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Type 2 diabetes (T2D) is often linked to metabolic syndrome, which assembles various risk factors related to obesity. Plasma levels of adiponectin are decreased in T2D and obese subjects. Aiming to develop a peptide able to bind adiponectin receptors and modulate their signalling pathways, a 12-amino acid sequence homologous in AdipoR1/R2 has been targeted by phage display with a linear 12-mer peptide library. The selected peptide P17 recognises AdipoR1/R2 expressed by skeletal muscle, liver and pancreatic islets. In HepaRG and C2C12 cells, P17 induced the activation of AMPK (AMPKα-pT172) and the expression of succinate dehydrogenase and glucokinase; no cytotoxic effects were observed on HepaRG cells. In db/db mice, P17 promoted body weight and glycaemia stabilisation, decreased plasma triglycerides to the range of healthy mice and increased adiponectin (in high fat-fed mice) and insulin (in chow-fed mice) levels. It restored to the range of healthy mice the tissue levels and subcellular distribution of AdipoR1/R2, AMPKα-pT172 and PPARα-pS12. In liver, P17 reduced steatosis and apoptosis. The docking of P17 to AdipoR is reminiscent of the binding mechanism of adiponectin. To conclude, we have developed an AdipoR1/AdipoR2-targeted peptide that modulates adiponectin signalling pathways and has therapeutic relevance for T2D and obesity associated pathologies.

Blood-brain barrier (BBB) crossing and brain penetration are really challenging for the delivery of therapeutic agents and imaging probes. The development of new crossing strategies is needed, and a wide range of approaches (invasive or not) have been proposed so far. The receptor-mediated transcytosis is an attractive mechanism, allowing the non-invasive penetration of the BBB. Among available targets, the low-density lipoprotein (LDL) receptor (LDLR) shows favorable characteristics mainly because of the lysosome-bypassed pathway of LDL delivery to the brain, allowing an intact discharge of the carried ligand to the brain targets. The phage display technology was employed to identify a dodecapeptide targeted to the extracellular domain of LDLR (ED-LDLR). This peptide was able to bind the ED-LDLR in the presence of natural ligands and dissociated at acidic pH and in the absence of calcium, in a similar manner as the LDL. In vitro, our peptide was endocytosed by endothelial cells through the caveolae-dependent pathway, proper to the LDLR route in BBB, suggesting the prevention of its lysosomal degradation. The in vivo studies performed by magnetic resonance imaging and fluorescent lifetime imaging suggested the brain penetration of this ED-LDLR-targeted peptide.

Excessive pollen harvesting by bees can compromise the reproductive success of plants. Plants have therefore evolved different morphological structures and floral cues to narrow the spectrum of pollen feeding visitors. Among "filtering" mechanisms, the chemical and mechanical protection of pollen might shape bee-flower interactions and restrict pollen exploitation to a specific suite of visitors such as observed in Asteraceae. Asteraceae pollen is indeed only occasionally exploited by generalist bee species but plentifully foraged by specialist ones (i.e., Asteraceae paradox). During our bioassays, we observed that micro-colonies of generalist bumblebee ( Bombus terrestris L.) feeding on Taraxacum pollen (Asteraceae) reduced their pollen collection and offspring production. Bees also experienced physiological effects of possible defenses in the form of digestive damage. Overall, our results suggest the existence of an effective chemical defense in Asteraceae pollen, while the hypothesis of a mechanical defense appeared more unlikely. Pre- and post-ingestive effects of such chemical defenses (i.e., nutrient deficit or presence of toxic compounds), as well as their role in the shaping of bee-flower interactions, are discussed. Our results strongly suggest that pollen chemical traits may act as drivers of plant selection by bees and partly explain why Asteraceae pollen is rare in generalist bee diets.

Sand deserts are common biotopes on the earth's surface. Some specialized vertebrate species have colonized these ecological habitats by living buried in the sand. Among these so called psammophilic species are the Scincidae sand dune living species Scincus scincus and Eumeces schneideri. These two skinks share a relatively similar behavioral ecology by living buried in sand, almost all the time for S. scincus and at least for some part of the day for E. schneideri. The visual system of these two lizards was investigated by histological, immunohistochemical, Magnetic Resonance Imaging (MRI) and morphometric techniques. Both skink species exhibit a retina lacking fovea, composed predominantly of cones presenting two types of oil droplets (pale blue-green and colorless). Both species possess a subset of rod like-photoreceptors (about 1 rod for 30 cones) evidenced by anti-rhodopsin immunoreactivity. A ratio 1:1-1:2 between ganglion cells and photoreceptors points to a linear connection (photoreceptors/bipolar neurons/ganglion cells) in the retina and indicates that both skinks more likely possess good visual acuity, even in the peripheral retina. The MRI analysis revealed differences between the species concerning the eye structures, with a more spherical eye shape for S. scincus, as well as a more flattened lens. The relative lens diameter of both species seems to correspond to a rather photopic pattern. Beside the fact that S. scincus and E. schneideri have different lifestyles, their visual capacities seem similar, and, generally speaking, these two psammophilic species theoretically exhibit visual capacities not far away from non-fossorial species., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Thyroid cancers are the most frequent endocrine cancers and their incidence is increasing worldwide. Thyroid nodules occur in over 19-68% of the population, but only 7-15% of them are diagnosed as malignant. Diagnosis relies on a fine needle aspiration biopsy, which is often inconclusive and about 90% of thyroidectomies are performed for benign lesions. Galectin-1 has been proposed as a confident biomarker for the discrimination of malignant from benign nodules. We previously identified by phage display two peptides (P1 and P7) targeting galectin-1, with the goal of developing imaging probes for non-invasive diagnosis of thyroid cancer. The peptides were coupled to ultra-small superparamagnetic particles of iron oxide (USPIO) or to a near-infrared dye (CF770) for non-invasive detection of galectin-1 expression in a mouse model of papillary thyroid cancer (PTC, as the most frequent one) by magnetic resonance imaging and fluorescence lifetime imaging. The imaging probes functionalized with the two peptides presented comparable image enhancement characteristics. However, those coupled to P7 were more favorable, and showed decreased retention by the liver and spleen (known for their galectin-1 expression) and high sensitivity (75%) and specificity (100%) of PTC detection, which confirm the aptitude of this peptide to discriminate human malignant from benign nodules (80% sensitivity, 100% specificity) previously observed by immunohistochemistry.

