p16INK4a as an adjunct test in cervical cytology.

BACKGROUND: This study investigated the correlation between cervical cytology, the expression of P16INK4a, and human papilloma virus (HPV) infection. MATERIALS AND METHODS: The study included 100 subjects with suspected pathological cervical lesions. Cervical smears were analyzed for malignancy and p16INK4a. Histological finding represented "the golden standard". RESULTS: Immunocytochemical analysis of protein p16INK4a expression on epithelial cells of cervical smear demonstrated increased p16lNK4a expression in 36.0% of subjects. There was statistically significant positive correlation (Spearman r = 0.70; p < 0.001) between the pathological findings and the intensity of p16INK4a protein expression inside the epithelial cells, as well as with the histological finding (Spearman r = 0.71; p < 0.001). The intensity of p16INK4a protein expression in cytology finding was significantly higher in HPV16 positive patients (Mann- Whitney test, p = 0.0065). CONCLUSION: Good correlation between the expression rate and the severity of lesions indicates that this test might improve the results of cytology and HPV screening, as well as the results of predicting the prognosis of the disorder of the cervix.

Regulation of immunoreactive GAP-43 expression in rat cortical macroglia is cell type specific.

Growth-associated protein 43 (GAP-43) is an abundant, intensely investigated membrane phosphoprotein of the nervous system (Benowitz, L.I., and A. Routtenberg. 1987. Trends Neurosci. 10:527-532; Skene, J. H. P. 1989. Annu. Rev. Neurosci. 12:127-156), with a hitherto unknown function. We have previously demonstrated that astrocytes, brain macroglial cells, contain GAP-43 (Steisslinger, H. W., V. J. Aloyo, and L. Vitkovic, 1987. Brain Res. 415:375-379; Vitkovic, L., H. W. Steisslinger, V. J. Aloyo, and M. Mersel. 1988. Proc. Natl. Acad. Sci. USA. 85:8296-8300; Vitkovic L., and M. Mersel. 1989. Metab. Brain Dis. 4:47-53). Results from double immunofluorescent labeling experiments presented here show that oligodendrocytes also contain GAP-43 immunoreactivity (GAP-43ir). Thus, all three macroglial cell types of the central nervous system (type I and type 2 astrocytes and oligodendrocytes) contain GAP-43. Whereas immunoreactive GAP-43 is expressed by progenitors of all macroglial cell types, the developmental regulation of its expression is cell type specific. Immunoreactive GAP-43 is downregulated in type 1 astrocytes, and constitutively expressed in both type 2 astrocytes and oligodendrocytes. These results may be relevant to potential function(s) of GAP-43.

Mast cells in the brain: evidence and functional significance.

For the past two decades the brain has been considered to be an immune-privileged site that excludes circulating cells from the parenchyma. New evidence indicates that some hematocytes reside in the brain, while others traffic through it. Mast cells belong to both of these functional types. Moreover, the appearance of mast cells in the CNS can be triggered behaviorally. After a brief period of courtship, for example, there is a marked increase in mast cells in the medial habenula of sexually active doves compared with controls. Exposure to gonadal steroids that occur endogenously or that are administered exogenously increases both the number of mast cells and their state of activation in the brain. These results show that hematopoietic cells can provide targeted delivery of neuromodulators to specific regions of the brain, thereby influencing neural-endocrine interactions.

Glucocorticoid receptor-mediated induction of glutamine synthetase in skeletal muscle cells in vitro.

We studied the regulation by glucocorticoids of glutamine synthetase in L6 muscle cells in culture. Glutamine synthetase activity was strikingly enhanced by dexamethasone. The dexamethasone-mediated induction of glutamine synthetase activity was blocked by RU38486 [11 beta-(4-dimethylaminophenyl)17 beta-hydroxy-17 alpha-(prop-1-ynyl)estra-4,9-dien-3-one], a glucocorticoid antagonist, indicating the involvement of intracellular glucocorticoid receptors in the induction process. RU38486 alone was without effect. Northern blot analysis revealed that dexamethasone-mediated enhancement of glutamine synthetase activity involves increased levels of glutamine synthetase mRNA. Increased enzyme activity was specific for glucocorticoids; other steroid hormones were essentially without effect. The induction of glutamine synthetase was selective, in that glutaminase activity was not induced by dexamethasone treatment of L6 cells. Thus, glucocorticoids regulate the expression of glutamine synthetase mRNA in cultured muscle cells via interaction with intracellular receptors. Such regulation may be relevant to control of glutamine production by muscle.

