Exogenous growth hormone attenuates cognitive deficits induced by intermittent hypoxia in rats.

Sleep disordered breathing (SDB), which is characterized by intermittent hypoxia (IH) during sleep, causes substantial cardiovascular and neurocognitive complications and has become a growing public health problem. SDB is associated with suppression of growth hormone (GH) secretion, the latter being integrally involved in the growth, development, and function of the CNS. Since GH treatment is able to attenuate neurocognitive deficits in a hypoxic-ischemic stroke model, GH, GH receptor (GHR) mRNA expression, and GH protein expression were assessed in rat hippocampus after exposures to chronic sustained hypoxia (CH, 10% O(2)) or IH (10% O(2) alternating with 21% O(2) every 90 s). In addition, the effect of GH treatment (50 µg/kg daily s.c. injection) on erythropoietin (EPO), vascular endothelial growth factor (VEGF), heme oxygenase-1 (HO-1), and GLUT-1 mRNA expression and neurobehavioral function was assessed. CH significantly increased GH mRNA and protein expression, as well as insulin-like growth factor-1 (IGF-1). In contrast, IH only induced a moderate increase in GH mRNA and a slight elevation in GH protein at day 1, but no increases in IGF-1. CH, but not IH, up-regulated GHR mRNA in the hippocampus. IH induced marked neurocognitive deficits compared with CH or room air (RA). Furthermore, exogenous GH administration increased hippocampal mRNA expression of IGF-1, EPO, and VEGF, and not only reduced IH-induced hippocampal injury, but also attenuated IH-induced cognitive deficits. Thus, exogenous GH may provide a viable therapeutic intervention to protect IH-vulnerable brain regions from SDB-associated neuronal loss and associated neurocognitive dysfunction.

Restorative effect of intracerebroventricular insulin-like growth factor-I gene therapy on motor performance in aging rats.

Insulin-like growth factor-I (IGF-I) is a powerful neuroprotective molecule in the brain and spinal cord. We have previously shown that intracerebroventricular (i.c.v.) IGF-I gene therapy is an effective strategy to increase IGF-I levels in the cerebrospinal fluid (CSF). Since aging in rats is associated with severe motor function deterioration, we implemented i.c.v. IGF-I gene therapy in very old rats (30-31 months) and assessed the beneficial impact on motor performance. We used recombinant adenovectors (RAds) expressing either green fluorescent protein (GFP) or rat IGF-I. Injection in the lateral or fourth ventricle led to high transgene expression in the ependymal cell layer in the brain and cervical spinal cord. RAd-IGF-I-injected rats but not RAd-GFP-injected controls, showed significantly increased levels of CSF IGF-I. Motor tests showed the expected age-related decline in aged rats. Seventeen-day IGF-I gene therapy induced a significant improvement in motor performance in the aged but not in the young animals. These results show that IGF-I is an effective restorative molecule in the aging brain and spinal cord. The data also reveal that the ependymal route constitutes a promising approach for implementing protective IGF-I gene therapy in the aging CNS.

Embarazo como factor de riesgo de hospitalización y muerte en la pandemia por influenza A (H1N1) en Paraguay / Pregnancy as a risk factor for hospitalization and death during influenza A (H1N1) pandemic in Paraguay

La influenza A (H1N1) se ha identificado como la causa de epidemia de Infección Respiratoria Aguda en Paraguay y en el mundo. Se analizaron los factores de riesgo asociados a la morbimortalidad en embarazadas con sospecha de infección por H1N1 notificadas a la Dirección General de Vigilancia de la Salud (DGVS) del MSPBS durante los primeros cuatro meses de la pandemia, en comparación con mujeres en edad fértil no embarazadas con sospecha de infección por H1N1. A partir del 28 de abril de 2009, la DGVS comenzó sistemáticamente la vigilancia de H1N1 en todas sus unidades notificadoras, siendo la notificación inmediata y por planilla individual basándose la notificación en la definición de casos confirmado establecidas por el país. Hasta el 25 de agosto del 2009 fueron notificadas 2268 mujeres con sospecha de H1N1, de las cuales 1120 tenían entre 15 a 40 años, y de ellas 117 estaban embarazadas. El 68% (79/117)de las embarazadas requirió hospitalización y la mortalidad fue de 21% (25/117), mientras que en grupo de las no embarazadas el 21% se hospitalizó (288 /1003) y la mortalidad fue de 1,5% (16/1003). Todos los casos fallecidos desarrollaron Infección Respiratoria Aguda Grave caracterizado por un síndrome de distrés respiratorio que en algunos casos requirieron asistencia respiratoria mecánica (ARM). Ninguna de las embarazadas refirió antecedente de co-morbilidad. El 22% (26/117) de las embarazadas y 2,9% (30/1003) de las no embarazadas recibieron Oseltamivir como tratamiento. Se evidencian que la infección produce alta morbimortalidad en embarazadas en comparación con mujeres del mismo grupo de edad, los cuales apoyan la recomendación de un tratamiento antiviral precoz en gestantes, así como un seguimiento clínico cercano.

