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.

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.

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.

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.

Selective expression of lysyl oxidase (LOX) in the stromal reactions of broncho-pulmonary carcinomas.

Lysyl oxidase (LOX) is the extracellular enzyme that initiates the main pathway of collagen and elastin cross-linking. LOX has also been correlated with the ras recision gene, a putative tumour suppressor isolated from revertants of ras-transformed fibroblasts. The present study investigates the potential correlation of LOX-dependent matrix protein cross-linking in the stromal reaction of lung carcinomas, with reference to the architecture of the main stromal reactions accompanying the neoplastic breast tissues. A strong LOX expression was associated with the hypertrophic scar-like stromal reaction found at the front of tumour progression in squamous carcinomas, adenocarcinomas, large cell carcinomas, or at sites of initial extense in bronchiolo-alveolar carcinomas. In contrast, little or no LOX expression was found within the stromal reaction of invasive carcinomas, small cell carcinomas, and neuro-endocrine carcinomas. The significance of LOX expression and of the stromal reaction are discussed, in light of data that associate LOX expression with tumours displaying a rather good prognosis.

CD40 expression by human bronchial epithelial cells.

CD40 is a 50-kDa protein expressed on B cells, dendritic cells, monocytes and epithelial cells, but the distribution of CD40 expression in humans is not completely known. It binds to a ligand (CD40L) which is expressed essentially on activated T cells. The interaction between CD40 and CD40L plays important roles in immune responses. CD40 expression was investigated on bronchial tissues and human bronchial cell lines using immunohistochemistry, immunofluorescence staining and analysis with a cytometer, respectively. Constitutive CD40 expression, but not that of CD40L, was slightly detectable on normal human bronchial epithelial cells (HBEC) in situ and on an adult lung adenocarcinoma (SKLU1) cell line, while another cell line, a bronchial transformed SV40 cell line (WI26VA4), was negative for CD40. Among the various cytokines tested, only interferon (IFN)-gamma was found to induce CD40 expression on WI26VA4. Tumour necrosis factor (TNF)-alpha was the best cytokine able to up-regulate CD40 in SKLU1 cells. A combination of IFN-gamma and TNF-alpha was slightly more effective than the cytokine alone at up-regulating CD40 expression on both cell lines. We further investigated the functional consequences of CD40 ligation on both cell lines. These bronchial cells expressed CD40, HLADR and CD54 under basal conditions or when stimulated by cytokines. Stimulation through CD40 did not affect cell-surface-antigen expression on either cell line. The production of cytokines such as interleukin (IL)-6 and granulocyte macrophage-colony stimulating factor (GM-CSF) by HBEC has been described. SKLU1 and WI26VA4 cells released IL-6 and GM-CSF spontaneously. Whatever the case, CD40 engagement did not modulate spontaneous or TNF-alpha-induced production of these two cytokines. These data indicate for the first time that normal HBEC express CD40 in situ. Further investigations are required in order to determine the role of CD40 on normal HBEC.

Lysyl oxidase-like protein localizes to sites of de novo fibrinogenesis in fibrosis and in the early stromal reaction of ductal breast carcinomas.

Lysyl oxidase (LO) initiates the first step in the crosslinking of collagens and elastin and has also been shown to function as a tumor suppressor. The purpose of the present work was to determine whether the products of a newly described LO-like gene (LOXL) that encodes a close homolog of LO, the LO-like (LOL) protein, is associated with extracellular matrix remodeling during fibrotic disorders. Specific antibody against LOL identified proteins of approximately 30, 42, 52 and 68 kd in various cells and in bovine aorta. These proteins were immunochemically distinct from the recombinant LO expressed by fibroblasts and from the bovine aorta LO. The LO gene (LOX) and LOXL were transiently up-regulated at early stages of liver granuloma development in Schistosoma mansoni-infected mice, although the peak of LOL mRNA synthesis preceded that of LO. LOL protein and LO were colocalized at sites of fibrogenesis in human lung fibrosis and in the stromal reaction of bronchiolo-alveolar carcinomas and of in situ ductal breast tumors. In conclusion, the LOL protein was identified as a secreted protein and localized in the extracellular matrix in active fibrotic diseases and in the early stromal reaction of breast cancer.

