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As the world's population ages, Alzheimer's disease is currently the seventh most common cause of death globally; the burden is anticipated to increase, especially among middle-class and elderly persons. Artificial intelligence-based algorithms that work well in hospital environments can be used to identify Alzheimer's disease. A number of databases were searched for English- language articles published up until March 1, 2024, that examined the relationships between artificial intelligence techniques, eye movements, and Alzheimer's disease. A novel non-invasive method called eye movement analysis may be able to reflect cognitive processes and identify anomalies in Alzheimer's disease. Artificial intelligence, particularly deep learning, and machine learning, is required to enhance Alzheimer's disease detection using eye movement data. One sort of deep learning technique that shows promise is convolutional neural networks, which need further data for precise classification. Nonetheless, machine learning models showed a high degree of accuracy in this context. Artificial intelligence-driven eye movement analysis holds promise for enhancing clinical evaluations, enabling tailored treatment, and fostering the development of early and precise Alzheimer's disease diagnosis. A combination of artificial intelligence-based systems and eye movement analysis can provide a window for early and non-invasive diagnosis of Alzheimer's disease. Despite ongoing difficulties with early Alzheimer's disease detection, this presents a novel strategy that may have consequences for clinical evaluations and customized medication to improve early and accurate diagnosis., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Glyphosate (Gly) and its principal degradation product, the aminomethylphosphonic acid (AMPA) were found in soils from a riparian environment in Argentina. Sixty-five actinobacteria were isolated from these soils, rhizosphere, and plants (Festuca arundinacea and Salix fragilis). The isolate Streptomyces sp. S5 was selected to be used as bioinoculant in a greenhouse test, in which plants, actinobacteria, and their combinations were assessed to bioremediate the riparian soil. The dissipation of both compounds were estimated. All treatments dissipated similarly the Gly, reaching 87-92 % of dissipation. AMPA, dissipation of 38 % and 42 % were obtained by Salix and Festuca, respectively, while they increased to 57 % and 70 % when the actinobacterium was added to each planted system. Regarding the total dissipation, the higher efficiencies for both compounds were achieved by the non-planted soils bioaugmented with the actinobacterium, with 91 % of Gly dissipated and 56 % for AMPA. According to our study, it could be suggested which strategy could be applied depending on the bioremediation type needed. If in situ bioremediation is necessary, the combination of phytoremediation and actinobacteria bioaugmentation could be convenient. On the other hand, if ex situ bioremediation is needed, the inoculation of the soil with an actinobacterium capable to dissipate Gly and AMPA could be the more efficient and easier alternative., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Alvarez, A reports financial support was provided by National Agency for the Promotion of Scientific Research. Alvarez, A reports financial support was provided by National University of Tucuman. Giaccio, G.C.M. reports financial support was provided by National Institute of Agricultural Technology., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Biopurification systems (BPS) or biobeds are bioprophylaxis systems to prevent pesticide point-source contamination, whose efficiency relies mostly on the pesticide removal capacity of the biomixture, the majority component of a BPS. The adaptation of the components of the biomixtures to local availabilities is a key aspect to ensure the sustainability of the system. In this work, the removal of atrazine (ATZ) was evaluated in biomixtures formulated with three sugarcane by-products as alternative lignocellulosic substrates. Based on the capacity of actinobacteria to tolerate and degrade diverse pesticides, the effect of biomixtures bioaugmentation with actinobacteria was evaluated as a strategy to enhance the depuration capacity of biobeds. Also, the effect of ATZ and/or the bioaugmentation on microbial developments and enzymatic activities were studied. The biomixtures formulated with bagasse, filter cake, or harvest residue, reached pesticide removal values of 37-41% at 28 d of incubation, with t 1/2 between 37.9 ± 0.4 d and 52.3 ± 0.4 d. The bioaugmentation with Streptomyces sp. M7 accelerated the dissipation of the pesticide in the biomixtures, reducing ATZ t 1/2 3-fold regarding the controls, and achieving up to 72% of ATZ removal. Atrazine did not exert a clear effect on microbial developments, although most of the microbial counts were less in the contaminated biomixtures at the end of the assay. The bioaugmentation improved the development of the microbiota in general, specially actinobacteria and fungi, regarding the non-bioaugmented systems. The inoculation with Streptomyces sp. M7 enhanced acid phosphatase activity and/or reversed a possible effect of the pesticide over this enzymatic activity., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background and Objectives: Frontotemporal dementia (FTD) is a highly heritable disorder. The majority of genetic cases are caused by autosomal dominant pathogenic variants in the chromosome 9 open reading frame 72 ( c9orf72 ), progranulin ( GRN ), and microtubule-associated protein tau ( MAPT ) gene. As motor disorders are increasingly recognized as part of the clinical spectrum, the current study aimed to describe motor phenotypes caused by genetic FTD, quantify their temporal association, and investigate their regional association with brain atrophy., Methods: We analyzed baseline visit data of known carriers of a pathogenic variant in the c9orf72 , GRN , or MAPT gene from the Genetic Frontotemporal Dementia Initiative cohort study. Principal component analysis with varimax rotation was performed to identify motor sign clusters that were compared with respect to frequency and severity between groups. Associations with cross-sectional atrophy patterns were determined using voxel-wise regression. We applied linear mixed effects models to assess whether groups differed in the association between motor signs and estimated time to symptom onset., Results: A total of 322 pathogenic variant carriers were included in the analysis: 122 c9orf72 (79 presymptomatic), 143 GRN (112 presymptomatic), and 57 MAPT (43 presymptomatic) pathogenic variant carriers. Principal component analysis revealed 5 motor clusters, which we call progressive supranuclear palsy (PSP)-like, bulbar amyotrophic lateral sclerosis (ALS)-like, mixed/ALS-like, Parkinson disease (PD) like, and corticobasal syndrome-like motor phenotypes. There was no significant group difference in the frequency of signs of different motor phenotypes. However, mixed/ALS-like motor signs were most frequent, followed by PD-like motor signs. Although the PSP-like phenotype was associated with mesencephalic atrophy, the mixed/ALS-like phenotype was associated with motor cortex and corticospinal tract atrophy. The PD-like phenotype was associated with widespread cortical and subcortical atrophy. Estimated time to onset, genetic group and their interaction influenced motor signs. In c9orf72 pathogenic variant carriers, motor signs could be detected up to 25 years before expected symptom onset., Discussion: These results indicate the presence of multiple natural clusters of motor signs in genetic FTD, each correlated with specific atrophy patterns. Their motor severity depends on time and the affected gene. These clinicogenetic associations can guide diagnostic evaluations and the design of clinical trials for new disease-modifying and preventive treatments., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Development of an aberrant vascular network is a hallmark of the multistep pathological process of tumor growth and metastasis. In response to hypoxia, several pro-angiogenic factors are synthesized to support vascularization programs required for cancer progression. Emerging data indicate the involvement of glycans and glycan-binding proteins as critical regulators of vascular circuits in health and disease. Galectins may be regulated by hypoxic conditions and control angiogenesis in different physiopathological settings. These β-galactoside-binding proteins may promote sprouting angiogenesis by interacting with different glycosylated receptors and triggering distinct signaling pathways. Understanding the role of galectins in tumor neovascularization will contribute to the design of novel anti-angiogenic therapies aimed at complementing current anti-cancer modalities and overcoming resistance to these treatments. Here we describe selected strategies and methods used to study the role of hypoxia-regulated galectins in the regulation of blood vessel formation., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Actinobacteria represent a ubiquitous group of microorganisms widely distributed in ecosystems. They have diverse physiological and metabolic properties, including the production of extracellular enzymes and a variety of secondary bioactive metabolites, such as antibiotics, immunosuppressants, and other compounds of industrial interest. Therefore, actinobacteria have been used for biotechnological purposes for more than three decades. The development of a biotechnological process requires the evaluation of its cost/benefit ratio, including the search for economic and efficient substrates for microorganisms development. Biodiesel is a clean, renewable, quality and economically viable source of energy, which also contributes to the conservation of the environment. Crude glycerol is the main by-product of biodiesel production and has many properties, so it has a commercial value that can be used to finance the biofuel production process. Actinobacteria can use glycerol as a source of carbon and energy, either pure o crude. A circular economy system aims to eliminate waste and pollution, keep products and materials in use, and regenerate natural systems. Although these principles are not yet met, some approaches are being made in this direction; the transformation of crude glycerol by actinobacteria is a process with great potential to be scaled on an industrial level. This review discusses the reports on glycerol as a promising source of carbon and energy for obtaining biomass and high-added value products by actinobacteria. Also, the factors influencing the biomass and secondary metabolites production in bioreactors are analyzed, and the tools available to overcome those that generate the main problems are discussed., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The scaling-up of lindane-contaminated soils bioremediation from microcosms to mesocosms bioaugmentated with an actinobacteria quadruple culture and biostimulated with sugarcane filter cake (SCFC) was surveyed. Mesocosms of silty loam soil, clayey soil, and sandy soil were polluted with the pesticide, bioaugmented with the mixed culture, biostimulated with adequate amounts of 0.5 mm SCFC particles, and assessed during 63 days maintaining environmental parameters with minimal intervention. Samples were taken to determine residual lindane, heterotrophic microorganisms, enzymatic activities, and bioremediation effectiveness using ecotoxicity tests with Raphanus sativus, Lactuca sativa, and Lycopersicon esculentum. The bioaugmentation and biostimulation of the three soils improved lindane removal, microbial counts, and enzymatic activities, and reduced pesticide T 1/2 , regarding the values obtained in non-bioremediated controls. The removal process was significantly affected by the soil type, and the highest pesticide dissipation (82.6%) was detected in bioremediated sandy soil. Ecotoxicity tests confirmed the bioremediation success through a rise in the vigor index of seedlings compared to non-treated soils (R. sativus: 12-22%; L. sativa: 12-20%; L. esculentum: 30-45%). Finally, scanning electron microscopy corroborated soil colonization by actinobacteria. Successful scaling-up of the combined application of an actinobacteria quadruple culture and SCFC as an appropriate strategy for restoring lindane-polluted soils at mesocosms-scale was confirmed., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In this study, we evaluated polyurethane foam (PF), volcanic rock (VR), and a modified plastic cap (MPC) as supports for the immobilization of organophosphorus (OP) pesticide-degrading actinobacterial strains. The colonization and activity of four streptomycetes were favoured by PF, which was selected as the carrier to use in a continuous stirred tank bioreactor (CSTR) that can be operated at increasing inflows of a pesticide mixture that contains the insecticides chlorpyrifos (CP) and diazinon (DZ). Our results demonstrate that the CSTR can be operated at flow rates of 10 and 40 mL h -1 with greater than 85% removal of the pesticides in the short term. A significant decrease in the efficiency of CP removal was observed at the highest inflows into the reactor. The CP and DZ loading rates in the bioreactor ranged from 0.44 to 1.68 mg L -1  h -1 and from 0.50 to 2.17 mg L -1  h -1 , respectively. Although the treated wastewater exhibited moderate toxicity for Raphanus sativus , a bioreactor inoculated with a mixed culture formed by Streptomyces spp. strains AC5, AC9, GA11 and ISP13 may provide an effective biotechnological strategy for the reduction of OP pesticide residues produced during agronomic and manufacturing practices and therefore prevent environmental pesticidal pollution., Competing Interests: Conflict of interestOn behalf of all authors, the corresponding author states that there is no conflict of interest., (© King Abdulaziz City for Science and Technology 2020.)

