N-acetylcysteine inhibits TNF-alpha, sTNFR, and TGF-beta1 release by alveolar macrophages in idiopathic pulmonary fibrosis in vitro.

BACKGROUND: N-acetylcysteine (NAC) can act as an antioxidant. NAC slows the rate of decline of lung function in idiopathic pulmonary fibrosis (IPF) patients concurrently treated with prednisone and azathioprine. OBJECTIVE: In this study we investigated the effect of NAC on the production of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-8, IL-10, IL-12 (p70), IL-18, transforming growth factor (TGF)-beta1, and the soluble TNF receptors (sTNFR1 and sTNFR2) by alveolar macrophages (AM) in IPF patients. DESIGN: AMs were harvested by bronchoalveolar lavage (BAL) from 16 IPF patients and were cultured for 24 h with RPMI medium alone, or with lipopolysaccharide (LPS) (100 ng/ml), in the presence or absence of NAC at various concentrations. RESULTS: NAC suppressed the production of TNF-alpha, its soluble receptors, and TGF-beta1 by AMs in a dose-dependent manner. At the highest concentration of NAC (10 mM), the spontaneous or LPS-stimulated production ofTNF-alpha, sTNFR1, sTNFR2, and TGF-beta1 were significantly reduced. The LPS-stimulated IL-1beta production was also suppressed by 10 mM NAC. CONCLUSIONS: NAC has the potential to down-regulate the production of TNF-alpha and their soluble receptors, as well as TGF-beta1 and LPS-stimulated IL-1beta, by AM in IPF in vitro. NAC may have anti-inflammatory and anti-fibrotic effects.

Stoichiometric shifts in the common bean mitochondrial genome leading to male sterility and spontaneous reversion to fertility

The plant mitochondrial genome is characterized by a complex, multipartite structure. In cytoplasmic male-sterile (CMS) common bean, the sterility-inducing mitochondrial configuration maps as three autonomous DNA molecules, one containing the sterility-associated sequence pvs-or f 239. We constructed a physical map of the mitochondrial genome from the direct progenitors to the CMS cytoplasm and have shown that it maps as a single, circular master configuration. With long-exposure autoradiography of DNA gel blots and polymerase chain reaction analysis, we demonstrate that the three-molecule CMS-associated configuration was present at unusually low copy number within the progenitor genome and that the progenitor form was present substoichiometrically within the genome of the CMS line. Furthermore, upon spontaneous reversion to fertility, the progenitor genomic configuration as well as the molecule containing the pvs-or f 239 sterility-associated sequence were both maintained at substoichiometric levels within the revertant genome. In vitro mitochondrial incubation results demonstrated that the genomic shift of the pvs-or f 239-containing molecule to substoichiometric levels upon spontaneous reversion was a reversible phenomenon. Moreover, we demonstrate that substoichiometric forms, apparently silent with regard to gene expression, are transcriptionally and translationally active once amplified. Thus, copy number suppression may serve as an effective means of regulating gene expression in plant mitochondria.

Immunocytochemical localization of the mGluR1b metabotropic glutamate receptor in the rat hypothalamus.

The mGluR1 metabotropic glutamate receptor is a G-protein-coupled receptor that exists as different C-terminal splice variants. When expressed in mammalian cells, the mGluR1 splice variants exhibit diverse transduction mechanisms and also slightly differ in their apparent agonist affinities. In the present study, we used an affinity-purified antiserum, specifically reactive to the mGluRlb splice variant, in combination with a highly sensitive preembedding immunocytochemical method for light microscopy to investigate the distribution of this receptor in the rat hypothalamus. An intense immunoreactivity for mGluRlb was observed in distinct hypothalamic nuclei. Thus, neuronal cell bodies and dendrites were stained in the preoptic area, suprachiasmatic nucleus, dorsal hypothalamus, lateral hypothalamus, dorsomedial nucleus, tuberomammilary nucleus, and lateral mammilary body. The ventromedial nucleus exhibited neuropil immunostaining but neuronal cell bodies were not labeled. Strong mGluRlb immunoreactivity was observed in magnocellular neurons of the neuroendocrine supraoptic, paraventricular, and arcuate nuclei. Also, neuronal cell bodies were heavily labeled in the retrochiasmatic nucleus, anterior commissural nucleus, and periventricular nucleus. These immunocytochemical observations, together with previous studies, suggest that mGluRlb is coexpressed with other class I mGluRs in some nuclei throughout the hypothalamus. However, mGluRlb is so far the only receptor of this class strongly expressed in the supraoptic, paraventricular, and arcuate nuclei, which might have relevant implications in the physiological control of the neuroendocrine hypothalamic-pituitary system.

