Increased expression and altered intracellular distribution of adhesion/growth-regulatory lectins galectins-1 and -7 during tumour progression in hypopharyngeal and laryngeal squamous cell carcinomas.

AIMS: To examine the level of expression of the pleiotropic regulators galectins-1 and -7 in relation to neoplastic progression of hypopharyngeal (HSCCs) and laryngeal (LSCCs) squamous cell carcinomas. METHODS AND RESULTS: The presence of galectins-1 and -7 was investigated using quantitative immunohistochemistry in (i) a series of 78 HSCCs by comparison with 17 normal epithelia (N_E), 26 low-grade dysplasia (low_D) and 27 high-grade dysplasia (high_D) and (ii) a series of 56 LSCCs by comparison with 50 N_E, 23 low_D and 29 high_D. Galectin-1 positivity expressed as a percentage of cells was significantly higher in carcinomas (HSCCs and LSCCs) than in N_E, low_D or high_D (P < 10(-6)). Galectin-7 expression was elevated in low_D (P = 0.0004) compared with N_E and in carcinomas (HSCC) compared with high_D (P = 0.0002). Tumour progression from high_D to carcinomas was associated with a shift of galectin-1 localization from the nucleus towards the cytoplasm. Increased expression of galectin-7 in dysplasias was accompanied by a shift from the cytoplasmic compartment (N_E) to the nucleus (low_D and high_D). CONCLUSIONS: Our data reveal an association between the level of presence of galectins-1 and -7 and neoplastic progression of HSCCs and LSCCs. Moreover, inverse shifts between nuclear and cytoplasmic positivity intimating functional divergence were detected.

Immunohistochemistry of the golden hamster pituitary during chronic administration of diethylstilbestrol: a quantitative analysis using confocal laser scanning microscopy.

The aim of this study was to examine by immunohistochemistry the morphologic changes affecting pituitary cell populations in male Syrian hamsters undergoing chronic exposure (3 days to 9 months) to diethylstilbestrol (DES). Cell proliferation in the hypophysis was monitored by the immunohistochemical demonstration of S-phase cells after pulse labeling with 5-bromo-2'-deoxyuridine. Cell proliferation analysis was combined with the identification of different cell populations by immunostaining with antisera raised against hypophyseal hormones. Sections processed for double-label immunofluorescence were examined by confocal microscopy. In the adenohypophysis, the relative surface occupied by gonadotrophs and thyrotrophs decreased rapidly during the first months of treatment while corticotroph and somatotroph populations remained unaffected. Accordingly, the incidence of S-phase cells in these four cell populations was lower than or similar to control values. In contrast, lactotrophs increased gradually during the first month of exposure to DES to reach a maximum value at 2-4 months. At the beginning, this increase was primarily due to hyperplasia but later on it also involved cellular hypertrophy. Somatomammotrophs did not seem to be involved in this model. In the pars intermedia, the labeling index of melanotrophs rose rapidly to reach values 5-6 times higher than controls. After 4 months, neoplasms originating from the pars intermedia were seen invading both the neuro- and the adenohypophysis. At the end of treatment, the pituitary was markedly enlarged resulting from the development of an adenoma of the pars intermedia.

Immunohistochemical analysis of diethylstilbestrol-induced renal tumors in adult male Syrian hamsters: evidence for relationship to peripheral nerve sheath tumors.

Estrogen-induced Syrian hamster kidney tumors (SHKT) are widely used as experimental models for the study of hormonal and renal carcinogenesis. In order to characterize the direction of differentiation of SHKT, kidney sections of diethylstilbestrol (DES)-treated hamsters (1-11 months) were analyzed by immunohistochemistry using a panel of lineage-specific markers. The first tumorous buds found in animals exposed to DES for 4-6 months exhibited prominent S100, Leu-7, and vimentin immunoreactivities. Immunopositivities for neuron-specific enolase, PGP 9.5, desmin, and glial fibrillary acidic protein were mostly detected in medium-sized and large tumors after prolonged exposure to DES (> 6 months). All neoplasms, irrespective of the size and the duration of treatment, appeared negative for cytokeratin, neurofilaments, synaptophysin, and CD99 antibodies. Western blotting confirmed to a large extent the immunohistochemical observations. The systematic analysis of serial kidney sections by confocal microscopy after double immunostaining for S100 and neurofilaments revealed that early neoplastic buds could stem from S100-positive cells associated with nerves bundles. Altogether, these observations suggest that DES-induced SHKT could be related to malignant peripheral nerve sheath tumor and originate from a yet unidentified precursor cell present in the sheath of peripheral nerves.