Transforming growth factor-beta 1 (TGF-beta 1) expression and regulation in rat cortical astrocytes.

Transforming growth factor-beta 1 (TGF-beta 1) is a potent modulator of immune and glial cells' functions and thus, could play an important role in neuro-immune interaction. However, published reports disagree on whether or not TGF-beta 1 is expressed in normal brain. We demonstrate here the constitutive expression of TGF-beta 1 mRNA but not protein in both cerebral cortex and primary rat cortical astrocytes. Steady-state TGF-beta 1 mRNA level increased 2-fold in adult compared to neonatal cortex and during proliferation and differentiation of astrocytes in primary culture. This response was not accompanied by the appearance of detectable TGF-beta protein either in vivo or in vitro. However, both intracellular immunoreactive TGF-beta and extracellular TGF-beta 1 activity were detected upon in vitro stimulation of astrocytes with interleukin-1 (IL-1). The extracellular TGF-beta 1 increased with time of exposure to and concentration of IL-1. In contrast, the amount of TGF-beta 1 mRNA remained unchanged during stimulation of astrocytes with IL-1. These results suggest that the production of TGF-beta 1 in astrocytes is regulated at both mRNA and protein levels. The former may occur during astrocytic development, and the latter during astrocytic response to injury in association with elevation of IL-1.

HIV-1 non-specifically stimulates production of transforming growth factor-beta 1 transfer in primary astrocytes.

HIV-1 expression in monocytes/macrophages can be controlled by transforming growth factor-beta 1 (TGF-beta 1). TGF-beta 1 is present in astrocytes surrounding HIV-1-infected monocyte/macrophages in brain tissue from patients with AIDS but not from seronegative, normal individuals. We sought to determine whether or not production of TGF-beta 1 can be directly stimulated by HIV-1 in astrocytes. Astrocytes from neonatal rat cortex grown in primary culture were exposed to HIV-1 virions for 24 h. One day later, TGF-beta 1 was measured in culture supernatants by a biological assay. HIV-1 caused 1.7-2.1-fold increase in extracellular concentration of TGF-beta 1. TGF beta 1 production also was stimulated by recombinant HIV-1 proteins gp120, p66 and p24. Gp120 labeled with fluorescein was visualized inside astrocytes and its stimulatory effect was not blocked by antibodies against rat CD4. The effect was not specific to HIV-1 and its proteins, because non-opsonized Latex particles and leucine methyl ester (LME) (known to be phagocytosed and endocytosed, respectively, by astrocytes) also stimulated TGF-beta 1 production. The effect was inhibited by two inhibitors of the phago/endocytotic pathway, chloroquine and leupeptin. These results may be relevant to the neuropathogenesis of HIV-1 infection.

Glial cell-specific mechanisms of TGF-beta 1 induction by IL-1 in cerebral cortex.

Transforming growth factor beta-1 (TGF-beta 1) immunoreactive product (IRP) has recently been detected in autopsied brains of individuals who died with central nervous system diseases and/or fever but not in normal individuals or in normal rodent brain. However, the mechanism(s) of induction of TGF-beta 1 in brain and the identity of cells expressing TGF-beta 1 need to be understood before a role, if any, for this potent pleiotropic cytokine in neuropathogenesis can be discerned. Towards this end we determined that IL-1 stimulated the production of TGF-beta 1 IRP in cells and TGF-beta 1 activity in culture fluids of all glial cells, astrocytes, microglial cells, and oligodendrocytes, derived from neonatal rat cortex and grown in cell type-enriched cultures. TGF-beta 1 production in vitro varied with the cell type and isoform of IL-1. Oligodendrocytes produced the most and astrocytes the least amount of TGF-beta 1. IL-1 alpha stimulated TGF-beta 1 production in all glial cell types, whereas IL-1 beta did not. In vivo, TGF-beta 1 IRP was detected in human tissues from cerebral frontal cortex and subcortical white matter only when interleukin-1 (IL-1) was elevated in the same tissues. Moreover, the amount of detectable TGF-beta 1 was positively correlated with the amount of detectable IL-1 (rho = 0.605; P = 0.003), as determined by morphometry. Double-labelling of cells for their phenotypic markers and expression of TGF-beta 1 indicated that all glial cells, but not neurons, expressed TGF-beta 1. IL-1 alpha and IL-1 beta IRPs were also detected in all three glial cell types, most frequently in astrocytes and least frequently in microglial cells. The cells containing both cytokine IRPs were also detected. These results indicate that TGF-beta 1 may be induced by IL-1 in all glial cells of the frontal cortex, by both autocrine and paracrine mechanisms.