Titanium dioxide (TiO 2 ) nanoparticles (NPs) are produced abundantly and are frequently used as a white pigment in the manufacture of paints, foods, paper, and toothpaste. Despite the wide ranges of uses, there is a lack of information on the impact of NPs on animal and human health. In the present study, rats were exposed to different doses of TiO 2 nanoparticles and sacrificed, respectively, 4 days, 1 month, and 2 months after treatment. Dosage of TiO 2 in tissues was performed by ICP-AES and revealed an important accumulation of TiO 2 in the liver. The nanoparticles induced morphological and physiological alterations in liver and kidney. In the liver, these alterations mainly affect the hepatocytes located around the centrilobular veins. These cells were the site of an oxidative stress evidenced by immunocytochemical detection of 4-hydroxynonenal (4-HNE). Kupffer cells are also the site of an important oxidative stress following the massive internalization of TiO 2 nanoparticles. Enzymatic markers of liver and kidney functions (such as AST and uric acid) are also disrupted only in animals exposed to highest doses. The metabonomic approach allowed us to detect modifications in urine samples already detectable after 4 days in animals treated at the lowest dose. This metabonomic pattern testifies an oxidative stress as well as renal and hepatic alterations.

Nowadays, nanoparticles (NPs) of titanium dioxide (TiO 2 ) are abundantly produced. TiO 2 NPs are present in various food products, in paints, cosmetics, sunscreens and toothpastes. However, the toxicity of TiO 2 NPs on the central nervous system has been poorly investigated until now. The aim of this study was to evaluate the toxicity of TiO 2 NPs on the central nervous system in vitro and in vivo . In cell cultures derived from embryonic cortical brain of rats, a significant decrease in neuroblasts was observed after 24 to 96 h of incubation with TiO 2 NPs (5 to 20 μg/ml). This phenomenon resulted from an inhibition of neuroblast proliferation and a concomitant increase in apoptosis. In the same time, a gliosis, characterized by an increase in proliferation of astrocytes and the hypertrophy of microglial cells, occurred. The phagocytosis of TiO 2 NPs by microgliocytes was also observed. In vivo , after intraperitoneal injection, the TiO 2 NPs reached the brain through the blood brain barrier and the nanoparticles promoted various histological injuries such as cellular lysis, neuronal apoptosis, and inflammation. A reduction of astrocyte population was observed in some brain area such as plexiform zone, cerebellum and subependymal area. An oxidative stress was also detected by immunohistochemistry in neurons of hippocampus, cerebellum and in subependymal area. In conclusion, our study demonstrated clearly the toxic impact of TiO 2 NPs on rat brain and neuronal cells and pointed about not yet referenced toxicity impacts of TiO2 such as the reduction of neuroblast proliferation both in vitro and in vivo .

The diagnosis of Alzheimer's disease (AD) is a critical step in the management of patients. We have developed a non-invasive diagnosis tool based on magnetic resonance molecular imaging (MRMI) of amyloid-β peptide using ultra-small particles of iron oxide (USPIO) functionalized with a disulfide constrained cyclic heptapeptide (PHO) identified by phage display (USPIO-PHO). After previously demonstrating the optimal pharmacologic properties of USPIO-PHO and its capacity to cross the blood-brain barrier (BBB), the ability of USPIO-PHO to target amyloid plaques (AP) by MRMI has been validated in the present work on AD transgenic mice. The immunohistochemistry and immunofluorescent detection of USPIO-PHO on brain sections collected after in vivo MRMI studies enabled its colocalization with AP, confirming the BBB passage and specific targeting. The AP targeting by USPIO-PHO has been moreover corroborated by the good correlation between the number of AP detected with anti-amyloid β antibody and Perls'-DAB staining. Finally, the crossing mechanism of USPIO-PHO through the BBB was elucidated, revealing the involvement of non-degradation pathway of caveolae, while the control contrast agent USPIO-PEG was not endocytosed by the human brain endothelial cells.

Alzheimer's disease (AD) is a neurodegenerative disorder most often diagnosed 10 years after its onset and development. It is characterized by the accumulation of amyloid-β peptide (ABP) into amyloid plaques between nerve cells, which produces a massive local neurodegeneration. Molecular magnetic resonance imaging allows diagnosis of AD by showing ABP accumulation in the brain. The ultrasmall particles of iron oxide (USPIO) derivatives proposed in the present work were functionalized with peptides that present an affinity for ABP, independently of its state of aggregation. Their nanomolar Kd * confirms the high affinity of our vectorized contrast agents (VCA) for ABP and therefore their high labeling potential, specificity and sensitivity. Their lack of toxicity has been demonstrated, both by in vitro studies using the MTT method on several cell types, and by in vivo investigations with assessment of renal and hepatic biomarkers and by histopathology evaluation. The results of biodistribution studies corroborated by MRI demonstrate that USPIO-PHO (USPIO coupled to peptide C-IPLPFYN-C) are able to cross the blood-brain barrier without any facilitating strategy, and accumulates in the brain 90 min after its injection in NMRI mice. None of the USPIO derivatives were found in any organs one week after administration. To conclude, USPIO-PHO seems to have a genuine potential for labeling amyloid plaques in the brain; it has a nanomolar binding affinity, no toxic effects, and its elimination half-life is about 3 h. Further tests will be made on transgenic mice, aimed at confirming the potential of early AD diagnosis using our VCA., (Copyright © 2014 John Wiley & Sons, Ltd.)

Idiopathic pulmonary fibrosis (IPF) is a chronic disease with a poor prognosis and is characterized by the accumulation of fibrotic tissue in lungs resulting from a dysfunction in the healing process. In humans, the pathological process is patchy and temporally heterogeneous and the exact mechanisms remain poorly understood. Different animal models were thus developed. Among these, intratracheal administration of bleomycin (BML) is one of the most frequently used methods to induce lung fibrosis in rodents. In the present study, we first characterized histologically the time-course of lung alteration in rats submitted to BLM instillation. Heterogeneous damages were observed among lungs, consisting in an inflammatory phase at early time-points. It was followed by a transition to a fibrotic state characterized by an increased myofibroblast number and collagen accumulation. We then compared instillation and aerosolization routes of BLM administration. The fibrotic process was studied in each pulmonary lobe using a modified Ashcroft scale. The two quantification methods were confronted and the interobserver variability evaluated. Both methods induced fibrosis development as demonstrated by a similar progression of the highest modified Ashcroft score. However, we highlighted that aerosolization allows a more homogeneous distribution of lesions among lungs, with a persistence of higher grade damages upon time.