Clara cell specific protein (CC16) expression after acute lung inflammation induced by intratracheal lipopolysaccharide administration.

Clara cell secretory protein (CC16, CC10, or CCSP), the major secretory protein of the Clara cell, presents several biologic properties, suggesting that it may play a protective role against intrapulmonary inflammatory processes. The aim of the present study was to investigate the changes of CC16 concentrations in the lung, bronchoalveolar lavage fluid (BALF), and serum of rats with acute lung injury induced by lipopolysaccharide (LPS). These changes were compared with Clara cell density, CC16 mRNA level in the lung and classic indices of inflammation in BALF. Injected at doses of 10, 100, or 200 microgram/100 g body weight, LPS induced an acute lung inflammation as estimated by an increased influx of cells and albumin in the BALF. This inflammatory response was associated with a marked reduction of CC16 concentrations in BALF and lung homogenate as well as of the CC16 mRNA levels in the lung. At the highest dose of LPS, the CC16-positive cell density in the bronchiolar epithelium was also decreased. In serum, by contrast, the concentration of CC16 was elevated as a consequence of increased airway permeability. Pretreating rats intraperitoneally with dexamethasone (2 mg/kg) significantly lowered the leukocyte influx and attenuated the albumin increase in BALF. Dexamethasone, however, failed to prevent the increased airway permeability to CC16, suggesting that during inflammation different mechanisms regulate the leakage of proteins across the alveolocapillary barrier depending on the direction of passage and/or the size of the protein. Our results show a marked decrease of the secretion and synthesis of CC16 during LPS-induced acute lung inflammation.

Demonstration of estrogen receptors and of estrogen responsiveness in the HKT-1097 cell line derived from diethylstilbestrol-induced kidney tumors.

This study was undertaken in order to examine the estrogen sensitivity of HKT-1097, an established cell line recently derived from diethylstilbestrol (DES)-induced kidney tumors in Syrian hamsters. Estrogen receptor (ER) level in HKT-1097, determined by enzyme-linked immunoassay, was 67 fmol/mg protein, i.e., a value approx. 30% lower than that found in Syrian hamster kidney tumors. ER immunostaining in cells fixed with Carnoy's mixture, as well as ER demonstration by Western blotting, suggested DES-induced nuclear translocation or stabilization of the receptor within the nucleus. Kinetic parameters of estrogen binding to ER in HKT-1097 cells were 8.4 x 10(-11) M and 60.8 fmol/mg protein for Kd and Bmax, respectively. The Kd of estrogen binding to ER in HKT-1097 was close to that evaluated for the receptor in breast cancer-derived MCF-7 cell line, whereas the Bmax value was approx. seven times lower in HKT-1097 as compared to MCF-7. In HKT-1097 cells, antiestrogens ICI 182,780 and RU 58,668 induced ER downregulation and competed with estrogen binding to the receptor. As demonstrated by Western blot analysis, DES exposure led to an increased expression of progesterone receptor (PgR) in HKT-1097 cells. Addition of DES to estrogen-free medium produced a stimulation of growth in both HKT-1097 and MCF-7 cells, but the mitogenic effect was less marked for HKT-1097. Despite the fact that ICI 182,780 and RU 58,668 clearly interact with HKT-1097 cell ER, they appeared unable to suppress DES-induced stimulation of growth and increase of PgR expression.

Characterization of a cell line established from diethylstilbestrol-induced renal tumors in Syrian hamsters.

This article describes HKT-1097, a new cell line established from renal tumors induced by the protracted administration of diethylstilbestrol (DES) to male Syrian golden hamsters. Cell culture was initiated from tumor samples obtained from two 14-mo.-old animals which had undergone exposure to DES for a period of 11 mo. The HKT-1097 cell line was characterized between Passages 16 and 22 with respect to cell morphology, growth properties, karyology, and the presence of estrogen receptors. Moreover, immunostaining with a panel of antisera was performed to identify the cytological profile of the cell line and establish a parallel with tumor tissue in vivo. HKT-1097 cells are fibroblastoid; their most distinctive feature is that they exhibit strikingly long processes. The HKT-1097 cell line grows as a monolayer with a tendency toward a less stringent density-dependent inhibition of growth. The modal chromosome number is 44, but more than 50% of the cells are aneuploid, suggesting a substantial degree of karyotype instability. HKT-1097 cells express estrogen receptors. They contain immunoreactive vimentin and desmin, but appear negative upon cytokeratin immunostaining. In addition, these cells express glial fibrillary acidic protein and other markers of the neuroectodermal lineage, but lack neurofilament protein. Insofar as the same lineage markers have been demonstrated in DES-induced Syrian hamster kidney tumors (SHKT), we conclude that HKT-1097 cells retain some of the original tumor cell phenotype. The current observations suggest that estrogen-induced SHKT derive from the renal interstitium and point to an involvement of neuroectodermal cells in the development of these neoplasms.