Influenza A (H1N1) has been identified as the cause of the Acute Respiratory Infection epidemic in Paraguay and the world. Risk factors associated to morbid-mortality in cases of pregnant women with suspicion of H1N1 infection and notified to the General Direction of Health Surveillance (DGVS in Spanish) of the Ministry of Public Health and Social Welfare (MSPBS in Spanish) during the fourth first months of the pandemic were analyzed in comparison to non-pregnant fertile women with suspicion of H1N1 infection. From April28, 2009 the DGVS started a systematic surveillance of H1N1 in all its notifying units, being the notification immediate and by individual spreadsheet basing the notification inthe case definition established by the country. Until August 25, 2009 2,268 cases of women with suspicion of H1N1 were notified, 1,120 of them were between 15 to 40 years and from them 117 were pregnant. Sixty eight percent (79/117) of the pregnant women required hospitalization and mortality was 21% (25/117) while in the non-pregnant women, 21% were hospitalized (288 /1003) and mortality was 1.5% (16/1003). All deceased cases developed Serious Acute Respiratory Infection characterized by a respiratory distress syndrome that, in some cases, required mechanical respiratory assistance (MRA). None of the regnant women referred history of co-morbidity. Twenty two percent (26/117) of the pregnant women and 2.9% (30/1003) of the non-pregnant women received Oseltamivir as treatment. These results show that the infection produces high morbid-mortality in pregnant women in comparison to women of the same age group, supporting the recommendation of an early antiviral treatment in pregnant women as well as a close clinical follow-up.

Glial cell line-derived neurotrophic factor gene therapy ameliorates chronic hyperprolactinemia in senile rats.

Progressive dysfunction of hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons during normal aging is associated in the female rat with chronic hyperprolactinemia. We assessed the effectiveness of glial cell line-derived neurotrophic factor (GDNF) gene therapy to restore TIDA neuron function in senile female rats and reverse their chronic hyperprolactinemia. Young (2.5 months) and senile (29 months) rats received a bilateral intrahypothalamic injection (10(10) pfu) of either an adenoviral vector expressing the gene for beta-galactosidase; (Y-betagal and S-betagal, respectively) or a vector expressing rat GDNF (Y-GDNF and S-GDNF, respectively). Transgenic GDNF levels in supernatants of GDNF adenovector-transduced N2a neuronal cell cultures were 25+/-4 ng/ml, as determined by bioassay. In the rats, serum prolactin (PRL) was measured at regular intervals. On day 17 animals were sacrificed and neuronal nuclear antigen (NeuN) and tyrosine hydroxylase (TH) immunoreactive cells counted in the arcuate-periventricular hypothalamic region. The S-GDNF but not the S-betagal rats, showed a significant reduction in body weight. The chronic hyperprolactinemia of the senile females was significantly ameliorated in the S-GDNF rats (P<0.05) but not in the S-betagal rats. Neither age nor GDNF induced significant changes in the number of NeuN and TH neurons. We conclude that transgenic GDNF ameliorates chronic hyperprolactinemia in aging female rats, probably by restoring TIDA neuron function.

Multi-luminescent hybrid gadolinium oxide nanoparticles as potential cell labeling.