Lindane is an organochlorine pesticide that, due to its persistence in the environment, is still detected in different matrices. Bioremediation using actinobacteria consortia proved to be promising for the restoration of contaminated soils. Another alternative to remove xenobiotics is to use agricultural residues, which stimulates microbial activity, increasing its capacity to degrade organic pollutants. The present work studies the coupling of sugarcane bagasse biostimulation and bioaugmentation with the actinobacteria consortium composed of Streptomyces sp. A2, A5, A11 and M7 on lindane removal in different soil types. In this sense, factorial designs with three factors (proportion and size of sugarcane bagasse particles, and moisture content) were employed. A response optimizer identified the combination of factors levels that jointly allowed obtaining the maximum lindane removal in the evaluated conditions. In the optimal conditions, the effect of the bioremediation process on soil microbiota was studied by evaluating different parameters. The highest lindane removal percentages were detected in biostimulated microcosms bioaugmented with the microbial consortium, which were accompanied by a decrease in lindane half-life respect to the controls. Also, the bioaugmentation of biostimulated microcosms increased the microbial counts and enhanced soil enzymatic activities, corroborating the bioremediation process efficiency. The survival of the four actinobacteria at the end of the assay confirmed the ability of all Streptomyces strains to colonize amended soils. Bioremediation by simultaneous application of biostimulation with sugarcane bagasse and bioaugmentation with the actinobacteria consortium, in the optimized conditions, represents an efficient strategy to restore lindane contaminated soils., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The biomixture is the major constituent of a biopurification system and one of the most important factors in its efficiency; hence the selection of the components is crucial to ensure the efficient pesticides removal. Besides, bioaugmentation is an interesting approach for the optimization of these systems. A mixed culture of the fungus Trametes versicolor SGNG1 and the actinobacteria Streptomyces sp. A2, A5, A11, and M7, was designed to inoculate the biomixtures, based on previously demonstrated ligninolytic and pesticide-degrading activities and the absence of antagonism among the strains. The presence of lindane and/or the inoculum in the biomixtures had no significant effect on the development of culturable microorganisms regardless the soil type. The consortium improved lindane dissipation achieving 81-87% of removal at 66 d of incubation in the different biomixtures, decreasing lindane half-life to an average of 24 d, i.e. 6-fold less than t 1/2 of lindane in soils. However, after recontamination, only the bioaugmented biomixture of silty loam soil enhanced lindane dissipation and decreased the t 1/2 compared to non-bioaugmented. The biomixture formulated with silty loam soil, sugarcane bagasse, and peat, inoculated with a fungal-actinobacterial consortium, could be appropriate for the treatment of agroindustrial effluents contaminated with organochlorine pesticides in biopurification systems., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Lindane is an organochlorine pesticide which persists in the environment and can cause serious health problems due to its chlorinated and hydrophobic nature. Microemulsions are isotropic and macroscopically homogeneous systems with high solubilization capacity of hydrophilic and hydrophobic compounds. The aim of this study was to evaluate the removal of high concentrations of lindane by the actinobacterium Streptomyces sp. M7 in aqueous and soil systems in the presence of stable microemulsions. Three stable microemulsions were successfully formed with Tween 80, 1-pentanol and three vegetable oils. In most cases, an increase in the cosurfactant/surfactant ratio in the microemulsions favored the solubilization of lindane, while an increase in the oil/surfactant ratio negatively affected the stability of the system. The microemulsion prepared with soybean oil allowed the solubilization of 66% of lindane added to the aqueous medium and 4.5 times more than the surfactant solution at the same concentration. This microemulsion increased the bioavailability of lindane in the aqueous medium and hence enhanced its removal by Streptomyces sp. M7 almost two times respect to the achieved with the surfactant solution. In loam soil system, the addition of the microemulsion allowed an 87% of lindane removal by Streptomyces sp. M7, increasing almost 50% the removal respect to the obtained without the addition of surfactant agents, although it did not present significant difference respect to the obtained with the surfactant solution. This is the first report on enhanced lindane removal by actinobacteria by using direct microemulsions as bioremediation tools., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Actinobacteria exhibit cosmopolitan distribution since their members are widely distributed in aquatic and terrestrial ecosystems. In the environment they play relevant ecological roles including recycling of substances, degradation of complex polymers, and production of bioactive molecules. Biotechnological potential of actinobacteria in the environment was demonstrated by their ability to remove organic and inorganic pollutants. This ability is the reason why actinobacteria have received special attention as candidates for bioremediation, which has gained importance because of the widespread release of contaminants into the environment. Among organic contaminants, pesticides are widely used for pest control, although the negative impact of these chemicals in the environmental balance is increasingly becoming apparent. Similarly, the extensive application of heavy metals in industrial processes lead to highly contaminated areas worldwide. Several studies focused in the use of actinobacteria for cleaning up the environment were performed in the last 15 years. Strategies such as bioaugmentation, biostimulation, cell immobilization, production of biosurfactants, design of defined mixed cultures and the use of plant-microbe systems were developed to enhance the capabilities of actinobacteria in bioremediation. In this review, we compiled and discussed works focused in the study of different bioremediation strategies using actinobacteria and how they contributed to the improvement of the already existing strategies. In addition, we discuss the importance of omic studies to elucidate mechanisms and regulations that bacteria use to cope with pollutant toxicity, since they are still little known in actinobacteria. A brief account of sources and harmful effects of pesticides and heavy metals is also given., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Anaplastic thyroid cancer (ATC) is the most aggressive solid tumor and almost uniformly lethal in humans. The Boards of the Thyroid Cancer Group of the Spanish Society of Endocrinology and Nutrition and the Grupo Español de Enfermedades Huérfanas e Infrecuentes of the Spanish Society of Oncology requested that an independent task force draft a more comprehensive consensus statement regarding ATC. All relevant literature was reviewed, including serial PubMed searches together with additional articles. This is the first, comprehensive Spanish consensus statement for ATC and includes the characteristics, diagnosis, initial evaluation, treatment goals, recommendations and modalities for locoregional and advanced disease, palliative care options, surveillance, and long-term monitoring. Newer systemic therapies are being investigated, but more effective combinations are needed to improve patient outcomes. Though more aggressive radiotherapy has reduced locoregional recurrences, median overall survival has not improved in more than 50 years.