Clara cell protein as a marker of Clara cell damage and bronchoalveolar blood barrier permeability.

The 16 kDa Clara cell protein (CC16), an abundant component of airway secretions, has recently been proposed in humans as a pulmonary marker measurable not only in bronchoalveolar lavage fluid (BALF) but also in serum. The aim of the present study was to investigate the changes and determinants of CC16 concentrations in these fluids in normal rats and rats with lung injury. Female Sprague-Dawley rats were given a single i.p. injection of arachis oil (n=20) or chemicals in arachis oil (n=10) that mainly damage Clara cells (4-ipomeanol (IPO) 8 mg x kg(-1) and methylcyclopentadienyl manganese tricarbonyl (MMT) 5 mg x kg(-1)) or endothelial cells (alpha-naphthylthiourea (ANTU) 5 mg x kg(-1)). CC16 concentration (mean+/-sD in microg x L(-1)), measured by a sensitive latex immunoassay, was significantly reduced in BALF of all treated groups (IPO 380+/-100; MMT 730+/-200; ANTU 1,070+/-200; controls 1,700+/-470). The same pattern of decrease was observed in the labelling of Clara cells with an anti-CC16 antiserum as well as in the CC16 messenger ribonucleic acid levels assessed by Northern enzyme-linked immunosorbent assay. In serum, by contrast, CC16 was significantly increased in all treated groups (IPO 31+/-7; MMT 22+/-12; ANTU 52+/-24; controls 15+/-6). This rise of CC16 in serum was associated with an elevation of albumin in BALF which is an index of increased bronchoalveolar/blood barrier permeability. In conclusion, lung injury induces a decrease of the 16 kDa Clara cell protein in bronchoalveolar lavage fluid owing to a reduced production by damaged Clara cells, and an increase in serum protein levels resulting from its enhanced leakage across the bronchoalveolar/blood barrier. This study provides new insights into the understanding of the changes of lung secretory proteins in bronchoalveolar lavage fluid and serum.

Exposure to hydrocarbons and renal disease: an experimental animal model.

The association between hydrocarbon exposure and chronic glomerulonephritis is still a controversial scientific issue. Recent epidemiological evidence suggests a role of exposure to hydrocarbons in the progression of glomerulonephritis towards chronic renal failure. The present experimental study on rats has been designed to assess the possible role of styrene in the progression of adriamycin (ADR) nephrosis, a well known model of renal fibrosis following nephrotic syndrome induced by ADR. Female Sprague-Dawley rats were exposed to styrene, 300 ppm, 6 h/day, 5 days/week for 12 weeks (group 1); treated with ADR, 2 mg/Kg, i.v., twice on day 1 and day 15 of the study (group 2); Additional groups of animals received both the styrene and ADR treatments (group 3) or served as controls (group 4). The urinary excretion of total and single proteins (albumin, Retinol-Binding Protein (RBP), Clara Cell 16 Kd protein (CC16), fibronectin) was measured monthly, whereas histopathology and determinations requiring blood sampling were carried out at the end of the experiment. A progressive increase in total proteinuria, falling in the nephrotic range already by the 6th week was observed in ADR-treated groups. Styrene exposure caused up to a 3- to 5-fold increase as compared to controls. Co-exposure to ADR and styrene also resulted in a proteinuria much greater than that caused by ADR alone. The interactive effect of styrene and ADR was statistically significant for albuminuria and urinary fibronectin. A similar response was observed for glomerular filtration rate at the end of the experiment, styrene-exposed animals showing hyperfiltration as compared to their respective control group. At the end of the experiment, histopathological scoring for interstitial infiltration and fibrosis was also significantly higher in styrene-treated animals as compared to their respective control groups. In ADR-treated rats, low molecular weight proteinuria (l.m.w.p.) was only slightly affected, suggesting minimal tubular dysfunction associated with extensive tubular atrophy. However, styrene-exposed animals showed l.m.w.p. higher than their respective controls. In summary, in this animal model we were able to confirm both styrene-induced microproteinuria, mainly albuminuria and minor increases in l.m.w.p., observed among occupationally exposed workers and the role of hydrocarbon exposure as a factor accelerating the progression of renal disease suggested by epidemiological investigations in patients suffering from chronic renal disease. Whereas in rats exposed to styrene only, microproteinuria was stable over time and minor histopathological changes were noted at the end of the experiment, evidence of a role of solvent exposure in the progression of ADR nephropathy was obtained in terms of both renal dysfunction and interstitial fibrosis. The mechanistic basis of styrene-ADR interaction is unclear. However, experimental evidence is consistent with epidemiological findings suggesting the need to avoid solvent exposure in patients suffering from renal diseases.