Changes in the pattern of glutamate-like immunoreactivity in rat superior colliculus following retinal and visual cortical lesions.

We have investigated the pattern of glutamate-like immunoreactivity in the superficial layers of the rat superior colliculus by means of postembedding immunocytochemical methods for light and electron microscopy. At the light microscopic level, labelling was faintly to moderately intense in most perikarya of the stratum zonale, stratum griseum superficiale and stratum opticum. Furthermore, strong glutamate-immunoreactive terminal-like elements were accumulated most densely in stratum zonale, stratum griseum superficiale and stratum opticum. At the electron microscopic level, a postembedding immunogold method revealed that the vast majority of those labelled elements corresponded to retinal and visual cortical terminals. These profiles were about twice as heavily labelled as their postsynaptic partners. To determine the contribution of retinal and cortical afferents to the pattern of glutamate-like immunoreactivity, rats were subjected to right retinal ablation, left cortical ablation or combined right retinal and left cortical ablations. After retinal ablation, strongly labelled perikarya were observed in the retinorecipient layers. Furthermore, a prominent loss of glutamate-immunoreactive terminal-like elements occurred in stratum zonale and stratum griseum superficiale. Ipsilateral superior colliculus to cortical ablation exhibited subtle changes characterized by a moderate increase in perikaryal immunostaining in stratum zonale, stratum griseum superficiale and stratum opticum and by an apparent discrete reduction of labelled dots in stratum griseum superficiale and stratum opticum. In cases with combined lesions, strongly immunoreactive cell bodies and dendrites were accompanied by a massive disappearance of labelled terminal-like elements in stratum zonale, stratum griseum superficiale and stratum opticum. The effect of retinal and visual cortical ablations on the pattern of glutamate-like immunoreactivity suggests that these afferents are the major sources for glutamate-immunoreactive terminals in the rat superior colliculus. In addition, these findings provide further evidence for glutamate as neurotransmitter in the visual pathways studied.

Glutamate-like immunoreactivity in synaptic terminals of the posterior cingulopontine pathway: a light and electron microscopic study in the rabbit.

A postembedding immunoperoxidase method for light microscopy was used to localize glutamate-like immunoreactivity in the rabbit basilar pontine nuclei. Labelled fibre bundles, neuronal cell bodies and numerous puncta of diverse size were heavily glutamate immunoreactive throughout all subdivisions of the pontine nuclei. To determine whether some of the glutamate-immunoreactive puncta were synaptic terminals of posterior cingulate cortical neurons, a double-labelling technique involving an anterograde tract-tracing method and a postembedding immunogold procedure for electron microscopy was used. A quantitative evaluation of gold particle densities revealed that anterogradely labelled cingulopontine synaptic terminals were about twice as immunoreactive as their postsynaptic dendrites, perikaryal and glial profiles and about three times more than symmetric synaptic terminals. The present results indicate that the posterior cingulopontine projection contains high levels of glutamate at its synaptic terminals. This observation provides further support to the role for glutamate as a neurotransmitter in the corticopontine pathway.

Immunohistochemical analysis of adrenal proliferation and corticosterone expression in experimental adrenal regeneration.

The proliferative activity, the organization and the corticosterone expression of adrenocortical cells in an experimental adrenal regeneration process after the transplantation of neonatal adrenal glands to adult hosts was investigated. Three days after transplantation, the medullar and the innermost adrenocortical cells of the neonatal adrenal glands showed degenerative and necrotic changes due to the lack of vascular supply. The remaining outermost adrenocortical cells did not display any PCNA immunoreaction. The first PCNA expression, pointing out the beginning of the proliferative cycle, was observed in a 45.4% of the adrenocortical cells, one week after transplantation. After three weeks, several regenerated adrenocortical nodules with a bigger size than the one observed in the previous periods were seen. In these nodules, while the outermost adrenocortical cells were disposed in parallel to the capsule or in rounded groups, the bulk of the regenerated mass width was composed of cells forming longitudinal cords. PCNA immunoreaction was almost exclusively restricted to subcapsular cells (62.5%) and to cells of the outermost portion of the cords (32.5%), the global percentage of PCNA immunopositive cells being 18.4%. Twelve weeks after transplantation, regenerated adrenocortical cells were arranged in three layers: glomerulosa, fasciculata and reticularis. Only 1.85% of the adrenocortical cells were PCNA immunopositive. Although in the early stages of the regeneration process, all the adrenocortical cells, both proliferating and non proliferating cells expressed corticosterone, a restriction of this immunoreactivity to the zonae fasciculata and reticularis was observed when cell zonation was apparent.