The Wistar-Furth (WF) rat strain is usually used in models of full major histocompatibility complex-mismatched kidney transplantation. Because these rats have been demonstrated to be resistant to several models of chronic kidney disease, the aim of the present study was to investigate their potential resistance to renal ischaemia-reperfusion (I/R) injury compared with another strain, namely Wistar-Hanover (WH) rats. Anaesthetized male WH and WF rats were submitted to I/R by occlusion of the left renal artery and contralateral nephrectomy. Urine, blood and tissue samples were collected at different time points after I/R to evaluate renal function, inflammation and tubular injury, along with determination of nitric oxide synthase (NOS) expression and thromboxane A2 (TxA2 ) production. Post-ischaemic renal function was better preserved in WF than WH rats, as evidenced by reduced levels of creatininaemia, urinary neutrophil gelatinase-associated lipocalin excretion and proteinuria. In addition, WF rats had less intrarenal inflammation than WH rats after I/R injury. These observations were associated with maintenance of neuronal NOS expression, along with lower induction of inducible NOS expression in WF versus WH rats. Moreover, WF rats excreted a significantly lower amount of TxB2 . The results indicate that WF rats are more resistant to an I/R injury than WH rats in terms of renal function and inflammation. These observations are associated with differential regulation of intrarenal NOS expression, as well as a reduction in thromboxane production, which could contribute to a better outcome for the postischaemic kidney in WF rats., (© 2014 Wiley Publishing Asia Pty Ltd.)

Agamid lizards use tongue prehension for capturing all types of prey. The purpose of this study was to investigate the functional relationship between tongue structure, both surface and musculature, and function during prey capture in Pogona vitticeps. The lack of a detailed description of the distribution of fibre-types in the tongue muscles in some iguanian lizards has hindered the understanding of the functional morphology of the lizard tongue. Three methodological approaches were used to fill this gap. First, morphological analyses were performed (i) on the tongue surface through scanning electron microscopy, and (ii) on the lingual muscle by histological coloration and histochemistry to identify fibre-typing. Secondly, kinematics of prey capture was quantified by using high-speed video recordings to determine the movement capabilities of the tongue. Finally, electromyography (EMG) was used to identify the motor pattern tongue muscles during prey capture. Morphological and functional data were combined to discuss the functional morphology of the tongue in agamid lizards, in relation to their diet. During tongue protraction, M. genioglossus contracts 420 ± 96 ms before tongue-prey contact. Subsequently, Mm. verticalis and hyoglossus contract throughout tongue protraction and retraction. Significant differences are found between the timing of activity of the protractor muscles between omnivorous agamids (Pogona sp., this study) and insectivorous species (Agama sp.), despite similar tongue and jaw kinematics. The data confirm that specialisation toward a diet which includes more vegetal materials is associated with significant changes in tongue morphology and function. Histoenzymology demonstrates that protractor and retractor muscles differ in fibre composition. The proportion of fast glycolytic fibres is significantly higher in the M. hyoglossus (retractor muscle) than in the M. genioglossus (protractor muscle), and this difference is proposed to be associated with differences in the velocity of tongue protrusion and retraction (5 ± 5 and 40 ± 13 cm s(-1) , respectively), similar to Chamaeleonidae. This study provides a way to compare fibre-types and composition in all iguanian and scleroglossan lizards that use tongue prehension to catch prey., (© 2014 Anatomical Society.)

Purpose: While macrophage migration inhibitory factor (MIF) has been extensively studied in the context of inflammation and inflammatory disorders, less work has been devoted to its involvement in cancer, notably in neoplastic progression. In a previous study, we have found evidence that MIF plays a role in head and neck squamous cell carcinomas (HNSCC). The current investigations were undertaken in order to estimate the importance of the MIF receptor, CD74 in the progression of HNSCC., Methods and Results: In a cohort of 46 cases of oral cavity carcinomas, immunohistochemical staining revealed an increase in CD74 expression during progression from benign lesions to carcinoma. As shown by cell culture experiments using squamous carcinoma cell line (SCCVII) transduced with anti-CD74 shRNA, the amount of cell-produced VEGF was lower in SCCVII CD74KD cell line compared with control SCCVII CD74sc cell line, suggesting that CD74 could be implicated in angiogenesis in vivo. Furthermore, knockdown of CD74 decreased proliferation of SCCVII cells in vitro. The migration of SCCVII cells, as well as the cell secretion of matrix metallopeptidase 9, was also negatively affected by CD74 knockdown. These observations in vitro were confirmed in an orthotopic mouse model of SCC where tumors produced by SCCVII CD74KD cell inoculation were found to grow more slowly than tumors generated by SCCVII CD74sc cells., Conclusion: The clinical observations and experimental data reported here suggest that CD74, as well as MIF, plays a pivotal role in HNSCC progression.

Recent clinical observations and experimental studies of our group indicate that macrophage migration inhibitory factor (MIF) may contribute to tumor progression in head and neck squamous cell carcinomas (HNSCC). The present study was undertaken to examine the effects of the irreversible MIF inhibitor 4-iodo-6-phenylpyrimidine (4-IPP) on proliferation and invasiveness of the squamous carcinoma cell line SCCVII. Cell counting, crystal violet assay and flow cytometry were used to analyze the effects of 4-IPP on SCCVII cell growth. The impact of 4-IPP on cell invasiveness was assessed by Boyden chamber assay. Knockdown of the MIF receptor CD74 was achieved by transduction with lentiviral vectors encoding anti-CD74 shRNAs. As shown by immunofluorescence staining, SCCVII cells express both MIF and CD74. Decreased MIF immunoreactivity as a result of exposure to 4-IPP suggested a covalent modification of the cytokine. 4-IPP inhibited SCCVII cell proliferation and invasiveness. Moreover, the cytostatic effect of 4-IPP was enhanced by CD74 knockdown. The inhibitory effects of 4-IPP on cell proliferation and invasiveness strongly suggest that MIF is involved in proliferative activity and invasive properties of squamous carcinoma cells. In conclusion, MIF inhibition may open possibilities for target-directed treatment of head and neck squamous cell carcinoma.