Apoptosis and cell proliferation in the seminal vesicles and coagulating glands of male Syrian hamsters exposed to diethylstilboestrol (DES).

Regression of the accessory sex glands was induced in male Syrian hamsters by chronic exposure to diethylstilboestrol (DES), an agonist of 17beta-oestradiol. Experimental groups (n = 4-5) were killed at increasing time intervals up to 6 months after initiation of treatment. Organ atrophy was evaluated by morphological examination. Apoptosis in the seminal vesicles and coagulating glands was visualized by in situ analysis of DNA fragmentation. Cell proliferation was monitored by immunostaining nuclei of S-phase cells after pulse labelling with BrdU. The volume of the seminal vesicles decreased drastically after 2 weeks of DES administration due to a marked reduction of secretions in the lumen of the glands. Cell proliferation in the seminal vesicles was stimulated by chronic administration of DES. Mitotic activity mostly increased during the first month of treatment, leading to epithelial hyperplasia associated with progressive hyperplasia of the fibromuscular stroma. Evidence of epithelial dysplasia and metaplasia, often associated with an infiltration of polymorphonuclear leukocytes, was observed in animals exposed to DES for 4 months or more. Regression of the seminal vesicles was also associated with apoptosis in the gland epithelium. Apoptosis appeared 3 days after starting DES administration and culminated at 1 month. Thereafter the number of apoptotic cells decreased progressively, but apoptosis remained present until the end of treatment. In contrast, coagulating glands were less sensitive to DES. No major morphological changes were observed in these glands except for a moderate reduction of protein secretion. The levels of the apoptotic and proliferating cells indices were very low in the coagulating glands during DES treatment. In conclusion, these data point to different sensitivities of the accessory sex glands to DES exposure because DES induces extensive alterations of the normal morphology of the seminal vesicles, but shows only a moderate impact on the coagulating glands.

Sublethal alterations and sustained cell proliferation associated with the diethylstilbestrol-induced renal carcinogenesis in male Syrian golden hamsters.

The current study was initiated to explore the sublethal alterations and the tissue damage occurring in the hamster kidney during diethylstilbestrol-induced renal carcinogenesis. A total of 49 male Syrian golden hamsters (35 treated and 13 control animals) was utilized in the experimental procedure. Chronic exposure to diethylstilbestrol was achieved by s.c. insertion of implants containing 25 mg diethylstilbestrol. For long-term observation, adequate blood level of diethylstilbestrol was insured by renewing the implant every 2 months. Experimental groups (n = 4 to 9) were terminated 1, 2, 4, 6, 9 and 11 months after initial implantation for morphological examination of the kidney. Diethylstilbestrol carcinogenicity in this experimental model was confirmed by the observation that most animals undergoing drug exposure for 6 months or more exhibited renal neoplasms. The most striking nonneoplastic morphological abnormality disclosed by histological and cytological examination consisted in the accumulation of granular inclusions in proximal tubule cells. In renal tissue, the extent of cell proliferation determined by PCNA labeling progressively increased along with the duration of diethylstilbestrol exposure and suggested a sustained proliferative response in altered proximal tubules. The present data suggest that an impairment of functional tubular regeneration could promote, as well as the estrogen genotoxic effect, the tumorigenicity of diethylstilbestrol in the kidney of male hamsters.

Quantification of Clara cell protein in rat and mouse biological fluids using a sensitive immunoassay.