Objectives: To present the state of knowledge and surgical practice concerning cleft lip and palate leading to the modern era., Design: Bibliographical review., Setting: Analysis of the scientific medical, surgical, and odontological literature from the 16th to 18th centuries., Results: Texts and art confirm the existence of cleft lip and palate from antiquity in all cultures; however, the first certain references in Western scientific literature did not appear until the works of Celsus (ca. 25 BC to AD 50) and Galen (AD 129 to 200). Indications for cleft lip surgery appear in the Middle Ages with Albucasis (936 to 1013) and Yperman (1260 to 1332). From the Renaissance period onward, and with the invention of the printing press, numerous authors described their own surgical methods., Conclusions: Given the limitations imposed by pain and infection, the authors of the Modern Age focus on correcting the functional and esthetic defect of the lip with techniques that do not differ greatly from those described in the Middle Ages. The treatment for cleft palate was limited to the creation of "obturators" and surgery for cleft palate was not possible until the 19th century.

Pesticides are normally used to control specific pests and to increase the productivity in crops; as a result, soils are contaminated with mixtures of pesticides. In this work, the ability of Streptomyces strains (either as pure or mixed cultures) to remove pentachlorophenol and chlorpyrifos was studied. The antagonism among the strains and their tolerance to the toxic mixture was evaluated. Results revealed that the strains did not have any antagonistic effects and showed tolerance against the pesticides mixture. In fact, the growth of mixed cultures was significantly higher than in pure cultures. Moreover, a pure culture (Streptomyces sp. A5) and a quadruple culture had the highest pentachlorophenol removal percentages (10.6% and 10.1%, resp.), while Streptomyces sp. M7 presented the best chlorpyrifos removal (99.2%). Mixed culture of all Streptomyces spp. when assayed either as free or immobilized cells showed chlorpyrifos removal percentages of 40.17% and 71.05%, respectively, and for pentachlorophenol 5.24% and 14.72%, respectively, suggesting better removal of both pesticides by using immobilized cells. These results reveal that environments contaminated with mixtures of xenobiotics could be successfully cleaned up by using either free or immobilized cultures of Streptomyces, through in situ or ex situ remediation techniques.

In the last few decades, highly toxic organic compounds like the organochlorine pesticide (OP) hexachlorocyclohexane (HCH) have been released into the environment. All HCH isomers are acutely toxic to mammals. Although nowadays its use is restricted or completely banned in most countries, it continues posing serious environmental and health concerns. Since HCH toxicity is well known, it is imperative to develop methods to remove it from the environment. Bioremediation technologies, which use microorganisms and/or plants to degrade toxic contaminants, have become the focus of interest. Microorganisms play a significant role in the transformation and degradation of xenobiotic compounds. Many Gram-negative bacteria have been reported to have metabolic abilities to attack HCH. For instance, several Sphingomonas strains have been reported to degrade the pesticide. On the other hand, among Gram-positive microorganisms, actinobacteria have a great potential for biodegradation of organic and inorganic toxic compounds. This review compiles and updates the information available on bacterial removal of HCH, particularly by Streptomyces strains, a prolific genus of actinobacteria. A brief account on the persistence and deleterious effects of these pollutant chemical is also given.

Lindane (γ-HCH) is an organochlorine insecticide that has been widely used in developing countries. It is known to persist in the environment and can cause serious health problems. One of the strategies adopted to remove lindane from the environment is bioremediation using microorganisms. Immobilized cells present advantages over free suspended cells, like their high degradation efficiency and protection against toxins. The aims of this work were: (1) To evaluate the ability of Streptomyces strains immobilized in four different matrices to remove lindane, (2) To select the support with optimum lindane removal by pure cultures, (3) To assay the selected support with consortia and (4) To evaluate the reusability of the immobilized cells. Four Streptomyces sp. strains had previously shown their ability to grow in the presence of lindane. Lindane removal by microorganisms immobilized was significantly higher than in free cells. Specifically immobilized cells in cloth sachets showed an improvement of around 25% in lindane removal compared to the abiotic control. Three strains showed significantly higher microbial growth when they were entrapped in silicone tubes. Strains immobilized in PVA-alginate demonstrated lowest growth. Mixed cultures immobilized inside cloth sachets showed no significant enhancement compared to pure cultures, reaching a maximum removal of 81% after 96 h for consortium I, consisting of the four immobilized strains together. Nevertheless, the cells could be reused for two additional cycles of 96 h each, obtaining a maximum removal efficiency of 71.5% when each of the four strains was immobilized in a separate bag (consortium III)., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In this paper, we address the problem of estimating the parameters of Gaussian mixture models. Although the expectation-maximization (EM) algorithm yields the maximum-likelihood (ML) solution, its sensitivity to the selection of the starting parameters is well-known and it may converge to the boundary of the parameter space. Furthermore, the resulting mixture depends on the number of selected components, but the optimal number of kernels may be unknown beforehand. We introduce the use of the entropy of the probability density function (pdf) associated to each kernel to measure the quality of a given mixture model with a fixed number of kernels. We propose two methods to approximate the entropy of each kernel and a modification of the classical EM algorithm in order to find the optimum number of components of the mixture. Moreover, we use two stopping criteria: a novel global mixture entropy-based criterion called Gaussianity deficiency (GD) and a minimum description length (MDL) principle-based one. Our algorithm, called entropy-based EM (EBEM), starts with a unique kernel and performs only splitting by selecting the worst kernel attending to GD. We have successfully tested it in probability density estimation, pattern classification, and color image segmentation. Experimental results improve the ones of other state-of-the-art model order selection methods.