Aims: We used a rat model of renal ischemia (35 min) to test the potential involvement of platelet/endothelial cell adhesion molecule 1 (PECAM-1/CD31) in the process of S3 tubule regeneration., Methods: A monoclonal antibody specific for murine PECAM-1 was injected i.p. immediately after kidney reperfusion or 48 h post-ischemia. One day before ischemia, each animal received an i.p. injection of 80 mg/kg 5-bromo-2'-deoxyuridine (BrdU). Experimental animals were sacrificed 1, 2, 3, 7 and 14 days post-ischemia. Renal sections were processed to characterize the histopathological alterations and the distribution of BrdU-immunopositive cells., Results: Our observations showed that anti-PECAM-1 administration was associated with an inhibition of S3 tubule regeneration along with a progressive cystic dilatation of renal tubules that was particularly prominent 2 weeks post-ischemia. Interestingly, injection of anti-PECAM-1 48 h post-ischemia failed to block renal regeneration and was followed by a normal re-epithelialization of S3 tubules., Conclusion: Our data showed that the blockade of PECAM-1 immediately after kidney reperfusion inhibits tubular regeneration. These observations suggest that transendothelial migration of extrarenal cells could be a precocious and pivotal step in kidney reparation, but also suggest that these extrarenal cells could be essential to the process of tubular regeneration., (Copyright © 2011 S. Karger AG, Basel.)

Estrogen receptor alpha (ERalpha) belongs to the superfamily of nuclear receptors and as such acts as a ligand-modulated transcription factor. Ligands elicit in ERalpha conformational changes leading to the recruitment of coactivators required for the transactivation of target genes via cognate response elements. In many cells, activated ERalpha also undergoes downregulation by proteolysis mediated by the ubiquitin/proteasome system. Although these various molecular processes have been well characterized, little is known as to which extent they are interrelated. In the present study, we used a panel of type I (estradiol derivatives and "linear", non-steroidal ligands) and type II ("angular" ligands) estrogens, in order to identify possible relationships between ligand binding affinity, recruitment of LxxLL-containing coactivators, ERalpha downregulation in MCF-7 cells and related transactivation activity of ligand-bound ERalpha. For type I estrogens, there was a clear-cut relationship between ligand binding affinity, hydrophobicity around C-11 of estradiol and ability of ERalpha to associate with LxxLL motifs, both in cell-free condition and in vivo (MCF-7 cells). Moreover, LxxLL motif recruitment by ERalpha seemed to be a prerequisite for the downregulation of the receptor. By contrast, type II ligands, as well as estradiol derivatives bearing a bulky side chain at 11beta, had much less tendency to promote ERalpha-LxxLL interaction or even behaved as antagonists in this respect, in agreement with the well known partial estrogenicity/antiestrogenicity of some of these compounds. Interestingly, some type II ligands which antagonized LxxLL motif recruitment were nonetheless able to enhance ERalpha-mediated gene transactivation., (2009 Elsevier Inc. All rights reserved.)

This work explores the distribution of various markers expressed by interstitial cells in rat kidneys after ischemic injury (35 minutes) during regeneration of S3 tubules of outer stripe of outer medulla (OSOM). Groups of experimental animals (n = 4) were sacrificed every two hours during the first 24 hours post-ischemia as well as 2, 3, 7, 14 days post-ischemia. The occurrence of lineage markers was analyzed on kidney sections by immunohistochemistry and morphometry during the process of tubular regeneration. In postischemic kidneys, interstitial cell proliferation, assessed by 5-bromo-2'-deoxyuridine (BrdU) and Proliferating Cell Nuclear Antigen (PCNA) labeling, was prominent in outer medulla and reach a maximum between 24 and 72 hours after reperfusion. This population was characterized by the coexpression of vimentin and nestin. The density of -Neural Cell Adhesion Molecule (NCAM) positive interstitial cells increased transiently (18-72 hours) in the vicinity of altered tubules. We have also localized a small population of alpha-Smooth Muscle Actin (SMA)-positive cells confined to chronically altered areas and characterized by a small proliferative index. In conclusion, we observed in the postischemic kidney a marked proliferation of interstitial cells that underwent transient phenotypical modifications. These interstitial cells could be implicated in processes leading to renal fibrosis.

The farnesoid X receptor (FXR, NR1H4), a member of the nuclear receptor superfamily of ligand-dependent transcription factors, is normally produced in the liver and the gastrointestinal tract, where it acts as a bile acid sensor. It has been recently detected in breast cancer cell lines and tissue specimens. The expression of FXR was scored (0-8) by immunohistochemistry on 204 breast cancer samples and correlated with established cancer biomarkers. Moreover, the effect of the FXR activator chenodeoxycholic acid (CDCA) was determined on cell proliferation and estrogen receptor regulation/activation in breast cancer cell lines. FXR was detected in 82.4% of samples with a high median expression score of 5. FXR expression significantly correlated with estrogen receptor (ER) expression (P = 0.009) and luminal-like markers. In ER-positive tumors, FXR expression was significantly correlated with the proliferation marker Ki-67 (P < 0.001) and the nodal status (P = 0.028), but only so in postmenopausal women, suggesting that lack of estrogens may disclose the association between FXR and cell proliferation. In vitro experiments confirmed clinical data since CDCA stimulated the proliferation of ER-positive cells only in steroid-free medium, a stimulation inhibited upon siRNA-silencing of FXR expression as well as ER blockade by antiestrogens. Moreover, co-immunoprecipitation experiments revealed that CDCA activated-FXR interacted with ER. These results suggest that ER-positive breast tumors could be stimulated to proliferate via a crosstalk between FXR and ER, particularly in a state of estrogen deprivation (menopause, aromatase inhibitors).

We describe the synthesis of an 11beta isomer 3 of the steroidal antiestrogen fulvestrant 2. Partial fluorination of the 11beta side chain in 3 leads to 4, which still shows strong antiproliferative activity on MCF-7 cells. However, unlike 2 and 3, compound 4 fails to down-regulate estrogen receptor alpha (ERalpha). This result suggests that ERalpha down-regulation is not a sine qua non condition for the antitumor activity of steroidal antiestrogens.