This manuscript analyses the use of newly developed hybrid gadolinium oxide nanoparticles as cell-labeling tracers. The nanoparticles are core-shell particles composed of a core of gadolinium oxide of [2-4] nm and a protecting shell of polysiloxane [1-3 nm] where different organic dyes (fluoresceine isothiocyanate (FITC) or rhodamine B isothiocyanate (RBITC)) are embedded. They are functionalized with poly(ethylene glycol)bis(carboxymethyl) to ensure their colloidal stability in biological buffers. These particles are potential multi-labeling tracers (magnetic and optical). In this paper, we show by optical imaging that they can be efficiently internalized in cells without cell alteration. The in-vitro uptake of the nanoparticles was followed in two cell lines (human fibroblasts and a human adenocarnima cell lines MCF7 cells). Nanoparticles distribution within cells was analysed by confocal analysis, and gadolinium concentration within cells was quantified by mass spectrometry (ICP-MS analysis). Nanoparticles uptake is found to be fast and efficient for both cell lines, with fluorescent labeling visible after 10 min of incubation whatever the nature of the fluorophore. The fluorescent intensity is mainly found as concentrated dots in the perinuclear region of the cells and decreases with the number of days in culture, but is still easily detectable after 3 days in culture. No significant effect on cell growth was detected. Finally, we show in this study the protective effect of the polysiloxane layer: encapsulation of RBITC within the polysiloxane shell, leads to a better photostability of this low cost dye than Cy3 and even reach a level comparable to Alexa 595. With their high photostability and long-lasting contrast properties, these hybrid luminescent nanoparticles appears thus as a versatile solution to assess multiple cell fate both in in-vitro cell model as well as in-vivo.

The ependymal route for insulin-like growth factor-1 gene therapy in the brain.

I.c.v. administration of the peptide insulin-like growth factor-1 (IGF-1) has been shown to be an effective neuroprotective strategy in the brain of different animal models, a major advantage being the achievement of high concentrations of IGF-1 in the brain without altering serum levels of the peptide. In order to exploit this therapeutic approach further, we used high performance recombinant adenoviral (RAd) vectors expressing their transgene under the control of the potent mouse cytomegalovirus immediate early (mCMV) promoter, to transduce brain ependymal cells with high efficiency and to achieve effective release of transgenic IGF-1 into the cerebrospinal fluid (CSF). We constructed RAd vectors expressing either a chimeric green fluorescent protein fused to HSV-1 thymidine kinase (TK/GFP)(fus), or the cDNA encoding rat IGF-1, both driven by the mCMV promoter. The vectors were injected into the lateral ventricles of young rats and chimeric GFP expression in brain sections was assessed by fluorescence microscopy. The ependymal cell marker vimentin was detected by immunofluorescence and nuclei were labeled with the DNA dye 4',6-diamidino-2-phenylindole. Blood and CSF samples were drawn at different times post-vector injection. In all cerebral ventricles, vimentin immunoreactive cells of the ependyma were predominantly transduced by RAd-(TK/GFP)(fus), showing nuclear and cytoplasmic expression of the transgene. For tanycytes (TK/GFP)(fus) expression was evident in their cytoplasmic processes as they penetrated deep into the hypothalamic parenchyma. I.c.v. injection of RAd-IGF-1 induced high levels of IGF-1 in the CSF but not in serum. We conclude that the ependymal route constitutes an effective approach for implementing experimental IGF-1 gene therapy in the brain.

Gene therapy in the neuroendocrine system.

The implementation of experimental gene therapy in animal models of neuroendocrine diseases is an area of growing interest. In the hypothalamus, restorative gene therapy has been successfully implemented in Brattleboro rats, an arginine vasopressin (AVP) mutant which suffers from diabetes insipidus, and in Koletsky (fa(k)/fa(k)) and in Zucker (fa/fa) rats which have leptin receptor mutations that render them obese, hyperphagic and hyperinsulinemic. In the above models, viral vectors expressing AVP, leptin receptor b and proopiomelanocortin, respectively, were stereotaxically injected in the relevant hypothalamic regions. In rats, aging brings about a progressive degeneration and loss of hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons, which are involved in the tonic inhibitory control of prolactin secretion and lactotropic cell proliferation. Stereotaxic injection of an adenoviral vector expressing insulin-like growth factor I corrected their chronic hyperprolactinemia and restored TIDA neuron numbers. Spontaneous intermediate lobe pituitary tumors in a retinoblastoma (Rb) gene mutant mouse were corrected by injection of an adenoviral vector expressing the human Rb cDNA and experimental prolactinomas in rats were partially reduced by intrapituitary injection of an adenoviral vector expressing the HSV1-thymidine kinase suicide gene. These results suggest that further implementation of gene therapy strategies in neuroendocrine models may be highly rewarding.

Cellular distribution of growth hormone-releasing hormone receptor in human reproductive system and breast and prostate cancers.