Glaucoma represent one of the most frequent causes of partial loss of the visual field. It comes along with an ongoing destruction of the optic nerve caused by an increased pressure of the eye liquid. The disease becomes obvious from investigations of the retina with scanning laser microscopes. In this report an image analysis and classification system based on independent component analysis and k-nearest-neighbor classification is proposed. The method is tested with 120 selected retina images collected with the Heidelberg Retina Tomograph and achieves a classification rate of 91%.

In DM (differentiation medium), Sol 8 myoblasts spontaneously form myotubes and express the betaMHC (beta-myosin heavy chain), their main marker of terminal differentiation. This marker is detectable at 24 h, and increases up to 72 h. Our aim was to define temporal effects of TGFbeta (transforming growth factor beta) on betaMHC expression in Sol 8 cells. TGFbeta1 (1 ng/ml) added at time zero to DM decreased MyoD expression and completely inhibited betaMHC expression in Sol 8 cells. This inhibition of betaMHC expression was progressively lost when TGFbeta1 was added from 8 to 34 h. After 34 h, the cells were irreversibly differentiated, and TGFbeta1 did not inhibit betaMHC accumulation any longer. Two independent approaches showed that a TGFbeta autocrine regulatory loop retarded and partially impaired Sol 8 cell terminal differentiation. First, permanent immunoneutralization of the active TGFbetas released by the cells into DM increased betaMHC levels at 72 h compared with controls. Secondly, a dominant-negative mutant of the TGFbeta type II receptor was overexpressed in Sol 8 cells under the control of the betaMHC promoter. Both the dominant-negative receptor and the betaMHC gene were expressed after 24 h in DM. The delayed blocking of the TGFbeta signalling pathway by the dominant-negative receptor was as effective as permanent immunoneutralization to promote betaMHC expression. To conclude, TGFbeta inhibits Sol 8 cell terminal differentiation within a narrow time interval (24-34 h) that coincides with the onset of betaMHC expression.

Retinoic acid (RA) was recently shown to modify testosterone secretion of the fetal testis in vitro. We characterized this effect by culturing rat testes explanted at various ages, from Fetal Day 14.5 to Postnatal Day 3. In basal medium, RA inhibited, in a dose-dependent manner, both basal and acute LH-stimulated testosterone secretion by testes explanted on Fetal Days 14.5, 15.5, and 16.5. It had no effect on testes from older animals. The negative effect of RA did not result from a diminution in the number of Leydig cells but from a decrease in P450c17 mRNA levels and in LH-stimulated cAMP production. However, the RA-induced decrease in P450C17 mRNA levels was also observed with neonatal testes, suggesting that this enzymatic step is no longer rate limiting at this developmental stage. To study the physiological relevance of RA effects, we used fetuses and neonates issued from mothers fed a vitamin A-deficient (VAD) diet, resulting in a threefold decrease of plasma retinol concentration. On Fetal Day 18.5 and on Posnatal Day 3, testosterone secretion by the testis ex vivo was significantly increased in VAD animals. This shows that the endogenous retinol inhibits differentiation and/or function of fetal Leydig cells before Fetal Day 18.5 and is required for the normal regression of fetal Leydig cell function that occurs after Fetal Day 18.5. In conclusion, our results show that retinoids play a negative role on the steroidogenic activity during the differentiation of rat fetal Leydig cells.

In Lyon hypertensive (LH) rats, a model of low-renin genetic hypertension, we investigated adrenal sensitivity to angiotensin II in terms of angiotensin II receptor (AT1 and AT2 receptors) regulation, morphological changes, and aldosterone and corticosterone secretion. Twelve-week-old LH rats, compared with normotensive LN and LL rats, were either untreated or treated for 4 weeks with AT1 receptor antagonist irbesartan (50 mg/kg/d), angiotensin-converting enzyme inhibitor perindopril (3 mg/kg/d), or perindopril (3 mg/kg/d) plus angiotensin II infusion (200 ng/kg/min). At 16 weeks, untreated LH rats had high systolic blood pressure (P<0.05), low aldosterone (P<0.05), and increased corticosterone (P<0.05) plasma levels. AT1-receptor binding density in the zona glomerulosa was similar in the three strains. In LH rats, angiotensin II infusion increased the relative adrenal weight from 10.5+/-0.3 to 16.7+/-0.7 mg/100g (P<0.05), whereas this change was very modest in normotensive rats. Zona glomerulosa enlarged and plasma aldosterone increased after angiotensin II infusion in the 3 strains, but more markedly in LH versus normotensive rats (2.4- versus 1.3- and 1.6-fold, respectively; 20- versus 10-fold in normotensive rats, P<0.05). Surprisingly, after angiotensin II infusion, despite the absence of angiotensin II receptors in the three strains, the zona fasciculata-reticularis enlarged 1.5-fold and plasma corticosterone increased 1.7-fold only in LH rats (P<0.05), suggesting an indirect control of this compartment by angiotensin II. The hypertrophy and hypersecretory activity of both zona glomerulosa and zona fasciculata-reticularis in LH rats in response to angiotensin II point to the adrenal cortex as a pivotal tissue in the pathophysiology of hypertension in LH rats.

Outer membrane proteins (OMP) are expressed in Gram-negative bacterial cell wall. OmpA from Klebsiella pneumoniae (KpOmpA) has been shown to bind and to activate selectively antigen presenting cells (APCs), eliciting protective CTL responses. In this study, we investigated whether OmpX, another member of the OMP family and structurally related to OmpA, exhibits the same immune properties. Using recombinant OmpX from Escherichia coli (EcOmpX), we report that EcOmpX binds to and is internalized by human APCs. However, EcOmpX does not activate APCs. EcOmpX acts as an efficient carrier protein as it induces a potent and Th1/Th2 mixed anti-TNP humoral response. However, adjuvant is required to generate a protective anti-tumoral immune response in mice injected with a tumor model antigen coupled to EcOmpX. Collectively, these data show that EcOmpX is recognized by innate cells but does not activate them, suggesting that EcOmpX does not provide a signal danger to APCs. In conclusion, this study provides information on the molecular mechanisms involved in the recognition and activation of innate cells by bacterial outer membrane proteins.

