACE activity was found in the zona glomerulosa, fasciculata, or reticularis. ACE immunoreactivity was found mainly but not exclusively on the luminal side of all the types of vessels in the adrenal medulla such as capillaries, venous sinuses, and arteriae medullae. In addition, ACE was localized on the plasma membrane of chromaffin cells but never in Schwann cells nor in nerve fibers. These results provide evidence for a local production of angiotensin II at the vascular level in the adrenal gland. In addition, the presence of ACE on the plasma membrane of chromaffin cells suggests that angiotensin II or other peptides, such as bradykinin, could be metabolized here.

Expression of endothelin and ETB receptors in the megakaryoblastic MEG-01 cell line.

The presence of endothelin (ET) receptors and the nature of the subtype and expression of ET were investigated in the human megakaryoblastic cell line MEG-01. By the RT-PCR procedure, we have shown that both ETA and ETB receptor subtype mRNAs are expressed in the cells. However, binding experiments have shown that the selective ETB receptor antagonist BQ788, but not the selective ETA receptor antagonist BQ123, competes with the specific binding of [125I]ET-1. Using immunocytochemistry, RIA, and RT-PCR Southern blot, we have shown that MEG-01 cells express ET-1. In addition, ET (1-21)-like immunoreactivity was released from the cells into the culture medium, and this release was modulated by thrombin. These data suggest that an ET-1-mediated autocrine loop could occur in the human megakaryoblastic MEG-01 cell line.