Farnesoid X receptor (FXR) is a metabolic nuclear receptor expressed in the liver and traditionally considered as a bile acid sensor. Yet, FXR has been recently demonstrated in other tissues and cells, such as the kidneys, the adrenals, and arterial smooth muscle cells. Immunohistochemical data reported in this study point to the expression of FXR in human breast cancer. In addition, FXR expression was also found by Western blotting and immunofluorescence microscopy in breast-cancer-derived cell lines MCF-7 (estrogen receptor [ER]-positive) and MDA-MB-231 (ER-negative). The FXR activator farnesol, a mevalonate pathway intermediate, exerts a mitogenic effect on MCF-7 cells. The growth stimulation is completely suppressed by antiestrogens. In contrast, MDA-MB-231 cells appear farnesol-insensitive, suggesting an involvement of ER in farnesol mitogenicity. In accordance with this interpretation, farnesol induces in MCF-7 cells a decrease of ER level, consistent with a phenomenon of receptor downregulation. Farnesol also increases progesterone receptor (PgR) expression in MCF-7 cells and stimulates ER-mediated gene transactivation in MVLN cells (MCF-7 cells stably transfected with an ER reporter gene). Of note, both effects of farnesol on ER expression and activity are completely suppressed by antiestrogens. In addition, farnesol-induced PgR is markedly reduced by FXR gene silencing (siRNA), demonstrating the involvement of FXR in the estrogenic effects of farnesol. Finally, coimmunoprecipitation experiments (FXR immunoprecipitation followed by Western blot analysis of ER in the immunoprecipitate) produced definite evidence that FXR interacts with ER. Altogether, these observations reveal the hitherto unreported presence of FXR in breast cancer and show that the latter receptor functionally interacts with ER. The occurrence of such a crosstalk calls for some caution regarding the pharmacological use of FXR agonists.

4,5-Diaryl-2-imidazolines (Im(s)) and 2,3-diarylpiperazines (Pip(s)) belong to the type II class of estrogens. These compounds enhance ERalpha-mediated transcription of ERE-driven reporter genes in MCF-7 cells but do not compete with [(3)H]estradiol (E(2)) for receptor binding, because of distinct anchoring modes. The present study examined whether the estrogenic action of Im(s) and Pip(s) is associated with a down regulation of ERalpha, as reported for conventional agonists. Im and Pip derivatives displaying a large spectrum of activities in three distinct ERE-dependent transactivation systems were selected for that purpose. ERalpha immunostaining as well as Western blotting analysis revealed that both classes of compounds down regulated ERalpha with an efficiency closely related to their transactivation potency. MG-132 abrogated this down regulation, pointing to a proteasomal degradation process. Im(s) and Pip(s) with strong transactivation potency also altered [(3)H]E(2) binding parameters, leading to a progressive decrease of cellular estrogen binding capacity. This property occurred largely before ERalpha down regulation and persisted even in presence of MG-132, indicating that it did not result from ERalpha breakdown but rather from a conformational change of the receptor. The additional finding that the most active agonist tested in this study enhanced the capacity of a purified ERalpha recombinant to recruit LxxLL co-activators, while its inactive counterpart failed to do so confirmed this hypothesis. Altogether, our data indicate that the association of Im(s) and Pip(s) with ERalpha elicits similar responses to conventional agonists, even if they interact with distinct residues of the binding pocket.

Calmodulin (CaM) contributes to estrogen receptor alpha (ER)-mediated transcription. In order to study the underlying mechanisms, we synthesized a peptide including the CaM binding site: ERalpha17p (P(295)-T(311)). This peptide inhibited ER-CaM association, unlike two analogs in which two amino acids required for CaM binding were substituted. Exposure of MCF-7 cells to ERalpha17p down regulated ER, stimulated ER-dependent transcription and enhanced the proliferation of ER-positive breast cancer cell lines. Interestingly, ERalpha17p analogs unable to bind to CaM induced similar responses, demonstrating that ERalpha17p-mediated effects are mainly relevant to mechanisms independent of ER-CaM dissociation. The P(295)-T(311) motif is indeed a platform for multiple post-translational modifications not necessarily CaM-dependent. The additional finding that deletion of the P(295)-T(311) sequence in ER produced a constitutive transcriptional activity revealed that this platform motif has autorepressive functions. With regard to cell function, association of CaM to ER would counteract this autorepression, leading thereby to enhanced ER-mediated transactivation.

A number of coumarins exhibit interesting pharmacological activities and are therefore of therapeutic use. We report here the synthesis and the structural analysis of new N-substituted 4-amino-3-(2-methylbenzyl)coumarins (compounds 8a-8e) that present structural analogies with estrothiazine and 11- or 7-substituted 17beta-estradiol. These derivatives were tested with respect to estrogenic activity on the estrogen receptor positive (ER+) human MCF-7 breast cancer cell line. Two of the reported compounds (8a and 8b) stimulated specifically the proliferation of MCF-7 cells, but not that of estrogen receptor negative (ER-) human MDA-MB-231 breast cancer cells, suggesting that their mitogenic activity is mediated by ER. Accordingly, the stimulating effect of 8a and 8b was suppressed by the pure antiestrogen fulvestrant. Besides, 8a and 8b induced ER down-regulation similar to that produced by classical ER agonists or pure antagonists. The effects of the compounds under study on ER-mediated transcription were assessed on (ER+) MVLN cells, that is, MCF-7 cells stably transfected with a pVit-tk-Luc reporter plasmid. Derivatives 8a and 8b, and surprisingly compound 8c, enhanced ER-mediated gene transactivation in that model. Finally, no coumarin was able to compete with tritiated 17beta-estradiol ([(3)H]E(2)) for ER binding, suggesting unconventional interactions with the receptor, such as interactions with the second binding pocket or with the coactivator-binding region. To conclude, observations performed in this study on compound 8c reveal that estrogenic activity can be dissociated from enhancement of cell proliferation. Furthermore, ERE-driven transactivation of transcription seems to be a condition necessary, but not sufficient, for estrogen-induced stimulation of cell growth.

Ischemia/reperfusion (I/R) injury in the kidney involves hemodynamic and cellular dysfunctions as well as leukocyte infiltration. Functional recovery occurs via cell proliferation and/or migration. To determine the roles of hyaluronan (HA) and its main receptor CD44 in renal postischemic processes, we compared their localization and expression with that of neutrophils, macrophages, and PCNA-positive (regenerative) cells as characterized by immunohistochemistry, up to 28 days after I/R in uninephrectomized rats. Observations covered all kidney zones, i.e. cortex (C), outer and inner stripes of outer medulla (OSOM, ISOM), and inner medulla (IM). In controls, HA was localized to the interstitium of IM and ISOM, and CD44 was mostly present on the basolateral membranes of collecting ducts in ISOM, the thin descending limb of Henle's loop and macula densa cells. After I/R, HA and CD44 staining appeared in C and OSOM at 12 h and persisted throughout the regenerative period, i.e. until day 7. Thereafter, they regressed but remained associated with remodeling areas. CD44 expression was found de novo on the apical pole of regenerating, not fully differentiated tubular cells and on some interstitial cells. It was prominent on all infiltrating neutrophils, as soon as 2 h post-I/R, and on 30% of the macrophages, including those in late HA-rich inflammatory granulomas. CD44 is probably involved in early leukocyte infiltration, in tubular regeneration, and in macrophage activity, while HA modifies the physico-chemical environment of interstitial and migrating cells. Based on its presence in remodeling areas, the HA-CD44 pair may be implicated in persistent postichemic inflammation as observed in chronic allograft nephropathy.