Clara cell protein is a 16-17 kDa protein (CC16) secreted by Clara cells in the bronchiolar lining fluid of the lung. In order to investigate the potential of this protein as a pulmonary marker in animals, CC16 was isolated from rat bronchoalveolar lavage fluid (BALF) and a sensitive latex immunoassay applicable to both rat and mouse CC16 was developed. The pattern of CC16 concentrations in rat biological fluids determined by the immunoassay was consistent with the hypothesis of a passive diffusion of the protein across the bronchoalveolar/blood barriers showing a difference of more than 5,000 fold between the concentration in the epithelial lining fluid (mean, 140 mg x L(-1)) and that in serum (20 microg x L(-1)) or urine (3 microg x L(-1)). In BALF, the CC16 concentration averaged 5,500 microg x L(-1) and was of the same magnitude as that determined on lung and trachea homogenates. CC16 was also detectable in amniotic fluid with a mean value of 800 microg x L(-1) before delivery. Damage of Clara cells produced by methylcyclopentadienyl manganese tricarbonyl resulted in a significant decrease of CC16 in BALF but did not affect the serum levels of the protein. The nephrotoxicant sodium chromate by contrast had no influence on the CC16 content of BALF but markedly increased CC16 levels in both serum and urine as a result of impaired glomerular filtration and tubular reabsorption, respectively. In conclusion, mouse or rat Clara cell protein of 16-17 kDa can easily be quantified, not only in bronchoalveolar lavage fluid, but also in extrapulmonary fluids such as serum or urine. Thus, in rodents, Clara cell protein of 16-17 kDa follows the same metabolic pathway as in humans, diffusing from the respiratory tract into serum where it is eliminated by the kidneys. This serum Clara cell protein of 16-17 kDa may be useful as a peripheral marker of events taking place in the respiratory tract.

In vitro demonstration of a mitogenic activity in renal tissue extracts during regenerative hyperplasia.

Normal rat kidney (NRK-52E) cells, an established cell line of renal origin, were used as a bioassay system to reveal a possible mitogenic activity in tissue extracts prepared from kidneys undergoing tubular regeneration. Acute tubular injury was induced in female Wistar rats by a 4-day treatment with gentamicin at daily doses of 50 or 100 mg/kg twice daily. Animals were killed either 1 or 4 days after cessation of gentamicin administration. Proximal tubule regeneration in treated animals was confirmed by morphological examination after proliferating cell nuclear antigen staining. Tissue extracts from regenerating kidneys stimulated DNA synthesis in growth-arrested cells to a higher extent than extracts from intact kidneys. Sera from treated and control animals showed no difference with respect to mitogenic activity. The mitogenic effect of tissue extracts was sensitive to the tyrosine kinase inhibitor tyrphostin A46. The cell proliferative response to regenerating kidney extracts, but not that to intact kidney extracts, was partly suppressed by the addition of anti-insulin-like growth factor I (anti-IGF-I) antiserum. These data indicate that nephrogenic repair entails an elevation of biologically active IGF-I in kidney tissue.

In situ demonstration of germinal cell apoptosis during diethylstilbestrol-induced testis regression in adult male Syrian hamsters.

Testis regression was induced in male Syrian hamsters by chronic exposure to diethylstilbestrol (DES), and estradiol-17 beta agonist. Experimental groups (n = 4-5) were killed at increasing time intervals over a period of 6 mo after initiation of treatment. Apoptosis in testes was demonstrated by in situ analysis of DNA fragmentation. Cell proliferation was monitored by immunostaining nuclei of S-phase cells after pulse labeling with 5-bromo-2'-deoxyuridine. Levels of FSH and testosterone, measured by RIA fell rapidly in DES-treated hamsters. In parallel, testis weight and seminiferous tubule area underwent an 80% decrease during the first 2 wk of DES administration. The composition of seminiferous epithelium was also drastically affected by DES, since it became progressively confined to Sertoli cells, spermatogonia, and spermatocytes. Testis regression was associated with an important increase of apoptosis, which started 3 days after the beginning of DES administration. Apoptosis was still 10- to 50-fold higher than in control testes by the end of treatment; it affected primarily spermatocytes and, to a much lesser extent, spermatogonia. Cell proliferation was not inhibited by chronic DES administration. In conclusion, these data indicate that apoptosis can by itself account for estrogen-induced testis regression.

Involvement of transforming growth factor-alpha and its receptor in a model of DES-induced renal carcinogenesis in the Syrian hamster.