Lysyl oxidase gene expression in the stromal reaction to in situ and invasive ductal breast carcinoma.

Lysyl oxidase is involved in the main pathway of collagen and elastin cross-linking: it has a role in the maturation of fibrillar matrix proteins in fibrosing processes and dictates their stability against metalloproteases. The stromal reaction patterns in ductal breast carcinoma are known to be morphologically varied. This has raised the hypothesis that there might be a differential expression of the lysyl oxidase gene as a function of stromal reaction pattern. The present study investigates this potential correlation and the role of matrix protein cross-linking in stromal differentiation. Lysyl oxidase was detected by immunohistochemistry and lysyl oxidase gene expression by in situ hybridization. Maximal expression was observed in myofibroblasts and myoepithelial cells around in situ tumors and in the reactive fibrosis facing the invasion front of infiltrating tumors. The lysyl oxidase substrates were observed in parallel, resulting in the stabilization of a scar-like peritumor barrier. In contrast, a lack of lysyl oxidase was associated with the loose or scirrhous stroma accompanying invading tumors; here, in situ hybridization revealed type I collagen synthesis, resulting in the deposition of non-cross-linked matrix proteins susceptible to degradation. The early development of a cross-linked matrix around ductal breast carcinoma suggests a possible bost defense mechanism, whereas the synchronous or late stromal reaction lacking lysyl oxidase favors tumor dispersion.

Fibrosing vasculitis in Wegener's granulomatosis: ultrastructural and immunohistochemical analysis of the vascular lesions.

This study of two cases of pulmonary Wegener's granulomatosis (WG) focuses on the ultrastructural aspects of the vascular wall injury and on the immunohistochemical characterization of the perivascular connective matrix. The iterative waves of endothelial cell necrosis and regeneration are demonstrated by the multilamellar appearance of the basal lamina. Neutrophils infiltrate the vessel wall and myofibroblasts are recruited to injured vessels. The perivascular connective matrix associates basement-membrane like and fibrillar material with fibrin deposits. The initiation of the fibrosing process was assessed by the visualization of matrix molecules involved in targeting (p-fibronectin), organizing (cellular fibronectin and tenascin) and stabilizing (lysyl-oxidase) the fibrogenic activity. These elementary lesions affect different levels of the vascular tree, and capillaritis is involved in the extension of the pathological process. Lysyl-oxidase labelling reveals the fibrosing front which is located on the border of dense fibrosis. The markers of fibrosing activity disappear in the areas of fibrosis following vasculitis and/or ischaemic necrosis and/or granulomatosis. Vasculitis plays a major role in both the genesis and progression of the fibrosis observed in the late stage of WG.

Transient expression of lysyl oxidase by liver myofibroblasts in murine schistosomiasis.

BACKGROUND: Murine schistosomiasis provides an experimental model of reversible fibrosis. The lysyl oxidase catalyzes the first step of collagen and elastin enzymatic cross-linking and appears to be a crucial factor in stabilizing the neosynthesized extracellular matrix in the liver. EXPERIMENTAL DESIGN: A cDNA probe encoding a portion of the murine lysyl oxidase was cloned, and antibodies were raised against the corresponding recombinant peptide expressed as a fusion protein. Both tools were used to examine the expression of the lysyl oxidase mRNAs and peptides, all within granulomas and cells extracted from infected liver. RESULTS: Transient up-regulation of two dominant 4.5 kb and 5.5 kb transcripts was observed among four mRNAs hybridizing with the lysyl oxidase cDNA probe during the development of granulomas. An identical time course was obtained for alpha 1(I) procollagen messenger expression. The lysyl oxidase expression was observed in type I collagen producing cells mainly localized at the periphery of fibroinflammatory granulomas and it disappeared in late granulomas. The lysyl oxidase and type I collagen expressing cells, located within granulomas, exhibited the characteristics of myofibroblasts, as judged by their expression of alpha-smooth muscle actin and desmin and by their ultrastructural morphology. Four antigenically related peptides were immunopurified from an enriched preparation of myofibroblast-like cells extracted from infected mouse liver. Two of these peptides had the molecular weight of prolysyl oxidase (50,000) and activated lysyl oxidase (32,000). The two others (28,000 and 66,000) might correspond to cleavage product or dimeric form respectively. CONCLUSIONS: This study demonstrates that the lysyl oxidase was transiently up-regulated at the transcriptional level parallely to alpha(1)I procollagen within developing granulomas. Myofibroblasts are involved in the expression of the lysyl oxidase which may be secreted as a proenzyme and an activated enzyme.