Growth hormone releasing hormone receptor (GHRH-R) mRNA and protein was first localized to the anterior pituitary gland, consequent with the action of its ligand on GH synthesis and release. Subsequent studies found GHRH-R also expressed in the hypothalamus and in systemic tissues including those of the reproductive system. In the present work, we studied the distribution of GHRH-R in human reproductive system of males and females by immunohistochemical method. GHRH-R immunostaining was localized in male reproductive system: Leydig cells, Sertoli and basal germ cells of the seminiferous tubules and prostate secretory cells. GHRH-R immunostaining was also demonstrated in the ovary: oocytes, follicular cells, granulosa, thecal and corpus luteum cells. Endometrial glands, placenta and normal mammary glands also showed GHRH-R immunostaining. Our results demonstrate the localization of GHRH-R in the reproductive system, which may mediate the direct action of GHRH in these tissues. Moreover, GHRH-R was demonstrated in prostate and breast carcinomas, opening a variety of possibilities for the use of GHRH antagonists in the treatment of prostatic and mammary tumors.

Suppressor of cytokine signalling gene expression is elevated in breast carcinoma.

Cytokines are important for breast cell function, both as trophic hormones and as mediators of host defense mechanisms against breast cancer. Recently, inducible feedback suppressors of cytokine signalling (SOCS/JAB/SSI) have been identified, which decrease cell sensitivity to cytokines. We examined the expression of SOCS genes in 17 breast carcinomas and 10 breast cancer lines, in comparison with normal tissue and breast lines. We report elevated expression of SOCS-1-3 and CIS immunoreactive proteins within in situ ductal carcinomas and infiltrating ductal carcinomas relative to normal breast tissue. Significantly increased expression of SOCS-1-3 and CIS transcripts was also shown by quantitative in situ hybridisation within both tumour tissue and reactive stroma. CIS transcript expression was elevated in all 10 cancer lines, but not in control lines. However, there was no consistent elevation of other SOCS transcripts. CIS protein was shown by immunoblot to be present in all cancer lines at increased levels, mainly as the 47 kDa ubiquitinylated form. A potential proliferative role for CIS overexpression is supported by reports that CIS activates ERK kinases, and by strong induction in transient reporter assays with an ERK-responsive promoter. The in vivo elevation of SOCS gene expression may be part of the host/tumour response or a response to autocrine/paracrine GH and prolactin. However, increased CIS expression in breast cancer lines appears to be a specific lesion, and could simultaneously shut down STAT 5 signalling by trophic hormones, confer resistance to host cytokines and increase proliferation through ERK kinases.

Leukemia inhibitory factor is a key signal for injury-induced neurogenesis in the adult mouse olfactory epithelium.

The mammalian olfactory epithelium (OE) is composed of primary olfactory sensory neurons (OSNs) that are renewed throughout adulthood by local, restricted neuronal progenitor cells. The molecular signals that control this neurogenesis in vivo are unknown. Using olfactory bulb ablation (OBX) in adult mice to trigger synchronous mitotic stimulation of neuronal progenitors in the OE, we show the in vivo involvement of a cytokine in the cellular events leading to the regeneration of the OE. We find that, of many potential mitogenic signals, only leukemia inhibitory factor (LIF) is induced before the onset of neuronal progenitor proliferation. The rise in LIF mRNA expression peaks at 8 hr after OBX, and in situ RT-PCR and immunocytochemistry indicate that LIF is upregulated, in part, in the injured neurons themselves. This rise in LIF is necessary for injury-induced neurogenesis, as OBX in the LIF knock-out mouse fails to stimulate cell proliferation in the OE. Moreover, delivery of exogenous LIF to the intact adult OE using an adenoviral vector stimulates BrdU labeling in the apical OE. Taken together, these results suggest that injured OSNs release LIF as a stimulus to initiate their own replacement.

High stromal and epithelial human gh gene expression is associated with proliferative disorders of the mammary gland.