Endogenous lectins act as effectors of cellular activities such as growth regulation, migration, and adhesion. Following their immunohistochemical localization in our previous study (Saussez et al. in Histochem Cell Biol 123:29-41, 2005) we purified several galectins and used them as tools for monitoring accessible binding sites. Herein, we report the use of galectin histochemistry for the analysis of diethylstilbestrol (DES)-induced renal tumors in male Syrian hamster kidney (SHKT). Sections of normal kidney and DES-treated kidney were analyzed with biotinylated galectins-1, -3 (full-length and truncated), and -7. Accessible binding sites were detected, localization was predominantly extracellular and confined to medium-sized and large tumors. Monitoring the SHKT-derived HKT-1097 line, processed in vitro or as xenograft material, cytoplasmic and nuclear staining for galectins-1, -3, and -3tr could be observed. Adaptation of SHKT cells to long-term growth in culture is thus associated with emergence of this signal. Our data set illustrates the feasibility to complement immunohistochemical data by application of the tissue lectins as probes, and to detect regulation of galectin reactivity with differential characteristics within tumor progression in vivo and unique features of the tumor cell line in vitro and in vivo.

Background: The Helicase-Like Transcription Factor (HLTF/SMARCA3) belongs to the family of SWI/SNF proteins that use the energy of ATP hydrolysis to remodel chromatin in a variety of cellular processes. Several SWI/SNF genes are disrupted in cancer, suggesting a role of tumor suppressor. Similarly, the HLTF gene was recently found to be inactivated by hypermethylation in a number of advanced colon and gastric tumors. However, other evidences indicated a 20-fold HLTF overexpression in cell lines derived from various neoplasms (ovary, breast, cervix, kidney...)., Results: In the present study, we investigated HLTF expression by immunohistochemistry in a model of kidney tumors induced by continuous administration of diethylstilbestrol to male Syrian golden hamsters. A strong labeling was already detected in small tumor buds, making HLTF an early cancer marker in this model. Although every cell stained for HLTF at this early stage, the number of HLTF-positive cells decreased to 10% with cancer progression, and these positive cells were dispersed in the tumor mass. HLTF expression was conserved in the HKT-1097 cell line established from kidney tumors, but again only 10% of positive cells were found in xenografts produced by HKT-1097 cells in nude mice., Conclusion: In conclusion, our data suggest that HLTF gene activation is linked to initial steps of carcinogenesis in this model and should be investigated in early stages of other neoplasms.

1. The renin-angiotensin system may be involved in the compensatory adaptations occurring after the reduction of renal mass and during the consecutive changes leading to chronic renal failure. We therefore investigated the regulation of angiotensin II receptors in two models of renal hypertrophy in the rat: hypertrophy following uninephrectomy (UNx) or subtotal nephrectomy (STNx). The level of angiotensin type 1 (AT1A-R and AT1B-R) and type 2 (AT2-R) receptor mRNA was quantified by competitive reverse transcription-polymerase chain reaction (RT-PCR) in specific renal zones and the intrarenal distribution of angiotensin II receptors was analysed by immunohistochemistry. 2. In the UNx rats, AT1-R mRNA expression was not modified in the cortex or in the inner stripe of the outer medulla of the residual kidney at any time after the surgery (1, 4 and 12 weeks). In contrast, AT1-R mRNA expression was significantly reduced in these zones in STNx rats (-33% and -40%, respectively). This downregulation was organ-specific, as AT1-R mRNA levels were not modified in the liver. The proportions of AT1-R subtype (AT1A and AT1B) mRNA were unchanged by UNx or STNx. Very low levels of AT2-R mRNA were found in the cortex of all groups. Immunostaining revealed a similar localization of AT1-R in mesangial cells, proximal tubule, basolateral membrane of thick ascending limb, in both models of hypertrophy. AT1-R labelling was also detected in the apical membrane of intercalated cells of cortical collecting ducts. 3. This differential mRNA expression of angiotensin II receptors during compensatory hypertrophy and renal injury suggests that the development of renal hypertrophy is independent of AT1-R and AT2-R gene expression levels.

Estrogen receptor alpha (ER) turnover in MCF-7 cells was assessed by pulse chase analysis and measurement of ER steady-state level. In untreated cells, degradation of (35)S-labeled ER was characterized by a slow phase followed by a more rapid decline. Without ligand, ER elimination was totally compensated by synthesis which maintained receptor homeostasis. Estradiol (E(2)) and the pure antiestrogen RU 58,668 abolished the slow phase of ER breakdown and enhanced the degradation of neosynthesized ER, producing a low ER steady-state level. By contrast, the partial antiestrogen OH-Tam was ineffective in this respect and caused ER accumulation. Regardless of the conditions, ER breakdown was abolished by proteasome inhibition (MG-132). ER ligands decreased cell capacity to bind [(3)H]E(2), even in the presence of MG-132, indicating that the regulation of ER level and E(2) binding capacity occurs through distinct mechanisms. MG-132 partially blocked the basal transcription of an ERE-dependent reporter gene and modified the ability of E(2) to induce the expression of the latter: the hormone was unable to restore the transactivation activity measured without MG-132. RU 58,668 and OH-Tam failed to enhance the inhibitory action of MG-132, suggesting that a loss of basal ER-mediated transactivation mainly affects the stimulatory effect of estrogens. Overall, our findings reveal that ER steady state level, ligand binding capacity and transactivation potency fit in a complex regulatory scheme involving distinct mechanisms, which may be dissociated from each other under various treatments.

The current study focused on galectins (-1, -3, -4, -7, and -8) and deliberately performed immunohistochemical fingerprinting to explore their complexity in a context of experimental renal carcinogenesis. The diethylstilbestrol (DES)-induced renal tumors in male Syrian hamster kidney (SHKT) represent a unique animal model for the study of estrogen-dependent renal malignancies. Kidney sections of DES-treated hamsters (3 days to 11 months of DES exposure) were analyzed by immunohistochemistry using a panel of non-crossreactive antibodies raised against galectins-1, -3, -4, -7, and -8. Levels of expression were quantitatively determined by using computer-assisted microscopy on immunostained tissue sections. Except for galectin-4, all above mentioned galectins were expressed in kidney tumors. Small clusters of galectin-1-positive, most likely preneoplastic cells at the corticomedullary junction were already evident 1 week after DES administration. Galectin-1 and -3 expression was apparently associated with the first steps of the neoplastic transformation, because small tumorous buds were found to be positive after 1 month of treatment. In contrast, galectins-7 and -8 were detected in large tumors and medium-sized tumors, respectively, thereby indicating an involvement in later stages of DES-induced SHKT. Galectins-1, -3, -7, and -8 were also detected by immunofluorescence staining in the HKT-1097 cell line established from SHKT, thus illustrating the stability of galectin expression in tumor cells. Our data document the presence and differential regulation of galectins in the course of renal tumorigenesis in the model of DES-induced SHKT.