This study explores the role played by TGF alpha in estrogen-induced renal tumors. Tumors were induced in male Syrian hamster by chronic administration of diethylstilbestrol (DES). Six experimental groups (n = 5-9) were chronically exposed to DES and sacrificed after 1, 2, 4, 6, 9 and 11 months, respectively. In the course of treatment, the nephrons were the site of an important increase of cell turnover, which was characterized by a process of hyperplasia/dysplasia in proximal tubules preceding the neoplastic transformation. In treated animals and in controls, the analysis of renal tissue by Western blot revealed the presence of a 6 kDa polypeptide crossreacting with anti-TGF alpha antibody. In controls, TGF alpha immunoreactivity was localized in proximal and in distal tubules. Before tumor development (1-4 months), TGF alpha RIA showed an increase of TGF alpha concentration in renal tissue, in parallel with the increased cell proliferation observed in proximal tubules. In addition, Western blot analysis also demonstrated in kidney tissue the presence of a 165 kDa protein displaying the immunoreactivity of EGF/TGF alpha receptor. The receptor immunoreactivity was localized in proximal tubular cells suggesting an involvement of TGF alpha in tubular epithelial growth through autocrine or paracrine pathways. In large neoplasms, immunocytochemistry revealed only clusters of transformed cells intensely stained by the anti-TGF alpha antibody. These cells displayed the appearance of stellate or polyhedric cells infiltrating adjacent neoplastic tissues. Antisera raised against intra- or extracytoplasmic domains of the EGF/TGF alpha receptor were assayed to localize this receptor in the tumors. In contrast with tubular structures, immunoreactivity to EGF/TGF alpha receptor was never detected in tumoral tissue. The apparent absence of EGF/ TGF alpha receptor immunoreactivity in malignant cells seems to exclude an involvement of this growth factor in the tumorigenic process, although it could be involved in tumor neovascularization.

Transforming growth factor-alpha and epidermal growth factor in hamster tissues: biochemical and immunohistochemical studies.

In Syrian golden hamster kidneys and submaxillary glands, the levels of EGF, determined by radioimmunoassay, were much lower than in the same organs of two other rodent species, mouse and rat. In submaxillary glands, the EGF/TGF-alpha receptor-binding activities were also much lower in hamster than in mouse and rat. In contrast, the TGF-alpha content of hamster kidneys, determined by radioimmunoassay, was higher than in the kidneys of the other animals, as was the EGF/TGF-alpha receptor-binding activity. Using immunohistochemistry, the TGF-alpha immunoreactivity in hamster kidneys was localized both in proximal and distal tubules with the exception of the macula densa area. The levels of TGF-alpha in the submaxillary glands were very low in all the animals tested. Hamster kidney extracts contained a specific immunoreactive protein with the M(r) and the N-terminal amino acid sequence (VVSHFNECPD) expected for mature hamster TGF-alpha. Western blot analysis of hamster renal solubilized membrane proteins using anti-EGF receptor antibodies revealed three immunoreactive protein bands of which one had the M(r) expected for the EGF/TGF-alpha receptor. The immunohistochemical pattern of this receptor in hamster kidneys proximal tubular cells was very similar to the other tested rodent species.

Morphological and kinematic study of the tongue and buccal cavity in the lizard Anguis fragilis (Reptilia:Anguidae).

BACKGROUND: The ability to detect chemical cues is highly developed in Scleroglossa, and particularly in anguid lizards. This ability was predicted because anguids possess a well-developed vomeronasal organ (VNO) (or Jacobson's organ) and rely largely on chemical cues in various behaviours as other active foragers. In this work, we have investigated the possible functional association between tongue flicking and the VNO in the lizard Anguis fragilis. METHODS: The morphology of the tongue and the buccal cavity was investigated by light and scanning electron microscopy. The kinematics of tongue and jaw movements was studied by high speed cinematography. RESULTS: The epithelial cells of the ventral aspect of the tongue tips show microstructures (microridges, microfacets, micropores) which are not present on other areas of the mouth. Beneath the tongue, the floor of the buccal cavity shows two concave-like elevations suggesting a structural analogy with the anterior processes described in snakes. The apex and the internal margin of these processes bear parallel oblique ridges. Taste buds occur anteriorly on the buccal floor and on the palate and are abundant on the internal side and on the edge on the anterior processes. The tongue showed three modes of tongue flicking: simple downward extension, single oscillation, and multiple oscillations. At each tongue flick, the ventral surface of the tips was observed contacting the substratum. Immediately after the tongue retraction, the buccal floor moved slightly upward. The observation of tongue flicking with the mouth open showed that the anterior processes moved upward when the tongue was retracted. CONCLUSIONS: These observations suggest that following: 1) during tongue flicking the ventral surface of the tongue tips invariably makes contact with the substratum; 2) the microstructures of the tongue tips and the ridges of the anterior processes might be helpful for collecting and receiving, respectively, chemicals during tongue flicking; 3) the anterior processes may be apposed on the roof of the mouth next to the ducts of VNOs when the buccal floor is fully elevated; 4) due to their localization, the taste buds could be equally stimulated by the molecules transferred during tongue flicking.