Astrocyte-conditioned medium stimulates HIV-1 expression in a chronically infected promonocyte clone.

Human promonocytic cells chronically infected with human immunodeficiency virus-1 (HIV-1) (clone U1.1.5) were grown in the presence of media conditioned by primary rat cortical astrocytes and HIV-1 expression was assessed by measuring reverse transcriptase activity. Media conditioned by non-stimulated and lipopolysaccharide (LPS)-stimulated astrocytes induced the expression of HIV-1 2.1-fold and 4.1-fold, respectively. LPS alone, media conditioned by the uninfected parental cell line of U1.1.5 (U937), and culture media from four other cell lines, had no effect on viral expression. The magnitude of induction was time- and dose-dependent. Tumor necrosis factor alpha (TNF-alpha) was detected in LPS-stimulated astrocyte-conditioned medium and the HIV-inducing capability of the medium was neutralized, in part, by an antibody to recombinant murine TNF-alpha. These results suggest a role for astrocytes in the induction of HIV expression and thus in the pathogenesis of HIV-1 infection in brain.

Central nervous system infection in a murine retrovirus-induced immunodeficiency syndrome.

Astrocyte-enriched primary glial cultures (AGC) from C57BL/6 mice were found to be highly susceptible to infection with the replication competent components of LP-BM5, consisting of the ecotropic and mink cell focus-inducing (MCF) helper murine leukemia viruses (MuLVs). The presence in infected AGC of defective LP-BM5 MuLV genome, a critical component for induction of the disease referred to as murine AIDS, was confirmed by Southern blot hybridization using a probe reactive with the p12 gag sequence of the 4.9 kb defective genome. Electron microscopic studies demonstrated C-type retrovirus particles in both astrocytes and microglial cells. In vivo studies demonstrated that the ecotropic MuLVs and the defective genome could be detected within AGC obtained form either 14-day-old mice following intraperitoneal inoculation or 7-day-old mice following intracranial inoculation. These findings suggest that: (1) the central nervous system (CNS) infection is present at an early stage in murine AIDS, (2) both astrocytes and microglial cells are possible CNS targets in which helper MuLVs replicate, and (3) these cells can harbor the defective genome that is a critical component for disease induction.

Animal models recapitulate aspects of HIV/CNS disease.

Neurobehavioral and pathological data indicate that the central nervous system (CNS) becomes infected with HIV-1 soon after the virus enters the body. However, neuropathogenesis of HIV-1 infection is difficult to investigate because the brain parenchyma is not accessible to sampling during the course of AIDS. The second compartment of the CNS, cerebrospinal fluid (CSF), is accessible to sampling but how changes in the CSF relate to the changes in the parenchyma is poorly understood. Thus, knowledge of the neuropathogenesis of HIV-1 infection predominantly stems from either postmortem or in vitro studies. This raises the need for animal models of HIV infection of the CNS. Such models have been developed and are briefly reviewed here. The models faithfully recapitulate some aspects of the HIV/CNS disease. Appropriate neuropathological changes and neurobehavioral dysfunction (e.g., cognitive and motor deficits) occur in SIV-infected macaques. Central sensory electrophysiological changes and sleep disturbances occur in FIV-infected cats. Infection of the brain and behavioral changes comparable to some of the changes seen in humans occur in mice infected with a mixture of murine leukemia viruses. Genetically immunodeficient mice (e.g., SCID) accept HIV-infected human organs and or cell grafts. Evidence summarized here indicates that these HuSCID animals undergo neuropathological changes similar to those observed in brains of individuals who died with AIDS. Thus, presently available animal models provide an opportunity to investigate HIV/CNS disease, and to develop and test therapeutic interventions to prevent or cure the disease.

Human astrocytes stimulate HIV-1 expression in a chronically infected promonocyte clone via interleukin-6.