Light microscopic comparison of the patterns of glutamate-like and homocysteate-like immunoreactivities in rat dorsal lateral geniculate after combined visual cortical and retinal ablations.

To study the contribution of retinal and cortical afferents to the patterns of glutamate- and homocysteate-like immunoreactivities in dorsal lateral geniculate, combined retinal and cortical ablations were performed in rats. In controls, glutamate immunoreactivity was in terminal-like dots and neurons. Homocysteate immunoreactivity was in small puncta. In lesioned animals, most glutamate-immunoreactive dots disappeared. In contrast, abundant puncta resembling parts of glial cells were immunoreactive for homocysteate.

Cell proliferation, nuclear ploidy, and EGFr and HER2/neu tyrosine kinase oncoproteins in infiltrating ductal breast carcinoma.

The purpose of this work was to study if the variations in the nuclear DNA content and the expression of EGFr and HER2/neu transmembrane oncoproteins were related and if this influences the rate of cell proliferation modifying, in each case, the potential aggressivity of the neoplasia. Thirty-four ductal breast carcinoma tissue samples of tumors of up to 2 cm in diameter were analyzed by flow cytometry. HER2/neu and EGFr were measured by immunohistochemical methods. Twenty cases were diploid (DNA index of 1) and 14 cases were aneuploid (DNA index other than 1). The expression of EGFr and HER2/neu was significantly higher in aneuploid tumors compared with diploid tumors. The cell proliferation rate was significantly higher in tumors with an aneuploid pattern. The expression of EGFr was associated with a higher rate of cell proliferation. The higher expression of EGFr and HER 2/neu oncoproteins in aneuploid tumors suggests that the increased proliferative activity of aneuploid carcinomas is influenced by the activity of such oncoproteins, which favors a more aggressive biological behavior.

Distribution of the group II metabotropic glutamate receptors (mGluR2/3) in the enteric nervous system of the rat.

We used affinity purified antisera specific for group II metabotropic glutamate receptors in combination with a pre-embedding immunocytochemical method for light microscopy to investigate the localization of mGluR2/3 receptors in the enteric nervous system of the rat small intestine. In the submucosal plexuses of the jejunum and ileum, strongly mGluR2/3 immunoreactive cells were distributed between the circular muscular layer and the muscularis mucosa. In addition, oval or rounded immunostained ganglion cells appeared located in the myenteric plexuses of both intestinal regions. Nerve fibers intensely stained were observed extending between adjacent myenteric ganglia. The localization of mGluR2/3 receptors in enteric neurons might have functional implications in the physiology and pathology of the gut.

Transgenic plants of cassava (Manihot esculenta) with resistance to Basta obtained by Agrobacterium-mediated transformation.

Transgenic plants of cassava (Manihot esculenta) resistant to the herbicide Basta were obtained through Agrobacterium-mediated transformation. The plants also expressed the uidA gene and two were positive for PCR- and/or Southern-based detection of the nptII gene. Somatic-embryo-derived cotyledons were used as source of explants. A non-disarmed Agrobacterium strain (CIAT 1182) was used to transfer the genes of interest into cassava cultivar MPer183. Greenhouse tests of resistance to Basta (Hoechst) showed three plant lines with different levels of tolerance to the herbicide. Based on Southern tests of transgenesis, the transformation efficiency was 1%. The results constitute the first report of the bar gene conferring herbicide resistance to cassava plants.

[Endoscopic surgery of nasopharyngeal angiofibroma by double embolization]. / Cirugía endoscópica del angiofibroma nasofaríngeo mediante doble embolización.

Juvenile nasopharyngeal angiofibroma is a source of controversy with respect to therapy because of the many therapeutic modalities that exist, such as classic surgical techniques and newer techniques like nasal endoscopic surgery, which have emerged with the advent of new and better diagnostic techniques. Endoscopic surgery is less aggressive and produces less morbidity, but its use often depends on the size and extension of the tumor. A clinical case is presented with the subsequent diagnostic steps and surgical treatment. Rigid endoscopy was used for double cynoacrylate embolization, by angiography before operation and intratumoral injection during surgery.

[Supernumerary nostril]. / Tercera ventana nasal.

Supernumerary nostril is an extremely rare congenital anomaly as a result of aberrant embryological development. Frequently it leads to a narrow cavity; in the end, opening into the nearest nasal cavity or just stops blindly after a short course. It can appear isolated or in association with other anomalies. Treatment is always surgical. We present a case of a triple nostril, associated to a congenital auricular hypoplasia and we also review the existing literature.