The human corticotropin-releasing factor receptor type 1 (hCRF-R1) functional transcript is mainly expressed in the anterior pituitary corticotrophs, a tissue usually not available for clinical investigation. Splice variants translated into defective isoforms of the receptor have been described in few peripheral tissues. The aim of this work was to determine whether peripheral white blood cells from healthy individuals, an accessible tissue for clinical investigation, were suitable for the analysis of the hCRF-R1 transcript and gene. We report that: (i) specific amplification of the hCRF-R1 transcript from peripheral white blood cells mRNAs is feasible; (ii) this transcript is similar to the functional transcript; (iii) the draft sequence of chromosome 17 and unrelated sequences allow direct sequencing of all 14 exons of the gene, adjacent splice sites and related branch points. In conclusion, these approaches would be suitable for studies in patients having isolated secondary glucocorticoids deficiency to implicate the hCRH-R1 in the etiology of the disease.

Microscopes and neuronavigators are often used in neurosurgical procedures. Our Mixed Reality prototype proposes to show neuronavigator imaging overlayed onto the surgical field of view. The proposal is to replace the traditional optical microscope with a digital one. The system is presented in this article. Firsts trials have been performed at the laboratory with satisfactory results.

The development and function of the primate adrenal cortex are characterized by rapid growth, high steroidogenic activity, and a particular morphological appearance. The fetal adrenal glands grow rapidly and exponentially and at term are similar in weight to adult adrenals. From birth to 1 year their mass is reduced as they undergo a process of differentiation. Growth then remains slow until age 7 years. Thereafter, growth accelerates and the adrenals reach adult weight by the end of puberty. In the first trimester of gestation, fetal adrenal growth is thought to be independent of adrenocorticotropic hormone (ACTH), but after 15 weeks, ACTH is absolutely required for normal morphological and functional development. Other factors of fetal and/or placental origin, acting independently of or in conjunction with ACTH, are also required. Basic fibroblast growth factor, epidermal growth factor/transforming growth factor beta, and insulin-like growth factor (IGF)-I and -II, all acting in an autocrine and/or paracrine fashion, have been postulated to stimulate fetal adrenal cell proliferation. Corticotropin-releasing hormone may also play an important role in primate fetal adrenal function, primarily at the end of gestation. Finally, the estrogens are also important in the development of the pituitary-adrenal axis in primates.

Recently it was shown that the cholinergic agonist carbachol stimulates cortisol production in bovine ZFR cells via muscarinic receptor M(3). In the present study, we investigated the effect of chronic cholinergic stimulation on steroidogenic response and muscarinic receptor regulation in ZFR cells. Cortisol secretion of ZFR cells treated with 10(-4) M of carbachol decreased in a dose- and time-dependent manner. The carbachol-elicited loss of response was associated with a decrease in M(3) receptor number, which was also time- and dose-dependent. The down-regulation of the receptors was not associated with the decrease of M(3) receptor mRNA level. The marked steroidogenic desensitization caused by pretreatment of carbachol did not alter ACTH or angiotensin II activated steroid response. Northern blot analysis showed that carbachol pretreatment did not change the gene expression of P450scc, P450cl7, 3betaHSD and StAR mRNAs. These results suggest that carbachol induces homologous steroidogenic refractoriness of ZFR cells.

Studies in Nur77-deficient mice have shown that the basal regulation of hypothalamic and pituitary functions as well as the adrenocortical steroidogenesis in these animals is normal. This indicates that Nur77-related orphan receptors may substitute Nur77 functions in the hypothalamo-pituitary-adrenal axis by a compensatory mechanism. Nor1 is the most recently cloned member of the NGFI-B/Nur77 subfamily, and its properties are still largely unknown. We demonstrate here that Nor1 is expressed in the pituitary gland and adrenal cortex, and that ACTH and angiotensin II (AngII) treatment of adrenal fasciculata cells induces Nor1 expression. Time-course analysis with both hormones on steroidogenic capacity and the specific gene expression in adrenal cells strongly suggest that Nor1 is an intermediate in the long-term consequences of ACTH or AngII treatment. The Nor1 and NGFI-B/Nur77 amino acid sequence homology and the analysis of the trans-activation properties of Nor1 show that the overall structural and functional organization of the two proteins is similar. As observed with NGFI-B/Nur77, Nor1 activates the expression of genes encoding steroidogenic enzymes as P450c21, through its interaction with NGFI-B response element promoter sequences. In contrast, binding experiments of Nor1 with the palindromic NurRE sequence suggest that Nor1 is not an efficient substitute for the NGFI-B/Nur77 activation of the POMC gene expression in pituitary glands. All these results indicate that Nor1 and NGFI-B/Nur77 may play similar albeit distinct roles in the hypothalamo-pituitary-adrenal axis. Further experiments also show that the mechanisms responsible for the transcriptional regulation of Nor1 in adrenal cells appear to depend on the protein kinase A and protein kinase C cascades.

The purpose of this study was to evaluate the time-course effect of a 36-h treatment with ACTH (10(-8) M), transforming growth factor-beta1 (TGFbeta1; 10(-10) M), angiotensin II (AngII; 10 (-7) M), and insulin-like growth factor I (IGF-I; 10(-8) M) on the steroidogenic capacity of bovine adrenocortical cells (BAC) and on messenger RNA (mRNA) levels of ACTH receptor, cytochrome P450c17, 3beta-hydroxysteroid dehydrogenase (3betaHSD), steroidogenic acute regulatory protein (StAR), and StAR protein. ACTH and IGF-I enhanced, in a time-dependent manner, the acute 2-h ACTH-induced cortisol production, whereas TGFbeta 1 and AngII markedly reduced it. ACTH, IGF-I, and AngII increased ACTH receptor mRNA, but the opposite was observed after TGFbeta1 treatment. ACTH and IGF-I increased P450c17 and 3betaHSD mRNAs, whereas AngII and TGFbeta1 had the opposite effects. However, the effects of the four peptides on ACTH-induced cortisol production appeared before any significant alterations of the mRNA levels occurred. The most marked and rapid effect of the four peptides was on StAR mRNA. The stimulatory effect of ACTH was seen within 1.5 h, peaked at 4-6 h, and declined thereafter, but at the end of the 36-h pretreatment, the levels of StAR mRNA and protein were higher than those in control cells. IGF-I also enhanced StAR mRNA levels within 1.5 h, and these levels remained fairly constant. The effects of AngII on StAR mRNA expression were biphasic, with a peak within 1.5-3 h, followed by a rapid decline to almost undetectable levels of both mRNA and protein. TGFbeta1 had no significant effect during the first 3 h, but thereafter StAR mRNA declined, and at the end of the experiment the StAR mRNA and protein were almost undetectable. Similar results were observed when cells were treated with ACTH plus TGFbeta1. A 2-h acute ACTH stimulation at the end of the 36-h pretreatment caused a higher increase in StAR mRNA and protein in ACTH- or IGF-I-pretreated cells than in control cells, which, in turn, had higher levels than cells pretreated with TGFbeta1, ACTH plus TGFbeta1, or AngII. These results and the fact that the stimulatory (IGF-I) or inhibitory (AngII and TGFbeta1) effects on ACTH-induced cortisol production were more pronounced than those on the ability of cells to transform pregnenolone into cortisol strongly suggest that regulation of StAR expression is one of the main factors, but not the only one, involved in the positive (IGF-I) or negative (TGFbeta1 and AngII) regulation of BAC for ACTH steroidogenic responsiveness. A high correlation between steady state mRNA level and acute ACTH-induced cortisol production favors this conclusion.