Renal tissue expression of EGF and EGF receptor after ischaemic tubular injury: an immunohistochemical study.

Rat kidneys undergoing tubular regeneration after ischaemic injury have been examined with regard to EGF, EGF receptor and vimentin, using immunohistochemical techniques. Renal ischaemia was induced in male Sprague-Dawley rats by 35-min clamping of renal arteries. Groups (n = 4-6) of experimental animals were killed at different time intervals (12, 24, 48, 72 h, 7 and 14 days) after reperfusion. One hour before sacrifice, each rat received i.p. 200 mg/kg 5-bromo-2'-deoxyuridine (BrdU) for the immunocytological demonstration of DNA synthesis. Renal necropsies were processed to reveal by immunohistochemistry EGF, EGF receptor, vimentin, and BrdU incorporated into DNA of S-phase cells. Tubular necrosis particularly involved proximal straight tubules in the outer stripe of the outer medulla and was followed by tubular regeneration, with a peak of cell proliferation at 48-72 h and an apparent dedifferentiation of tubular epithelium. As soon as 12 h after ischaemia, there was a substantial reduction of EGF immunostaining and the incidence of distal tubules showing EGF immunoreactivity reached a nadir at 48 h. In control kidneys, EGF receptor was mostly immunolocalized in proximal tubules although juxtaglomerular cells also exhibited immunolabelling. EGF receptor immunostaining in tubular epithelium showed no major change during the episode of tubular necrosis (12-24 h) but disappeared in tubular profiles undergoing regenerative hyperplasia (48-72 h). No vimentin immunostaining was found in tubules of control kidneys. Tubular epithelium remained mostly vimentin negative during the early phase of tubular necrosis/regeneration (12-72 h). By contrast, 7 days after ischaemia numerous dedifferentiated tubules expressed vimentin. In conclusion, tubular regeneration after ischaemia is associated with a typical sequence of transient events: (1) reduction of EGF immunostaining; (2) disappearance of EGF receptors during the mitogenic response; (3) expression of vimentin in tubular epithelium, and (4) return to a normal appearance.

Potentiation of gentamicin nephrotoxicity in the rat by infusion of aprotinin.

The present study was undertaken to examine a possible effect of aprotinin, a 6.5-kDa polypeptide with an inhibitory effect on proteolysis, on aminoglycoside nephrotoxicity. Experimental animals (female Sprague-Dawley rats, 175-200 g body wt) were treated for 4 days with 40 mg/kg gentamicin given ip at 12-hr intervals. Aprotinin (40,000 kIU per animal) was infused i.v. over a period of 8 days, using subcutaneously implanted miniosmotic pumps. In protocol A, infusion pumps were placed 4 days before starting gentamicin treatment. In protocol B, pumps were implanted 15-18 hr prior to first gentamicin administration. In addition to rats exposed to both gentamicin and aprotinin (GAP), animals were treated with gentamicin ip+saline i.v. (G), saline ip+aprotinin i.v. (AP), or received only saline by both routes of administration (C). All rats were terminated 4 days after the end of gentamicin dosing. One hour before sacrifice, 200 microCi of [3H]thymidine was given ip to each animal in order to monitor cell turnover in renal tissue. The kidneys were analyzed with respect to (i) histopathological alterations and renal dysfunction, (ii) aminoglycoside tissue accumulation, and (iii) tubular regeneration (measurement of cell proliferation). In animals receiving aprotinin alone, histological examination of renal cortex on paraffin sections disclosed mild tubular injury with focal cell necrosis. In plastic-embedded tissue, proximal tubule epithelium was characterized by the presence of numerous inclusions densely stained with toluidine blue. At the ultrastructural level, these inclusions appeared filled with amorphous electron-dense material. In gentamicin-treated animals, cortical drug accumulation reached values higher than 0.3 mg/g renal tissue, but a significant 30-40% decrease of gentamicin accumulation was noted in GAP groups, compared to G groups. Histological examination of renal cortex (paraffin sections) revealed the development of acute tubular necrosis in both G and GAP groups. Tubular injury was accompanied by mild renal dysfunction, as shown by the level of serum creatinine which was increased almost 3-fold in the G group, compared to C and AP groups. Aprotinin infusion produced a further increase of serum creatinine, particularly in protocol A where it was 72% higher for the GAP group than for the G group. In both G and GAP groups, postnecrotic tubular regeneration was evidenced by determining the rate of DNA synthesis and the frequency of S-phase cells in renal cortex. Both methods gave consistent results and showed a 8- to 13-fold increase of cell proliferation in groups receiving gentamicin alone, compared to C groups.(ABSTRACT TRUNCATED AT 400 WORDS)

Tongue structure and function in Oplurus cuvieri (Reptilia: Iguanidae).