Characterization of oral double balloon endoscopy in the dog.

Exploration of the canine small intestine using conventional endoscopy is restricted to the duodenum and/or the ileum. Double balloon endoscopy (DBE) is a 'push and pull' technique that has been described in humans and permits a complete exploration of the small intestine. In this study, oral DBE was performed on 12 healthy dogs (10-34kg) to characterize for the first time the efficiency, exploration dynamics and safety of the technique. DBE was successful in 83% of dogs; the average estimated insertion depth of the endoscope was 287±36cm, and the average duration of the exploration was 84±8min. No complications or relevant adverse clinical effects were observed, and there was no indication of post-procedure pancreatitis based on serology of two specific markers of pancreatitis (amylase and lipase) and the immediate nonspecific inflammatory mediator C-reactive protein. The study showed that oral DBE is viable and safe in the dog, allowing for the diagnosis and treatment of gastrointestinal diseases deep in the small intestine to an extent that has not previously been possible using conventional endoscopy.

Anatomical study of fibrous structures of the medial aspect of the canine elbow joint.

Forces exerted by fibrous structures on the medial aspect of the canine elbow joint have been reported to be involved in elbow pathology. The purpose of this paper is to assess the relationships of the ligamentous and muscular structures of this region of the canine elbow joint, and how they relate to the medial coronoid process. Six cadavers of adult German shepherd cross-breed dogs were used in this study to make intra-articular and vascular injections of this region. Some joints were dissected and some were frozen to saw sagittal or dorsal cryosections to assess the relationships of the myotendinous structures. The brachialis muscle tendon passed through the division of the bicipital tendon of insertion which formed a fibrous tendon sheath that was reinforced by the oblique ligament. The biceps' brachii's main insertion is the radial tuberosity where it inserts along with the cranial branch of the oblique ligament and the cranial branch of the medial collateral ligament. Rotational and compression forces exerted by the insertion of the biceps brachii-brachialis tendon complex onto the ulna might influence medial coronoid disease. Therefore, sectioning these tendons could be considered as a treatment for medial coronoid disease.

Morphometric study of the layers of the canine small intestine at five sampling sites.

The histology of the canine intestine has not been accurately defined. To establish the precise thickness of its different layers, whole wall samples of the small intestine were removed from 41 cadavers at five standardised sampling sites (duodenum, proximal jejunum, distal jejunum, proximal ileum and distal ileum). The total thickness was estimated by morphometry, as was the thickness of the mucosa, muscularis mucosae, submucosa and muscularis externa. In addition, the size of the lymphoid aggregates in the submucosa and the thickness of the circular and longitudinal layers within both the muscularis mucosae and the muscularis externa were estimated. The total intestinal thickness depended very much upon the thickness of the mucosa and submucosa. The mucosa decreased progressively from proximal to distal parts of the small intestine (47% reduction). The thickness of the submucosa, however, changed little from the duodenum to the distal jejunum, but increased significantly in the ileum; this change was positively correlated with the amount of lymphoid tissue. Sex influenced the thickness of the intestinal wall, with males displaying higher thickness values along the small intestine. Conversely, no correlation between bodyweight and intestinal thickness was found for any of the five sampling sites. This study gives absolute and relative values for the thickness of the layers of the dog intestine which might help in the diagnosis of small intestinal pathology from postmortem samples and/or endoscopic biopsies.

Morphometry of the canine intestine with reference to the use of double balloon endoscopy.

Double balloon endoscopy (DBE) enables the diagnosis and treatment of small intestinal disease. The dog is a potential animal model for DBE training and also a candidate for the clinical application of this technique. However, the anatomy of the canine small intestine may limit the use of DBE by restricting the push-and-pull manoeuvres required for DBE. To establish what these limitations are, the length and width of different portions of the small intestine as well as the mesenteric dependences were measured in 55 dogs. Several external parameters related to body size plus the age, weight, sex and breed were recorded. Potential restriction of the progress of the enteroscope due to narrow intestinal diameter is expected in the jejunum of mid- and small size dogs.

Immunocytochemical localization of metabotropic (mGluR2/3 and mGluR4a) and ionotropic (GluR2/3) glutamate receptors in adrenal medullary ganglion cells.