Somatotropic adenomas without acromegaly.

Seventeen somatotropic adenomas removed from patients without acromegaly were studied. Thirteen of them presented as a prolactinoma with amenorrhea and/or galactorrhea and elevated serum PRL levels. According to basal serum GH levels, the patients were divided into two groups, namely Group I: GH slightly elevated (n = 4) and group II: GH less than or equal to 5 micrograms/l (n = 13). The tumoral GH secretion was proved by immunocytochemistry in all cases and by intratumoral RIA, in vitro study and/or in situ hybridization in five of them. Pathological, clinical and biochemical relationships suggested two anatomoclinical aspects. In group I, the tumors were small, well-differentiated somatotropic adenomas with clinically silent GH hypersecretion. It is probably an early stage of the disease. In group II, the tumors were large with normal GH serum levels. They were poorly differentiated and secreted very low amounts of GH. In nine of them, PRL and/or PRL mRNA expression were also detected. These tumors do not secrete enough GH to increase serum levels and cause acromegaly. The somatotropic adenomas without acromegaly correspond to two anatomoclinical aspects of the disease.

Immunostaining of a cell type in the islets of Langerhans of the monkey Macaca irus by antibodies against S-100 protein.

S-100 protein-immunoreactive cells were demonstrated by immunocytochemical procedures in the pancreatic islets of Langerhans in the monkey Macaca irus. By use of antibodies against human S-100 protein or bovine S-100 protein, these cells were observed in all islets in the head and tail portions of the pancreas. Immunostained cells were usually located in the center of the islets or sometimes found in a more widely distributed form, but they were never arranged in a regular concentric fashion. The number of immunoreactive cells varied from one islet to another but it was relatively limited making up only 0.75%-6.3% of all insular cells. With the use of the double-immunoenzymatic procedure for demonstration of the four main endocrine cell types (insulin-, glucagon-, somatostatin- and pancreatic polypeptide producing elements), it was possible to establish that S-100 protein-immunoreactive cells represent a distinct cell type. Antibodies against S-100 protein-stained neuroinsular complexes. The present findings speak in favor of a new cell type to be added to the large variety of S-100 protein-immunoreactive cells outside the central nervous system.

Immunocytochemical localization of S-100 protein in stellate cells (folliculo-stellate cells) of the adenohypophysis in the monkeys Macaca irus and Cercopithecus aethiops.

With the use of an antibody against bovine S-100 protein, it was possible to reveal a characteristic cell type in the pars distalis and the pars tuberalis of the monkey Macaca irus. In the adenohypophysis of Cercopithecus aethiops, labeled cells were present in the pars distalis, pars tuberalis, and pars intermedia. These cells, so-called folliculo-stellate cells, were found in all pituitaries studied. Surprisingly, an antibody against human S-100 protein did not label the stellate cells of the adenohypophysis. However, in Macaca irus, this antibody gave a strong positive reaction with various other cell types (interstitial cells of the pineal gland, Müller cells of the retina, autonomic ganglionic cells, glial cells of the central nervous system, Schwann cells, Bergmann glia of the cerebellum, fat cells, reticular cells of lymphoid organs). By use of double immunoenzymatic labeling, it was evident that stellate cells are spatially related either to somatotropes, prolactin cells, "corticotropes", or to glycoprotein-containing cells. Thus, a specific relationship to a particular endocrine-cell type could not be observed.