Transforming growth factor beta (TGFbeta) has been reported to be a potent growth inhibitor of epithelial cells. The purpose of the present work was to study in vitro and in vivo the effects of overexpression of a dominant-negative type II TGFbeta receptor on the proliferation and differentiation of Y-1 cells. Stable transfections were performed with a mutant TbetaRII (TbetaRII-KR) fused with the Enhanced Fluorescent Green Protein (EGFP). The expression of this fusion protein and its overexpression were demonstrated by northern blot and immunoblot with EGFP and TbetaRII probes and antibodies respectively. The membrane localization of this fusion protein was confirmed by confocal microscopy. The functionality of this fusion protein was demonstrated by its blocking effects on TGFbeta action on DNA synthesis and on Y-1 expression of steroidogenic acute regulatory protein (StAR) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD). Moreover, in nude mice the tumorigenicity of cells stably transfected with the fusion protein was higher than that of cells stably transfected with EGFP alone. Taken together, the present results show that TbetaRII-KR/EGFP blocks the effects of TGFbeta1 on Y-1 cells and acts as a potent dominant-negative receptor preventing TGFbeta signaling.

The regulatory effects of angiotensin-II (AngII) and several growth factors, including insulin-like growth factor 1 (IGF-1), basic fibroblast growth factor (bFGF), and transforming growth factor beta1 (TGFbeta1) on the AngII subtype 2 (AT2) receptor were studied using R3T3 cells, a mouse fibroblast cell line that expresses only AT2 receptors. AngII increased (in a time- and dose-dependent manner) AT2 binding sites but had no effects on AT2 messenger RNA (mRNA) levels. At maximal concentration (10(-7) M) AngII caused a 4-fold increase of AT2 receptor number. In contrast, IGF-1 increased (3- to 4-fold), whereas bFGF and TGFbeta1 decreased (by about 90% and 80%, respectively) AT2 receptor and mRNA levels. Moreover, AngII potentiated the effect of IGF-1 on receptor number, but not on AT2 mRNA levels, and significantly reduced the inhibitory action of bFGF and TGFbeta1 on AT2 binding sites but not on AT2 mRNA levels. None of these factors modified AT2 mRNA half-life. The potential effects of these factors on transcription of the AT2 gene were measured by means of nuclear run-on assays. IGF-1 increased the rate of transcription by about 2.5-fold, whereas bFGF and TGFbeta1 reduced it by 90 and 80%, respectively. In contrast, AngII did not modify either the basal or IGF-1-stimulated transcription rate. Finally, AngII alone or together with IGF-1, but not IGF-1 alone, increased the attachment of AT2 mRNA to polysomal fractions. The present findings demonstrate that the main mechanism by which AngII regulates the AT2 receptor is by increasing the rate of AT2 mRNA translation, whereas the stimulatory (IGF-1) or inhibitory (bFGF and TGFbeta1) effects of these growth factors on AT2 expression are exerted at the transcriptional level.

The localization of transforming growth factor beta3 (TGFbeta3) in the fetal and neonatal testis (from fetal day 13.5 to postnatal day 6) was investigated by immunohistochemical staining with a specific polyclonal antibody raised against a synthetic peptide corresponding to residues 50-75 of TGFbeta3. This antibody recognized 0.5 ng TGFbeta3 in western blot analysis, but did not detect 25 ng TGFbeta1 or TGFbeta2. The immunolocalization of TGFbeta3 in the fetal and neonatal testis changed throughout development. Immunostaining was present in the gonocytes by fetal day 13.5, persisted until postnatal day 3, and was heterogeneous in spermatogonia on postnatal day 6. The Sertoli cells contained no immunoreactivity at any age. The fetal-type Leydig cells were first immunostained for TGFbeta3 on day 16.5 and staining became very intense from day 18.5 onward. Staining disappeared when the antibody was presaturated with the synthetic peptide, but persisted when the antibody was presaturated with a tenfold excess of the corresponding peptide from TGFbeta2. Furthermore, we researched whether TGFbeta3 could act as a local regulator of fetal Leydig cell function. In a dispersed fetal testicular cell system, TGFbeta3 inhibited the LH-stimulated testosterone production by Leydig cells from 20.5-day-old fetuses. The inhibitory effect of TGFbeta3 was equal to that observed with TGFbeta1 or TGFbeta2. When compared with our previous studies showing the immunolocalization of TGFbeta1 and TGFbeta2, the present study shows that TGFbeta3 may have a specific role in the developing rat testis, but may also overlap the action of TGFbeta1 and TGFbeta2.

The expression and the maintenance of specific differentiated function of Leydig cells are regulated not only by gonadotropin but by locally produced factors, which may act as autocrine regulators. Many factors, in particular growth factors, have been postulated to have such a type of effect on testicular cells, but very few fulfilled the three criteria required to establish a paracrine/autocrine role: (a) presence of receptors and biological action on local cells; (b) local secretion regulated by physiological signals; and (c) blockade of the factor or its receptors must modify the function of local cells. In the present work we demonstrate that two factors, insulin-like growth factor 1 (IGF-I) and transforming growth factor beta1 (TGFbeta1) fulfilled the three criteria: (a) IGF-I stimulates the transcription of the genes encoding Leydig cell differentiated function, leading to an enhanced steroidogenic responsiveness to LH/hCG; (b) Leydig cells (LC) express and secrete IGF-I and this secretion is enhanced by hCG; and (c) incubation of LC with IgG anti-IGF-I, but not with IgG-control, markedly reduced the steroidogenic responsiveness to LH/hCG. In contrast to IGF-I, TGFbeta is a potent inhibitor of LC differentiated function. Moreover, LC express TGFbeta1 mRNA and secrete this peptide. To prove that the locally produced TGFbeta has an autocrine role, LC were transfected with 10 microM of an antisense oligonucleotide (AON) complementary to the translation initiation region of TGFbeta1 mRNA. Transfection with AON but not with sense deoxynucleotide induces a complete disappearance of TGFbeta immunoreactivity in LC and an enhanced hCG-induced testosterone production by LC. This increased steroidogenic responsiveness was associated with a significant enhancement of both LH/hCG receptor mRNA and P450c17 mRNA. Taken together, the above results show that both factors play an autocrine role, although opposite, on Leydig cell function.

In the present work, the presence of gastric inhibitory polypeptide (GIP) receptors and their functional role in the adrenal cells of three patients with food-dependent Cushing's syndrome were studied. RT-PCR and in situ hybridization studies demonstrated the presence of GIP receptor in the adrenals of the three patients. The presence of this receptor was also demonstrated in two human fetal adrenals, but not in two normal adult human adrenals or in the adrenals of one patient with nonfood-dependent Cushing's syndrome. Freshly isolated cells from patient adrenals responded in a dose-dependent manner to the steroidogenic action of both ACTH and GIP, whereas cells from normal adrenals responded only to ACTH. Treatment of cultured normal adrenal cells with ACTH, but not with GIP, increased the messenger ribonucleic acid (mRNA) levels of cholesterol side-chain cleavage cytochrome P-450, P450c17, and 3beta-hydroxysteroid dehydrogenase, whereas both hormones enhanced these mRNAs in patients' adrenal cells, although the effects of ACTH were greater than those of GIP. Moreover, pretreatment with ACTH enhanced the steroidogenic responsiveness of both normal and patient adrenal cells, whereas GIP caused homologous desensitization, and this was associated with a marked reduction of GIP receptor mRNA levels, as demonstrated by RT-PCR and in situ hybridization. Finally, both ACTH and GIP inhibited DNA synthesis in one patient's adrenal cells, whereas in normal adrenal cells only ACTH had this effect. In conclusion, the present data demonstrate that ectopic expression of functional GIP receptors is the main cause of food-dependent Cushing's syndrome.