The localization of metabotropic glutamate receptors of groups II (mGluR2/3) and III (mGluR4a) and the subunits 2 and 3 of alfa-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) ionotropic glutamate receptors (GluR2/3) was investigated with immunocytochemical methods in the rat adrenal gland. MGluR2/3, mGluR4a and GluR2/3 immunoreactivities were observed in large-sized, centrally located type I adrenal medullary ganglion neurons. Furthermore, the small-sized type II adrenal ganglion neurons identified by their immunoreactivity to brain nitric oxide synthase (bNOS), also expressed mGluR2/3, mGluR4a and GluR2/3. These cells were disposed in the peripheral portion of the adrenal medulla. None of the type I neurons were positively labeled for bNOS. These morphological observations suggest that activation of glutamate receptors in ganglion neurons may be instrumental in the control of adrenal endocrine systems as well as blood regulation.

Thalidomide reduces IL-18, IL-8 and TNF-alpha release from alveolar macrophages in interstitial lung disease.

Thalidomide exhibits diverse actions of anti-inflammation, immunomodulation and anti-angiogenesis. The efficacy of thalidomide treatment in sarcoidosis with lupus pernio is thought to be due to inhibition of tumour necrosis factor (TNF)-alpha. The mechanisms that underlie the properties of thalidomide are still unclear in interstitial lung disease. The current authors investigated the potential inhibitory effects of thalidomide at concentrations of 0.1, 0.01 and 0.001 mM on the production of transforming growth factor-beta, TNF-alpha, interleukin (IL)-1beta, IL-6, IL-8, IL-10, IL-12p70, IL-12p40 and IL-18 by alveolar macrophages from bronchoalveolar lavage in patients with sarcoidosis (n = 8), hypersensitivity pneumonitis (HP; n = 8) and idiopathic pulmonary fibrosis (IPF; n = 12). In sarcoidosis and HP patients, thalidomide induced a dose-dependent, partial suppression of lipopolysacchride (LPS)-stimulated TNF-alpha, IL-12p40 and IL-18 release. At the highest thalidomide concentration (0.1 mM), LPS-stimulated IL-8 production was also suppressed. In IPF patients, although spontaneous production of TNF-alpha, IL-12p40, IL-18 and IL-8 was lower than in sarcoidosis and HP patients, with LPS stimulation the cytokines were significantly elevated and also partially inhibited by thalidomide. In conclusion, thalidomide has the potential to improve the therapeutic regimens for sarcoidosis, hypersensitivity pneumonitis and idiopathic pulmonary fibrosis by reducing tumour necrosis factor-alpha, interleukin-12p40, interleukin-18 and interleukin-8 production.

Utility of an excluded and everted segment of small intestine placed under the skin of the abdominal wall as an adrenal transplant site in rats.

The feasibility of excluded and everted small intestine segments as reservoirs for adrenal tissue transplantation was compared between biadrenalectomized adult rats with isogenic transplants of adrenal quarters or allogenic transplants of neonatal adrenal glands. Small clusters of adrenocortical cells, located between the adipocytes and the mesenteric vessels of the segments' lumen, were observed following isogenic transplantation. Several large regenerated adrenocortical nodules, surrounded by a connective capsule, were present in each of the rats transplanted with neonatal adrenal glands. Medullary tissue did not regenerate. It was concluded that the excluded and everted segments of small intestine were optimal sites for adrenal tissue transplantation and that the regeneration process of the adrenocortical tissue takes place more quickly after the transplantation of neonatal adrenal glands than after the transplantation of adrenal quarters.

Analysis of the pituitary-thyroid axis in bilaterally adrenalectomized or adrenal transplanted rats.

The percentage, distribution, shape, intensity of staining and morphometrical parameters of the pituitary TSH immunoreactive cells and the histological features of the thyroid glands, were compared between adult rats with intact adrenals, without adrenals and biadrenalectomized animals with neonatal adrenal grafts. After the removal of the adrenal glands, TSH immunoreactive cells increased in percentage and exhibited a higher complexity of the cellular outline, than that of the intact animals. The nuclear, cytoplasmic and cell areas were significantly increased. However the bigger enhancement of the cytoplasmic area relative to the nuclear area, produced a decrease in the nuclear/cell area ratio. The thyroid glands showed some histological evidences of activation. After the transplantation of neonatal adrenal glands to adult rats, several adrenocortical nodules were present in the lumen of the small bowel segment. These adrenal masses induced a great decrease in the TSH cell area, which coupled with a smaller but significant variation of the nuclear area, led to an increase in the nuclear/cell area ratio relative to that observed in adrenalectomized animals. In addition, the distribution, shape and intensity of the immunoreactive material was similar to that observed in intact animals. In this experimental group, thyroid histology was observed to be similar to that of the intact animals.