We have demonstrated and localized human GH (hGH) gene expression in surgical specimens of normal human mammary gland and in proliferative disorders of the mammary gland of increasing severity using sensitive in situ RT-PCR methodology. hGH mRNA identical to pituitary hGH mRNA was first detected by RT-PCR of RNA derived from samples of normal human mammary gland. Cellular localization of hGH gene expression in the normal mammary gland exhibited restriction to luminal epithelial and myoepithelial cells of the ducts and to scattered stromal fibroblasts. We subsequently examined the expression of the hgh gene in three progressive proliferative disorders of the human mammary gland, i.e. A benign lesion (fibroadenoma), a pre-invasive stage (intraductal carcinoma) and an invasive ductal carcinoma. hGH mRNA was readily detected in the tumoral and non-tumoral epithelial components and also in cells of the reactive stroma including fibroblasts, myofibroblastic and myoepithelial cells, inflammatory infiltrate lymphocytes and endothelial cells in areas of neovascularization. In all three proliferative disorders examined, the intensity of the cellular labeling observed in both the epithelial and stromal compartments was always stronger compared with that in adjacent normal tissue. hGH protein was also present in significantly higher concentration in extracts derived from proliferative disorders of the mammary gland compared with extracts derived from normal mammary gland. We also examined hGH gene expression in axillary lymph nodes not containing and containing metastatic mammary carcinoma. hGH gene expression was evidenced in metastatic mammary carcinoma cells and in reactive stromal cells by both in situ hybridization and in situ RT-PCR. In contrast, in lymph nodes not containing metastatic mammary carcinoma, hGH mRNA was detected only by use of in situ RT-PCR. Thus, increased expression of the hGH gene in the epithelial component and the de novo stromal expression in proliferative disorders of the mammary gland are suggestive of a pivotal role for autocrine hGH in neoplastic progression of the mammary gland.

Intrauterine oxytocin system. Compartmental distribution of oxytocin and oxytocin receptors in rat endometrial epithelium.

At term, uterine epithelial cells express oxytocin (OT) as well as the OT receptor (OTR). Like other epithelial cells, uterine epithelial cells are polarized and sort secretory and membrane components to the apical or the basolateral cell surface. We have studied the subcellular localization of OT-like immunoreactivity (OT-IR) and OTR-IR in rat uterine epithelium by immuno-gold labelling of ultrathin frozen sections. Our observations indicate that OT and OTR are both distributed preferentially to the apical surface of rat uterine epithelial cells. OT-IR showed a 6-fold apical versus basolateral preference and was localized in apical secretory vesicles, suggesting that uterine OT is released by apical exocytosis. OTR-IR was localized to the apical surface with a 9-fold apical versus basolateral preference and was found specifically in association with apical microvilli. The present findings represent the first example of a G protein-coupled receptor that is preferentially localized on the microvillar compartment and support the concept of an autocrine uterine OT system at the apical side of the uterine epithelium.

Cellular localization of orexin receptors in human pituitary.

Orexins-A and -B are hypothalamic peptides derived from a precursor called prepro-orexin and relationated with the stimulation of food intake. They act on G protein receptors named orexin receptor 1 (OX(1)R) and orexin receptor 2 (OX(2)R), respectively. In the present study, we used RT-PCR and immunohistochemical techniques to detect the presence of OX(1)R and OX(2)R in human pituitary. A band of the expected size for both OX(1)R and OX(2)R was shown in human pituitary by RT-PCR. The cellular localization of OX(1)R and OX(2)R was carried out using histological techniques. By consecutive sections we demonstrated that OX(1)R was present in acidophil, diffusely distributed cells, which represent the half of the total adenohypophysis cell population. As was expected, these cells were shown to coexpress GH. OX(2)R was found in the pars intermedia and in clusters of basophil cells of the anterior pituitary, which coexpress ACTH. These results were confirmed by double immunofluorescence techniques. We also found focal positivity in axon terminals of neurohypophysis, more intense for OX(2)R than for OX(1)R. In conclusion, these results demonstrated for the first time that OX(1)R and OX(2)R were expressed by somatotrope and corticotrope cells, respectively.

Autocrine human growth hormone (hGH) regulation of human mammary carcinoma cell gene expression. Identification of CHOP as a mediator of hGH-stimulated human mammary carcinoma cell survival.