The anatomy of the hyo-lingual apparatus in the iguanid lizard Oplurus cuvieri has been studied by light microscopy and scanning electron microscopy. Four areas were observed on the dorsal lingual epithelium of the lizard. Tongue tips are covered with a smooth epithelium. Closely packed flattened and cylindriform papillae cover the foretongue. The surface of the midtongue bears an unpapillose epithelium. Short conical papillae are arranged on the two lateral posterior bundles of the tongue. At high magnification, microvilli and microridges are widely distributed over the surface of the papillae. The epithelium of the papillae is composed of cells filled with secretory granules. Each surface plays successive roles during food ingestion, intra-buccal transport, and swallowing. The mucous interpapillary spaces would serve the adherence between the tongue and the food, the smooth epithelium of the midtongue should facilitate movements of the prey toward the pharynx, and conical papillae of the hindtongue present a rough surface which should act on the prey during the swallowing phase. The intrinsic morphology of the tongue is rather similar to that previously described for iguanids, but fibers of M. verticalis encircles ventrally the lingual process. These fibers could act in tongue protrusion as previously suggested for agamids. The morphology and function of the extrinsic tongue musculature and the hyoid musculature, analysed by electrical stimulations, are similar to the previous descriptions in iguanids and agamids either for feeding or displaying functions.

Modification of immunoreactive EGF and EGF receptor after acute tubular necrosis induced by tobramycin or cisplatin.

Acute tubular necrosis induced by aminoglycoside antibiotics and various other nephrotoxins is followed by a regenerative process which leads to the restoration of damaged tubules. Several lines of evidence indicate that tubular regeneration is mediated by polypeptide growth factors such as epidermal growth factor (EGF). Previous studies devoted to cisplatin nephrotoxicity have shown that this agent causes tubular cystic degeneration possibly related to an impairment of renal tissue repair. Thus, we examined on a comparative basis the time course of the regenerative response subsequent to tubular damage induced by tobramycin or cisplatin, particular attention being paid to renal EGF and its receptor. Female Sprague-Dawley rats (160-180 g body weight) were treated during 4 consecutive days with daily doses of 200 mg/kg tobramycin i.p. (BID) or 2 mg/kg cisplatin (once a day). Sham-treated rats were given 0.9% NaCl i.p. following the same protocol. Groups of experimental animals (n = 5-10) were terminated at increasing time intervals (1, 4, 7, 14, 21, 60 days) after cessation of treatment. One hour prior to sacrifice, each individual received i.p. 200 mg/kg 5-bromo-2'-deoxyuridine (BrdU) for the immunohistochemical demonstration of cell proliferation. Blood was collected at the time of sacrifice in order to assess glomerular filtration rate by measuring serum creatinine and BUN levels. Kidneys were analyzed with respect to total EGF determined by RIA in renal tissue homogenates, and soluble EGF was assayed in extracts prepared by centrifugation. Renal tissue was processed for the immunohistochemical detection of S-phase cells, of EGF, of EGF receptors, and of the intermediate filament vimentin, the latter being used as a marker of epithelium dedifferentiation. In absence of nephrotoxic alterations, EGF was immunolocalized in distal tubules, whereas EGF receptor immunostaining was seen in proximal tubules cells. Vimentin immunostaining was confined to glomeruli and blood vessels. Tobramycin and cisplatin caused acute tubular necrosis in proximal convoluted tubules and proximal straight tubules, respectively. Tissue damage was accompanied by renal dysfunction reflected by an elevation of serum creatinine and BUN levels. Tubular necrosis was followed by a proliferative response indicative of tubular regeneration. Regenerative hyperplasia was associated with a reduction of total immunoreactive EGF due to a decrease of tissue-bound proEGF. Tubules undergoing regenerative repair were characterized by a disappearance of EGF receptors and the presence of immunoreactive vimentin. In tobramycin-treated rats, renal dysfunction lasted for 4-7 days and was fully reversible, as indicated by the return of serum markers to normal values.