Human promonocyte cells chronically infected with human immunodeficiency virus type (HIV-1) (clone U1.1.5) were grown in the presence of media conditioned by human astrocytes and glioma cell lines U251 and 253. HIV-1 expression was assessed by measuring reverse transcriptase activity. All media conditioned by unstimulated and lipopolysaccharide (LPS) stimulated glial cells induced HIV-1 expression and contained detectable levels of interleukin-6 (IL-6) but not tumor necrosis factor-alpha (TNF-alpha). An antibody against IL-6, but not against TNF-alpha, reduced the induction of HIV-1 by the conditioned media in a concentration-dependent manner. The magnitude of HIV-1 induction by the conditioned media was proportional to the concentration of IL-6 in them. The data indicate that normal and transformed human astrocytes are capable of stimulating HIV-1 expression in chronically infected promonocytic cells by secreting IL-6. The results demonstrate that cytokines secreted by neural cells could play an important role in regulating HIV-1 expression in the brain.

The ontogeny and localization of glutamine synthetase gene expression in rat brain.

A 2.4 kb cDNA clone containing the coding sequence for glutamine synthetase (GS) was isolated from a rat brain cDNA library, and a probe constructed from this cDNA was utilized in Northern analysis of total RNA to study the tissue distribution and the ontogeny of GS mRNA expression in the rat brain from embryonic day 14 (E14) to adulthood. The levels of GS mRNA were highest in the brain, followed by kidney and liver. In the brain, the GS message was detected as early as E14, earlier than it can be detected by either enzymatic assays of GS activity or by immunocytochemical localization of GS. The relatively low levels of GS mRNA seen at E14 increase to a peak around the time of birth, and in the second postnatal week another rise in GS message occurs approaching adult levels by P15. Localization of GS to astrocytes in the brain was confirmed using both immunocytochemistry and in situ hybridization.

Characterization of two plasma membrane proteins abundant in rat brain.

Plasma membranes were isolated from rat brain cortices and their proteins characterized by two-dimensional electrophoresis. Approximately 500 polypeptides with relative molecular weights (mol. wt.) between 20 kDa and 120 kDa and isoelectric points (pI) between 4.2 and 8.5 were visualized by silver staining. Two proteins, MP1 and MP2, comprised about 5% each of the total by mass. Their mol. wts. were 56 kDa and 43 kDa, and their pIs were 4.2 and 4.3, respectively. The two proteins were present in membranes of cultured granule neurons and cortical astrocytes and absent in liver and kidney. They were both substrates for phosphorylation by protein kinase C. MP2 is similar, if not identical, to a major phosphoprotein in growth cones, pp46 (also termed GAP-43, B-50, F1).

Control of astrocytosis by interleukin-1 and transforming growth factor-beta 1 in human brain.

Astrocytosis is a common neurocellular manifestation of brain pathology in individuals with a variety of diseases. It is comprised of astrocytic hyperplasia (an increase in number of astrocytes) and astrocytic hypertrophy (an increase in size of astrocytes). The precise cause(s) of astrocytosis remains unknown. We morphometrically measured the relative extent of astrocytosis in brains of 22 individuals who died with seven different diseases. The relative amounts of interleukin-1 (IL-1) and transforming growth factor-beta 1 (TGF-beta 1) immunoreactive products (IRPs) were next assessed in sections serial to those in which astrocytosis was measured because these cytokines were shown in animal and in vitro experiments to be associated with astrocytosis. The data demonstrate that astrocytosis and these cytokines were co-localized in all examined human tissues. Relative increase in density of astrocytes was correlated with the increase in total IL-1 but not TGF-beta 1. In contrast, the increase in size of astrocytes was correlated with TGF-beta 1 associated only with astrocytes but not with total IL-1. Both IL-1 and TGF-beta 1 IRPs were present in GFAP IRP-containing and other cells, as assessed by double label immunocytochemistry. These observations suggest that IL-1 acts on astrocytes by both, paracrine and autocrine mechanisms whereas, TGF-beta 1 only acts by an autocrine mechanism. Because these correlations were statistically significant and also because a change in number and size of astrocytes constitutes the most frequent response of astrocytes to several diseases or injury, we conclude that these cytokines may mediate the most common pathological change in human brain.

Gliosis in human brain: relationship to size but not other properties of astrocytes.