By use of cDNA array technology we have screened 588 genes to determine the effect of autocrine production of human growth hormone (hGH) on gene expression in human mammary carcinoma cells. We have used a previously described cellular model to study autocrine hGH function in which the hGH gene or a translation-deficient hGH gene was stably transfected into MCF-7 cells. Fifty two of the screened genes were regulated, either positively () or negatively (), by autocrine production of hGH. We have now characterized the role of one of the up-regulated genes, chop (gadd153), in the effect of autocrine production of hGH on mammary carcinoma cell number. The effect of autocrine production of hGH on the level of CHOP mRNA was exerted at the transcriptional level as autocrine hGH increased chloramphenicol acetyltransferase production from a reporter plasmid containing a 1-kilobase pair fragment of the chop promoter. The autocrine hGH-stimulated increase in CHOP mRNA also resulted in an increase in CHOP protein. As a consequence, autocrine hGH stimulation of CHOP-mediated transcriptional activation was increased. Stable transfection of human CHOP cDNA into mammary carcinoma cells demonstrated that CHOP functioned not as a mediator of hGH-stimulated mitogenesis but rather enhanced the protection from apoptosis afforded by hGH in a p38 MAPK-dependent manner. Thus transcriptional up-regulation of chop is one mechanism by which hGH regulates mammary carcinoma cell number.

Cellular localization of orexin receptors in human pituitary.

Orexins-A and -B are hypothalamic peptides derived from a precursor called prepro-orexin and relationated with the stimulation of food intake. They act on G protein receptors named orexin receptor 1 (OX(1)R) and orexin receptor 2 (OX(2)R), respectively. In the present study, we used RT-PCR and immunohistochemical techniques to detect the presence of OX(1)R and OX(2)R in human pituitary. A band of the expected size for both OX(1)R and OX(2)R was shown in human pituitary by RT-PCR. The cellular localization of OX(1)R and OX(2)R was carried out using histological techniques. By consecutive sections we demonstrated that OX(1)R was present in acidophil, diffusely distributed cells, which represent the half of the total adenohypophysis cell population. As was expected, these cells were shown to coexpress GH. OX(2)R was found in the pars intermedia and in clusters of basophil cells of the anterior pituitary, which coexpress ACTH. These results were confirmed by double immunofluorescence techniques. We also found focal positivity in axon terminals of neurohypophysis, more intense for OX(2)R than for OX(1)R. In conclusion, these results demonstrated for the first time that OX(1)R and OX(2)R were expressed by somatotrope and corticotrope cells, respectively.

Prenatal and adult growth hormone gene expression in rat lymphoid organs.

Growth hormone (GH) exerts its immune effects on mature lymphocytes through an autocrine/paracrine mechanism. We investigated the prenatal synthesis of GH mRNA in rat lymphoid organs using the sensitive in situ RT-PCR methodology. We show that GH transcripts are detectable in the thymus and liver of the 18-day fetus. At this stage, all thymocytes are immature and express the GH gene. In fetal liver, GH gene expression was localized in circulating lymphocytes and in hematopoietic cells surrounding GH mRNA-negative hepatocytes. In situ GH gene expression in fetal lymphoid organs was confirmed by in vitro RT-PCR showing that the amplified product from fetal lymphoid tissues was similar to the product obtained from the pituitary. Moreover, GH gene expression was detected in the thymus, spleen, and ileum Peyer's patches of adult rat, with a localization restricted to the lymphocytes and endothelial and smooth muscle cells of blood vessels. The autocrine/paracrine expression of the GH gene by lymphoid and hematopoietic cells during fetal growth might influence the generation of regulatory cells involved in immunity and hematopoiesis.

HCaRG, a novel calcium-regulated gene coding for a nuclear protein, is potentially involved in the regulation of cell proliferation.

Since a negative calcium balance is present in spontaneously hypertensive rats, we searched for the gene(s) involved in this dysregulation. A cDNA library was constructed from the spontaneously hypertensive rat parathyroid gland, which is a key regulator of serum-ionized calcium. From seven overlapping DNA fragments, a 1100-base pair novel cDNA containing an open reading frame of 224 codons was reconstituted. This novel gene, named HCaRG (hypertension-related, calcium-regulated gene), was negatively regulated by extracellular calcium concentration, and its basal mRNA levels were higher in hypertensive animals. The deduced protein showed no transmembrane domain, 67% alpha-helix content, a mutated calcium-binding site (EF-hand motif), four putative "leucine zipper" motifs, and a nuclear receptor-binding domain. At the subcellular level, HCaRG had a nuclear localization. We cloned the human homolog of this gene. Sequence comparison revealed 80% homology between rats and humans at the nucleotide and amino acid sequences. Tissue distribution showed a preponderance in the heart, stomach, jejunum, kidney (tubular fraction), liver, and adrenal gland (mainly in the medulla). HCaRG mRNA was significantly more expressed in adult than in fetal organs, and its levels were decreased in tumors and cancerous cell lines. We observed that after 60-min ischemia followed by reperfusion, HCaRG mRNA declined rapidly in contrast with an increase in c-myc mRNA. Its levels then rose steadily to exceed base line at 48 h of reperfusion. HEK293 cells stably transfected with HCaRG exhibited much lower proliferation, as shown by cell count and [(3)H]thymidine incorporation. Taken together, our results suggest that HCaRG is a nuclear protein potentially involved in the control of cell proliferation.