Transforming growth factor beta1 (TGFbeta1) has been reported to be a potent inhibitor of differentiated functions of many steroidogenic cells. Porcine Leydig cells (LC), as well as Sertoli cells (SC), express TGFbeta1 mRNA and secrete this peptide, suggesting that it might play an autocrine role. Moreover, many studies have suggested a possible paracrine regulation of LC by SC-secreted factors. To assess whether TGFbeta1 plays an autocrine/paracrine role on these steroidogenic cells, we attempted to inhibit TGFbeta1 protein synthesis by transfecting LC, SC and LC+SC for 24 h with 10 microM of an unmodified antisense oligonucleotide (AON) complementary to the translation-initiation region of the TGFbeta1 mRNA and, as controls, with the corresponding sense (SON) or scrambled (SCRON) oligonucleotides. First, we determined at which level, transcriptional or translational, the TGFbeta1 AON acts. Neither TGFbeta1 AON, SON nor SCRON modified TGFbeta1 mRNA levels in LC, SC or LC+SC. However, TGFbeta1 AON caused the disappearance of TGFbeta1 immunoreactivity in both cell types. In addition, TGFbeta1 AON reduced the attachment of TGFbeta1 mRNA in ribosomal and polyribosomal fractions. Then, we showed that the decrease of the TGFbeta1 protein induced by the AON results in an increase of the expression of LC specific genes and of LC steroidogenic capacity. In LC and LC+SC, TGFbeta1 AON increased the mRNA levels of both LH/hCG receptor (1.9-fold and 3.5-fold, respectively) and P450 c17 (5-fold and 8-fold, respectively). This was associated with an enhancement of hCG-induced testosterone production by both LC and LC+SC (1.6-fold and 2.2-fold, respectively) when compared with untransfected cells. The TGFbeta1 AON effects were always more pronounced on LC+SC than on LC. The present findings show that TGFbeta1 has an autocrine/paracrine inhibitory effect on cultured porcine Leydig cells, an effect that can be overcome by TGFbeta1 AON.

Since recombinant hormones are considered as safer and more reliable in their bioactivity than extractive hormones, the recently available human recombinant luteinizing hormone (r-hLH), will probably replace hCG in the near future, for clinical purposes. This prompted us to investigate whether or not, and by which mechanisms, r-hLH can induce a desensitization of signal transduction and/or an up-regulation of steroidogenic capacity in Leydig cells. The effects of a 30 min to 24 h exposure to r-hLH (10(-9) M) on the differentiated functions of cultured immature porcine Leydig cells were studied by measuring the following parameters: LH/hCG receptor number and mRNA, hCG-, cholera toxin- and forskolin-induced cAMP production, G protein alphas subunit content of the membrane, hCG-, cholera toxin-, forskolin-, 8Br-cAMP-, 22R-OH-cholesterol-, progesterone-, 170H-progesterone-, DHEA-, delta4-androstenedione-induced testosterone secretion and StAR, 3beta-HSD, cytochrome P-450scc and P-450c17 mRNAs. hCG binding sites and LH/hCG receptor mRNA were slowly down regulated by r-hLH, reaching 47+/-1 and 18+/-7% of control at 24 h, respectively. Down-regulation of both hCG- and cholera toxin-induced cAMP production occurred earlier and was more marked, and at 24 h represented only 2.7+/-0.5 and 12.5+/-3.6% of control. Due to the synergistic effect of r-hLH and forskolin on cAMP production, the forskolin-induced cAMP was higher in r-hLH treated than in control cells, but this response also declines with time and was, at 24 h, only 32% of that observed at 30 min. This decreased cAMP production was associated with a less marked decline in the amount of membrane content of Galphas protein. The testosterone production in response to hCG, cholera toxin, forskolin and 8Br-cAMP declined to reach a nadir at 6 h but increased thereafter and at 24 h was significantly higher than in control cells. In contrast, the conversion of several precursors into testosterone remained stable or increased slightly during the first hours of r-hLH treatment and significantly increased at 24 h and this was associated with an increase of StAR, 3beta-HSD, P-450scc and P-450c17 mRNAs. Taken together, the present results indicate that, despite the marked down-regulation of transmembrane signaling, r-hLH increased the steroidogenic capacity of Leydig cells by increasing the expression of several genes encoding the proteins involved in testosterone synthesis.

Receptors for insulin-like growth factors (IGF-I and IGF-II) are expressed in mammalian intestinal epithelium. No information on the presence of IGF receptors in intestinal endocrine cells is available. We tested for IGF-I receptors the endocrine cell line STC-1, which synthesizes and processes cholecystokinin (CCK) among other peptides, and assessed the effects of IGF-I on cell growth and CCK content. Cell monolayers in serum-free culture medium specifically bound [125I]IGF-I. Scatchard analysis was consistent with a single class of high affinity binding sites (KD = 0.91 nM; Bmax = 4,700 sites/cell). In competitive binding assays, unlabeled IGF-I, IGF-II and insulin displaced in a dose-dependent manner [125I]IGF-I binding with the following potencies (KI): IGF-I (0.74 nM) > IGF-II (3 nM) >> insulin (1 microM). Affinity cross-linking with [125I]IGF-I using disuccinimidyl suberate and SDS-PAGE under reducing conditions yielded a polypeptide band with apparent Mr 130,000, consistent with the alpha-subunit of the IGF-I receptor. IGF-I and IGF-II (0.3-30 nM) dose-dependently stimulated [3H]thymidine incorporation, with a maximal response of 110% above basal. IGF-II was approximately 10-fold less potent than IGF-I, suggesting a mediation through IGF-I receptors. In addition, the numbers of cells treated with 3 nM IGF-I amounted to 116, 130 and 159% of control values after 1, 2 and 4 days of incubation, respectively (p < 0.05). A significant increase in the cell CCK contents was observed after a 48-hour exposure to 3 or 30 nM IGF-I. These results demonstrate IGF-I receptor expression by the enteroendocrine cell line STC-1. IGF-I stimulates proliferation in short-term experiments, and increases intracellular levels of CCK.

The effects of insulin-like growth factor 1 (IGF-1), basic fibroblast growth factor (bFGF), transforming growth factor beta1 (TGFbeta1), fetal calf serum (FCS) and angiotensin II (AngII) on cell proliferation, (3H-thymidine incorporation and cell number) and AT2 receptor number and mRNA levels in R3T3 cells have been studied. All growth factors as well as FCS markedly increased cell proliferation, whereas AngII increased slightly 3H-thymidine incorporation, but not cell number. TGFbeta1, bFGF and FCS reduced by more than 80% both AT2 receptor number and mRNA, by inhibiting the transcription rate. In contrast, IGF-1 and AngII increased about 4-fold AT2 receptor number, but only IGF-1 increased AT2 mRNA. When added together the effects of IGF-1 and AngII were more than additive on AT2 receptor number, but not on mRNA level. None of the factors studied modified AT2 mRNA half-life. In conclusion, the present results demonstrated that: 1/ cell proliferation is not correlated with AT2 expression; 2/ growth factors regulate, positively or negatively, AT2 transcription rate, whereas AngII regulates the translation rate of AT2 mRNA; 3/ all the effects of AngII on R3T3 are mediated by AT2 receptors since they are mimicked by the AT2 agonist CGP42112 and blocked by the AT2 antagonist PD123177.