Gliosis is the most frequent and therefore important neurocellular reaction to brain insult occurring in diseases ranging from AIDS to infarction. Neuropathological diagnosis of gliosis is based on morphological changes of brain glial cells. Changes commonly agreed to reflect gliosis are qualitative increases in size, number and glial fibrillary acidic protein (GFAP) immunoreactivity of astrocytes. These parameters were morphometrically quantified in brain tissues of 22 individuals who died with 7 diseases and statistically compared to the extent of gliosis independently determined by 3 qualified observers. The data indicate that the extent of gliosis correlated with the increase in size of astrocytes in white matter (p = 0.67) and this relationship was statistically significant (P = 0.0006). In contrast, the extent of gliosis was not correlated with the density of astrocytes nor the intensity of GFAP staining.

A vasoactive peptide, endothelin-3, is produced by and specifically binds to primary astrocytes.

Primary rat astrocytes were found by immunohistochemistry to display positive staining for endothelin-3, located predominantly in the perinuclear area. The ability of these cells to produce and release endothelin-3 was confirmed by a combination of reverse-phase HPLC and radioimmunoassay, specific for endothelin-3, which demonstrated immunoreactive peptide in cellular extracts and astrocyte-conditioned medium. In addition, astrocytes were shown to possess a single class of binding sites for endothelin with comparable high affinity for endothelin-1, -2 and -3. These results suggest that astrocytes, by virtue of their ability to produce and secrete endothelin-3, serve as a potential extravascular source of intracerebral vasoregulation capable of influencing regional cerebral blood flow.

Glutamine synthetase expression and activity are regulated by 3,5,3'-triodo-L-thyronine and hydrocortisone in rat oligodendrocyte cultures.

Glutamine synthetase plays a central role in the detoxification of brain ammonia. Previously, we demonstrated that in vitro glutamine synthetase is expressed by all macroglial cell types and is developmentally regulated in oligodendrocyte lineage. Furthermore, glutamine synthetase is increased in secondary cultures of oligodendrocytes following a 72 h treatment with 30 nM 3,5,3'-triodo-L-thyronine [Baas, D., Bourbeau, D., Sarliève, L. L., Ittel, M. E., Dussault, J. H. and Puymirat, J., Oligodendrocyte maturation and progenitor cell proliferation are independently regulated by thyroid hormone. Glia, 1997, 19, 324-332]. Hydrocortisone also increases glutamine synthetase activity after 72 h [Fressinaud, C., Weinrauder, H., Delaunoy, J. P., Tholey, G., Labourdette, G. and Sarliève, L. L., Glutamine synthetase expression in rat oligodendrocytes in culture: regulation by hormones and growth factors. J. Cell. Physiol., 1991, 149, 459-468]; however, it is still unknown whether these increases in glutamine synthetase expression in oligodendrocytes after 3,5,3'-triodo-L-thyronine and hydrocortisone application are dose- and time-dependent. To further investigate this issue, we measured glutamine synthetase levels by Northern analysis, immunostaining and determination of glutamine synthetase activity after 3,5,3'-triodo-L-thyronine or hydrocortisone stimulation. We find that in rat oligodendrocyte secondary cultures, 3,5,3'-triodo-L-thyronine and hydrocortisone cause a dose- and time-dependent increase in glutamine synthetase mRNA, protein and activity. However, these hormones do not exert an additive or synergistic effect. Because purines, pyrimidines, and certain amino acids necessary for the synthesis of myelin components, are, in part, provided by the glutamine synthetase pathway, 3,5,3'-triodo-L-thyronine effect on myelination development and maturation could be mediated in part, through the glutamine synthetase gene regulation.

Developmental regulation of GAP-43, glutamine synthetase and beta-actin mRNA in rat cortical astrocytes.

Steady-state levels of mRNA encoding growth-associated protein 43 (GAP-43), glutamine synthetase (GS) and beta-actin were measured during development of neonatal rat cortical astrocytes in primary culture. GAP-43 mRNA and protein decreased rapidly during the first 2 weeks and slowly thereafter. In contrast, GS mRNA increased approximately 3-fold during the first 2 weeks and reached maximum by day 15. Actin mRNA first increased up to 8 days and decreased thereafter reaching a constant amount of 15 days, similar to the initial low value. Thus, GAP-43, GS and beta-actin mRNA levels are differentially regulated during development of astrocytes in primary culture. Because the patterns of expression of astrocytic markers GS and GFAP (shown previously) in vitro and in vivo are similar to each other, primary cultures of astrocytes may be an excellent system for investigating mechanisms of developmental regulation of these genes.