Molecular chaperones and co-chaperones, such as heat-shock proteins (Hsp's), play a pivotal role in the adequate folding and the stability of steroid hormone receptors. As shown by immunofluorescence staining and immunoblot analysis, the Hsp90 inhibitor radicicol induced a rapid (within hours) depletion of estrogen receptor-alpha (ER) in MCF-7 and IBEP-2 breast carcinoma cells. Inhibition of proteasomes (MG-132, LLnL) or of protein synthesis (cycloheximide), which both suppressed E(2)-induced downregulation of ER, failed to modify ER degradation caused by radicicol. On the other hand, partial antiestrogens, such as hydroxytamoxifen (a triphenylethylene) and LY 117,018 (a benzothiophene) stabilized ER, making it immune to radicicol-induced degradation. Furthermore, radicicol did not interfere with ER upregulation induced by hydroxytamoxifen. Thus, the current study points to possible variation in the mechanism/pathway of ER breakdown. Besides, the protective effect of partial antiestrogens suggests that ER stability is only compromized by Hsp90 disruption when the receptor is in its native, unliganded form.

IBEP-2, an established cell line recently derived from breast carcinoma, was characterized with regard to estrogen receptor (ER) expression, cell mitogenic response to estrogenic stimulation and sensitivity to antiestrogens. In addition, we examined ER modulation following binding of agonist and antagonists, and the ER-mediated induction of progesterone receptor (PgR). ER level in IBEP-2 cells, determined by enzyme-linked immunoassay (EIA), was slightly higher than that measured in MCF-7 cells (662 v.s. 595 fmol/mg protein). When tested on IBEP-2 and MCF-7, various agonists stimulated cell growth with EC50's reflecting different estrogenic potencies (E(2) approximately diethylstilbestrol > E(1) > genistein). IBEP-2 appeared slightly more sensitive than MCF-7, especially to E(2) (at least 4-fold difference between EC50 values). By contrast, IBEP-2 and MCF-7 were equally sensitive to the growth inhibitory effect of antiestrogens 4-hydroxy-tamoxifen (OH-Tam) and ICI 182,780. As revealed by immunoblotting and immunofluorescence using anti-ER alpha antibodies, ER expression in IBEP-2 cells was modulated by E(2) and estrogen antagonists like it has been shown in other ER-positive cell lines, that is, E(2) and ICI 182,780 caused ER downregulation, whereas OH-Tam induced ER accumulation. Ligand-induced downregulation of ER involved degradation in proteasomes, since it was suppressed by the proteasome inhibitor MG-132. Exposure of IBEP-2 cells to E(2) resulted in a marked (at least 25-fold) induction of PgR, documented by EIA, immunoblotting and immunofluorescence. PgR induction due to E(2) was not modified by MG-132. Interestingly, MG-132 alone produced an ER-independent increase of PgR expression. IBEP-2 might prove to be valuable to study ER-mediated induction of PgR.

This study aimed at a better understanding of estrogen receptor alpha (ER) up regulation induced by partial estrogen antagonists. Effect of treatment with hydroxytamoxifen (OH-Tam) on ER level in MCF-7 cells was investigated by an approach combining ER measurement (enzyme immunoassay) and morphological demonstration (immunofluorescence). Furthermore, the influence of drug exposure on the rates of ER synthesis and degradation was assessed by determining [35S]methionine incorporated into the receptor in different experimental conditions (measurement of synthesis or pulse-chase experiments). ER up regulation was already induced by a 1-h pulse treatment with OH-Tam, thus a continuous exposure was not required. This process appeared reversible (i.e. ER accumulation due to OH-Tam rapidly vanished upon subsequent exposure to 17beta-estradiol (E2) or the pure antiestrogen RU 58668). While OH-Tam did not affect the rate of [35S]methionine incorporation into ER, it clearly caused an impairment of ER degradation (pulse-chase experiments) indicating that up regulation results from a stabilization of the receptor associated with the maintenance of its synthesis. Various tamoxifen derivatives, as well as a few related partial antiestrogens, were compared on the basis of binding ability and propensity to induce ER up regulation. A close relationship was found between both properties. Structure-activity analysis revealed that the capacity of these compounds to induce ER up regulation is associated with characteristics of their aminoalkyle side-chain, similar to those required for antiestrogenicity.

In the vestibular nuclei of the awake guinea pig, all neurones are spontaneously active. After unilateral labyrinthectomy, this activity virtually disappears on the ipsilateral side, but is completely restored one week later. In a recent study, we observed that the restoration of spontaneous activity was correlated with an increase in pacemaker activity. In the current study, we found that the ratio of medial vestibular nucleus (MVN) neurones endowed with one of the currents known to play a role in pacemaker activity (i.e. low-threshold calcium current; LTCC) increased from 29% in control guinea pigs to 65% in animals labyrinthectomised on the ipsilateral side one week earlier. Yet this change was not correlated with a modification of the ratio of neurones expressing any of the three related protein-channels (alpha1G, alpha1H and alpha1I).

This study is a first step in an attempt to identify the factors which determine and maintain the electrophysiological phenotype(s) of mature neurons of the medial vestibular nucleus (MVN). We cultured MVN microexplants obtained from slices of the brainstem of newborn rats, using a hollow punching needle. The electrophysiological maturation of the neurons was followed by analyzing their responses to 1 s steps of current of increasing amplitude. The maximal number of spikes that was generated in response to such stimuli increased dramatically over time in vitro. However, even after 28 days in vitro, it did not exceed about 60 spikes/s. At this stage of culture, the input-output properties of the spike generator of the MVN neurons were similar to those observed in brainstem slices of newborn rats, but clearly inferior to those of adult neurons which can generate sustained firing up to 150-200 spikes/s., (Copyright 2002 Elsevier Science Ireland Ltd.)