Three main criteria are required to postulate that a factor has an autocrine action in any cell: presence of receptors and biological effects of the factor; local production which must be regulated by physiological signals and the inhibition of its secretion or action should cause effects opposite to those caused by the exogenous factor. In adrenal cells, transforming growth factor beta1 (TGFbeta1) fulfilled these criteria: 1) Adrenal cells possess TGFbeta1 receptors and this factor regulates the expression of adrenal cell specific genes, including ACTH and angiotensin II (AngII) receptors and several steroidogenic enzymes, leading to changes in the steroidogenic responsiveness; 2) Adrenal cells express and secrete TGFbeta1 and its secretion is regulated by the two adrenal specific hormones ACTH and AngII; 3) The blockade of TGFbeta1 protein synthesis by an antisense oligonucleotide produces changes in adrenal cell functions opposed to those induced by the exogenous factor.

We have shown previously that long-term testosterone secretion, which decreases when human Leydig cells are cultured alone, increases when purified human Leydig and Sertoli cells are cultured together. In this work, human Leydig cell functions were studied further during in vitro culture, either alone or with human Sertoli cells, on a basal membrane derived from bovine corneal endothelial cells. The secretion of testosterone increased during the first week of co-culture and remained elevated up to day 12 of culture. In one prolonged co-culture, testosterone secretion decreased progressively after day 12 and, after 1 month of culture, was at a level similar to that observed during the first 48 h. After culture for 48 h, testosterone secretion in the co-culture was enhanced by 162 +/- 5% (p < 0.0001) compared with values observed when Leydig cells were cultured alone (42.6 +/- 10.6 ng/10(6) Leydig cells/48 h; mean +/- SEM). This change was associated with increase in mRNA levels for 3 beta-hydroxysteroid dehydrogenase delta 5-delta 4-isomerase (2.49 +/- 0.58-fold), cytochrome P450c17 (2.81 +/- 0.99-fold), cytochrome P450scc (5.20 +/- 0.13-fold) and cytochrome P450 aromatase (1.73 +/- 0.21-fold) when Leydig cells were co-cultured with Sertoli cells (p < 0.05 for each enzyme). IGF-I mRNA levels were higher (2.77 +/- 0.72-fold for 7.6 kb and 1.41 +/- 0.07-fold for 1.1-1.3 kb transcripts) in the Leydig-Sertoli cell co-cultures than the sum of the levels in Leydig and in Sertoli cells cultured alone. Both basal and hCG-induced testosterone secretion were enhanced by treatment of the co-culture with human recombinant FSH (50 mIU/mL). For basal testosterone secretion, this increase amounted to 163 +/- 5% compared with Leydig cells cultured alone (p < 0.0001) and by 112 +/- 4% compared with non-FSH treated co-cultures (p < 0.01); for hCG-stimulated testosterone secretion this increase was 220 +/- 12% compared with Leydig cells cultured alone (p < 0.0001) and 132 +/- 8% compared with non-FSH treated co-cultures (p < 0.01). This study confirms the enhancement of long-term testosterone secretion by adult human Leydig cells by co-culture with adult human Sertoli cells and shows that this effect is associated with an enhancement of the expression of several steroidogenic enzymes; it might be mediated, as in other species, through increased production of IGF-I by co-culture.

Insulin like growth factor I (IGF-I) is believed to be a potent para/autocrine stimulator of Leydig cell function in adult testis. We investigated whether IGF-I is also an intratesticular regulator of fetal Leydig cell function by measuring its production in the fetal testis and its ability to affect testicular steroidogenesis during fetal development. Northern blot analysis revealed one major IGF-I transcript of 7-7.5 kb and two minor transcripts of 3.8 and 1.8 kb in 20.5 day fetal testis. IGF-I was detected by RIA in 16.5 fetal day testes, and the amounts of IGF-I secreted by 16.5 and 20.5 fetal day testes in vitro were much greater than the amounts contained in the testes, indicating active synthesis in culture. The secretion of IGF-I by the fetal testis in vitro was increased with testicular age and time in culture. It was not modified by gonadotropins or (Bu)2cAMP. Testosterone secretion by fetal testes explanted 13.5, 16.5, 18.5, and 20.5 days after conception and cultured in the presence or absence of 100 ng/ml LH for 3 days was not affected by the addition of 50 ng/ml IGF-I to the medium. In contrast, the addition of IGF-I to dispersed fetal testicular cells cultured for 3 days in the presence or absence of LH increased the number of Leydig cells identified by a positive cytochemical reaction for 3beta-hydroxysteroid dehydrogenase (3betaHSD). This was more pronounced with cells from 16.5- day-old fetuses (stage when the fetal Leydig cells are differentiating in vivo) than with 20.5-day-old fetuses cells (stage when the number and the function of fetal Leydig cells are stable or decreasing). It results from both an increased differentiation of mesenchymal cells in fetal Leydig cells and an increase in the mitotic index of the fetal Leydig cells, as inferred from the small increase in the percentage of bromodeoxyuridine/3betaHSD-positive cells. Both LH and IGF-I increased significantly testosterone production by day 16.5 cells. In the presence of LH, a high amount of testosterone was produced per 3betaHSD-positive cell; IGF-I further increased this production. This effect was not observed with day 20.5 cells. The amounts of testosterone produced per 3betaHSD-positive cell cultured in the presence of both LH and IGF-I were more than additive. Like IGF-I, insulin (50 ng/ml) increased testosterone secretion per 3betaHSD-positive cells in cultures of day 16.5 cells, but not in those of day 20.5, cells. Lastly, IGF-I also increased the steroidogenic activity of each Leydig cell in cultures containing (Bu)2cAMP, but its effects were weaker than those observed in the presence of LH. This suggests that IGF-I has sites of action both upstream and downstream cAMP generation. These results suggest that IGF-I acts as paracrine/autocrine factor in the differentiation and activity of fetal Leydig cells.

There is increasing evidence that in the fetal and postnatal development of the adrenal gland, trophic and differentiating effects of ACTH are locally modulated by a species-specific pattern of growth factors. As we have shown previously in human adult adrenocortical cells (HAC) in culture, IGF-I and, even more, IGF-II enhance the steroidogenesis and ACTH responsiveness. We now examined the secretion of IGFs and their binding proteins (IGFBP) in the medium of 12 serum free primary cultures of HAC by specific RIAs and [125I]IGF ligand blot or by immunoblot, and their long-term regulation by ACTH. HAC secrete 0.41 and 0.91 ng IGF-I and IGF-II/5 x 10(5) cells per day, respectively, and their secretion is significantly stimulated 2- and 1.6-fold, respectively, by ACTH. HAC secrete at least three IGFBPs. The 43-46 kDa and the 29 kDa proteins correspond to glycosylated and fragmented forms of IGFBP-3, and the 36 kDa protein to IGFBP-2. The most abundant protein is the 24 kDa IGFBP, with identical electrophoretic mobility to IGFBP-4. IGFBP-3, as measured by RIA, is in the range of 1 ng/day. None of the IGFBPs is significantly changed by ACTH. Thus, we have evidence for a local IGF system, and the IGF-levels are in a range compatible with a physiological auto or paracrine action on steroidogenesis.