Increased expression of growth hormone and prolactin receptors in hepatocellular carcinomas.

The liver is an essential target tissue for growth hormone (GH) and prolactin (PRL). The aim of this study was to determine the in situ expression of growth hormone receptor (GHR) and prolactin receptor (PRLR) in hepatocellular carcinomas and to compare the results with normal liver. For this purpose, in situ hybridization (ISH) and immunohistochemical techniques were performed and several tests were conducted to validate the results. By radioactive ISH, all the hepatocellular carcinomas studied showed labeling for GHR and PRLR mRNAs. Relative expression levels, determined by computer-assisted microdensity, were higher in hepatocellular carcinomas than in normal liver. Immunohistochemistry led us to confirm the constant expression of both receptor proteins in hepatocellular carcinomas and normal liver and to demonstrate their localization not only in the cytoplasm but also in the nucleus. These results confirm that the liver is a major GH and PRL target tissue and suggest that in hepatocellular carcinomas the proliferative effects of these hormones may be increased by a higher expression of their receptors.

Internalization and nuclear translocation of IFN-gamma and IFN-gammaR: an ultrastructural approach.

IFN-gamma signalling involves the Jak-STAT pathway. However, several hypothesis have been proposed where the receptor and its ligand itself took an active role within the cell. Using a quantitative immunogold approach, we found that both IFN-gamma and its receptor are rapidly internalized and translocated in the nucleus. We found that cell surface heparan sulfate, which binds IFN-gamma, delayed the nuclear accumulation of IFN-gamma suggesting that these molecules serve as storage depot around the cell for local delivery of the cytokine.

[Detection of human papillomavirus in cutaneous extragenital Bowen's disease in immunocompetent patients]. / Détection de papillomavirus humains dans la maladie de Bowen cutanée extragénitale chez le sujet immunocompétent.

INTRODUCTION: A specific link between human papillomavirus (HPV) types 16, 18, 31, and 33 and genital carcinomas and between HPV type 5 and cutaneous extragenital carcinomas in patients with epidermodysplasia verruciformis and renal transplant has been previously found. The aim of this prospective study was to detect HPV in cases of cutaneous extragenital Bowen's disease (BD) from non-immunosuppressed patients. PATIENTS AND METHODS: Twelve cases of cutaneous extragenital BD or Bowen's carcinoma (BC), seen in the period 1994-1996 and confirmed by histologic examination, were included in the study. Tissue sections were studied by in situ hybridization with a mixture of HPV DNA probes and specific HPV DNA probes. In addition, study on fresh materiel from 1995 included: Southern blot hybridization with various usual HPV probes (6, 11, 16, 18, 31, 33, 35, 39, 42), polymerase chain reaction (PCR) with hybridization using consensus HPV probes and probes specific for HPV types 6, 11, 16, 18 and 33. In positive samples with conventional PCR, in situ PCR with probes specific for HPV types 6/11 and 16 was performed on tissue sections. RESULTS: In situ hybridization was negative in all the cases. Southern blot hybridization was negative in our 9 studied cases. Three cases studied by consensus PCR were positive. PCR with specific HPV probes revealed positivity on two of these cases: HPV 6 in one, and HPV 16 in another. In situ PCR was positive with a mixed 6/11 HPV probe in the third positive consensus PCR case. DISCUSSION: Our study revealed the presence of HPV in 3 out of 12 cases of cutaneous extragenital BD and BC. HPV type 16, found in BC of skull, was the most usually found type in the literature. HPV types 6/11, detected in 2 cases, were rarely found in cutaneous extragenital BD and BC and these results are in favor of the oncogenic effect of these virus types. In our study, in situ hybridization and Southern blot hybridization were negative in all the cases; HPV was only found in 3 cases by conventional PCR and in 1 case by in situ PCR. The low range of detection of HPV in cutaneous extragenital BD may be due to the used methods, to difficulties related to sampling and/or to a low number of copies of the HPV genoma.