Linkage of a long CH(2 )side chain ('spacer') onto C-7 alpha of estradiol-17 beta (E(2)) does not abrogate the binding affinity of this hormone for its receptor. Our purpose was to assess whether the linkage of a CF(2 )side chain, which is more bulky and rigid, could also be accommodated by the estrogen receptor (ER). We describe here the synthesis of 7 alpha-perfluorohexylestradiol 7 by perfluoroalkylation of a key silylenolether 2 with FITS-6 (perfluorohexyl-phenyliodoniurn trifluoromethanesulfonate). 7 alpha-Trifluoromethylestradiol 10a was prepared as a fluorinated control compound by UV-light induced trifluoromethylation of 2 with Umemoto reagent (S-trifluoromethyldibenzo[b,d]thiophenium trifluoromethanesulfonate). Endocrine properties of these two E(2 )derivatives were tested on the MCF-7 breast cancer cell line. Our data reveal that rigidity of the side chain of 7 affected the association of its hormone moiety with the ER to the same extent as a long alkyl side chain. Rigidity also failed to abrogate estrogenicity, as demonstrated by the ability of 7 to enhance ERE-dependent transcription and cell growth. Compound retained the capacity of inducing down regulation of the receptor. Interestingly, no evidence of antiestrogenicity was recorded since this compound behaved like a weak estrogen, exerting a mitogenic effect at high concentration. Of note, control 10a displayed a higher binding affinity than 7 for ER and consequently acted like the latter, albeit with a higher efficiency. Selection of appropriate residues to be linked at the end of a long 7 alpha alkyl side chain is known to be essential for generating strong antiestrogenicity. One may hope that such a property may also hold for perfluorinated chains to produce antiestrogens with strong metabolic stability.

This work explores the phenotypic changes affecting transformed cells in an experimental model of diethylstilbestrol (DES)-induced renal tumours in male Syrian hamster. This estrogen-induced neoplasm presents an important cytological pleomorphism and its origin remains largely controversial. In order to characterize phenotypic variations during tumour progression, the occurrence of seven lineage markers was analysed by a morphometric approach in kidney sections of DES-exposed hamsters (6-11 months). S100 protein, neuron-specific enolase (NSE) and vimentin are expressed by a large percentage of malignant cells during tumour development. Glial fibrillary acidic protein (GFAP), protein gene product 9.5 (PGP 9.5) and desmin are mostly evidenced in advanced neoplasm whereas Leu 7 always presents a focal expression. As evidenced by double-label immunofluorescence, the coexpression of three important neuroectodermal lineage markers (S100, NSE and PGP 9.5) in earliest tumour buds points to a peripheral nerve sheath origin for this neoplasm thus confirming previously published data. For each marker, the fluctuations of expression levels during tumour progression as well as the spatial heterogeneity of distribution suggest variable phenotypic differentiation of transformed cell populations. This observation is largely corroborated by double-label immunofluorescence showing coexpression modification of several markers during tumour progression. This points to a complex dynamic and spatial self-organization of different phenotypes within neoplasms. Altogether, these results support the concept that DES-induced kidney tumours are not made of unstructured cell populations but represent adaptive complex dynamic biosystems.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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)

The metanephric mesenchyme is considered a homogeneous population of predetermined, but pluripotent cells with a nephrogenic bias. By an inductive stimulus, the mesenchyme is programmed to differentiate into the various epithelial phenotypes of the secretory nephron. A fraction of the mesenchymal cells, however, remains in the interstitium between the nephrons and differentiates into spindle-shaped, clear-cytoplasmic renal stroma. We have analyzed the molecular nature of these cells in order to discover the specific cell types that could be involved in the morphogenetic processes during kidney differentiation. In situ hybridization reveals neurofilament light protein mRNA, and immunohistology shows neurofilament light and medium proteins in the stromal cells around kidney tubules. By immunohistochemistry these peritubular stromal cells can be distinguished from the neuronal cells of the renal microganglion: the peritubular stromal cells are neurofilament-positive but L1 neural cell adhesion protein-negative, whereas the neuronal cells with axonal extension are both neurofilament-positive and L1 neural cell adhesion protein-positive. Proliferation index of the stromal cells was low as compared to tubular cells, as shown by bromodeoxyuridine incorporation.

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.

Cisplatin, a widely used chemotherapeutic agent, is characterized by a dose-limiting renal toxicity. Cystic tubular dilatation is the most typical histopathological alteration encountered in cisplatin-treated rats. The purpose of the present study was to explore by a morphometric approach the development of cystic degeneration and, in particular, to analyse, by computer-assisted tridimensional reconstructions, the spatial structure and the tubular origin of cisplatin-induced renal cysts. This study was performed on rats given 8 mg/kg cisplatin i.p. for four days and sacrificed 4, 7, 14, 21, 50 and 60 days after last drug administration. The relative area occupied by cystic tubules increased rapidly in the outer stripe of outer medulla (OSOM) and reached a maximum 21 days after the end of treatment. Cystic dilatations appeared later in the kidney cortex and the inner stripe of outer medulla (ISOM). The tridimensional study of cystic tubules located in OSOM confirmed previous reports indicating that they arise from proximal straight tubules and showed that cystic degeneration was not associated with atrophy or degeneration in more proximal parts of the nephron. Moreover, cystic tubules located in ISOM were found to originate from distal straight tubules and/or the loop of Henle, an observation which, to our knowledge, has not been reported so far in cisplatin-treated rats.

The present study was undertaken to analyze, at the cytological level, the intrarenal distribution of epidermal growth factor (EGF) in normal conditions and after drug-induced tubular injury. Female Sprague-Dawley rats were injected with gentamicin (50 mg/kg.day) for 4 days to induce tubular necrosis and were terminated 4 days after the last drug administration. For light microscopy, EGF immunoreactivity was demonstrated by immunogold-silver staining. In the kidneys of control rats EGF was found associated with distal tubules (cortex and outer medulla). Kidney exposure to gentamicin resulted in a drastic decrease of EGF immunoreactivity. For the electron microscopy study, immunoreactive EGF was detected on ultrathin sections by immunogold labeling. Within distal tubules epithelium of control animals, immunoreactive material was predominantly seen on the basolateral membrane, and to a much lesser extent in the cytoplasm or on the apical membrane. After treatment with gentamicin, there was a reduction of the density of gold particles observed in distal tubule cells. Interestingly, gold particles also appeared, but at a lower density, in proximal tubule cells. In these cells, EGF immunoreactivity also seemed membrane-bound and was mostly observed on (or next to) the basolateral membrane.