Second premolar agenesis as a subclinical phenotype of isolated cleft palate.

OBJECTIVE: To compare the prevalence of dental anomalies in patients with isolated cleft palate with or without hypodontia of the second premolar. SETTING AND SAMPLE POPULATION: A total of 653 patients with isolated cleft palate aged 8-12 years were divided into two groups: G1-subjects without hypodontia of second premolar (n = 546) and G2-subjects with hypodontia of at least one-second premolar (n = 107). The control group consisted of 107 non-cleft orthodontic patients. MATERIALS & METHODS: Panoramic and periapical radiographs and dental casts were used to analyze the presence of dental anomalies. Intergroup comparisons were performed using the chi-square test complemented by Proportion test and Bonferroni test. RESULTS: G2 presented higher prevalence of hypodontia of other permanent teeth compared to G1 and control. Ectopic eruption of the maxillary canine and tooth transposition were more frequent in patients with a cleft compared to patients without a cleft, without statistical differences. Deciduous molar infra-occlusion and second premolar disto-angulation were less prevalent in G1 and G2 compared to the control group. G2 showed a higher prevalence of complete cleft palate than G1. CONCLUSION: Patients with cleft palate and second premolar agenesis showed increased prevalence of tooth agenesis and palatally displaced canines.

Pigment epithelium-derived factor induces pro-survival genes through cyclic AMP-responsive element binding protein and nuclear factor kappa B activation in rat cultured cerebellar granule cells: Implication for its neuroprotective effect.

Pigment epithelium-derived factor (PEDF) protects immature cerebellar granule cell neurons (CGCs) against apoptosis induced by K+ and serum deprivation. However, the precise mechanism of this protection remains unknown. We recently reported that the transcription factor nuclear factor kappa B (NF-kappaB) is activated in PEDF-treated CGCs. Although it is well known that NF-kappaB blocks apoptotic cell death through the induction of pro-survival factors, the effects of PEDF on the expression of these factors are not fully understood. In this study, we employed the use of reverse transcriptase-polymerase chain reaction to analyze the gene expression of certain pro-survival genes and found that genes such as c-IAP1, c-IAP2, FLIPs, A1/Bfl-1 and Mn-SOD were induced in PEDF-treated neurons. On the other hand, no induction was observed of the pro-apoptotic Bcl-2 family members Bax and Bid at any time from 3 to 24 h following PEDF addition. Furthermore, phosphorylation of cyclic AMP-responsive element binding protein (CREB) and increment of nuclear cyclic AMP-response element (CRE)-like DNA binding were observed in PEDF-treated CGCs. The anti-apoptotic effect of PEDF was blocked by overexpression of dominant negative CREB or a mutated form of IkappaBalpha. These results suggested that induction of both CRE- and NF-kappaB-dependent genes is required for the observed neuroprotective effects of PEDF on CGCs.

Closed-head minimal traumatic brain injury produces long-term cognitive deficits in mice.

Victims of minimal traumatic brain injury (mTBI) do not show clear morphological brain defects, but frequently suffer lasting cognitive deficits, emotional difficulties and behavioral disturbances. In the present study we adopted a non-invasive closed-head weight-drop mouse model to produce mTBI. We examined the effects of 20, 25, or 30 g weight drop 7, 30, 60 and 90 days following injury on mice's ability to perform the Morris water maze. The mice suffered profound long-lasting learning and memory deficits that were force- and time-dependent. Although the injured mice could acquire the task, they could not improve their initial escape latency by more than 50%, while normal mice improved by up to 450% (P<0.001). In order to directly compare the learning ability of individual mice following our mTBI we have devised a new measure which we term learning rate. We define learning rate as the rate the mouse improved its own performance in consecutive trials in a given experimental day. The learning rate of control mice increased linearly throughout the testing period with a slope of approximately 0.9. Injured mice that sustained 20 and 25 g weight drop could also improve their learning rate linearly but with a slope of only 0.2. Mice who sustained 30 g weight drop could not improve their learning rate linearly and reached a plateau after the third experimental learning day. These results indicate that the severity of injury may correlate with the degree of integration of the learning task. These cognitive deficits occurred without any other clear neurological damage, no evident brain edema, no notable damage to the blood-brain barrier and no early anatomical changes to the brain (observed by magnetic resonance imaging imaging). These results demonstrate that persistent deficits of cognitive learning abilities in mice, similar to those observed in human post-concussive syndrome, can follow mTBI without any anatomical damage to the brain and its surrounding tissue.

NFkappaB activation is required for the neuroprotective effects of pigment epithelium-derived factor (PEDF) on cerebellar granule neurons.

Pigment epithelium-derived factor (PEDF) protects immature cerebellar granule cells (1-3 days in vitro) against induced apoptosis and mature cells (5+ days in vitro) against glutamate toxicity, but its precise mechanism is still unknown. Because the transcription factor NFkappaB blocks cell death, including neuronal apoptosis, we have investigated the ability of PEDF to exert its effects via NFkappaB activation. PEDF induced an increased phosphorylation of IkappaBalpha, decreased levels of IkappaB proteins, and translocation of p65 (RelA) to the nucleus followed by a time-dependent increase of NFkappaB-DNA binding activity in both immature and mature neurons. The protective effects of PEDF against both induced apoptosis and glutamate toxicity were blocked by the addition of either the IkappaB kinase inhibitor BAY 11-7082, which inhibits the phosphorylation of IkappaB, or N-acetyl-Leu-Leu-norleucinal, which blocks proteosome degradation of IkappaB, demonstrating that NFkappaB is required for the neuroprotective effects of PEDF. Reverse transcription-polymerase chain reaction analysis revealed that up-regulation of the anti-apoptotic genes for Bcl-2, Bcl-x, and manganese superoxide dismutase was observed in PEDF-treated immature but not mature neurons. Up-regulation of nerve growth factor, brain-derived neurotrophic factor, and glial cell-derived neurotrophic factor mRNA was long-lasting in mature neurons. These results suggest that PEDF promotes neuronal survival through activation of NFkappaB, which in turn induces expression of anti-apoptotic and/or neurotrophic factor genes.

Survival effects of pigment epithelium-derived factor expressed by a lentiviral vector in rat cerebellar granule cells.

We have previously shown that pigment epithelium-derived factor (PEDF) acts as a survival factor for cerebellar granule cells (CGCs), by blocking apoptotic death, and can also protect these cells against glutamate-induced neurotoxicity. In preparation for gene therapy studies, pseudotyped HIV-1-based lentiviral vectors containing the PEDF gene, as well as either green fluorescent protein or beta-galactosidase, were prepared. These bicistronic vectors are unique in that they express two genes efficiently under one promoter. Primary cell cultures of CGCs from postnatal day 8 rats were infected with the vectors encoding PEDF. RT-PCR demonstrated expression of mRNA and Western blot analysis confirmed that infected CGCs secrete PEDF protein to the medium. Assays for cell survival demonstrated that PEDF-infected cells were significantly more protected compared with mock-infected controls for 6-8 days in culture, as well as against induced apoptosis. The PEDF vectors expressing tat (trans-acting transcription factor) provided more protection than the tat(-) vectors. These results demonstrate that while the lentiviral vectors expressing PEDF are as neuroprotective as the protein itself for CGCs, the vectors have the advantage of providing long-lasting expression of PEDF protein, which will be more effective in in vivo studies. The present results suggest that this system may be useful for gene therapy for neurodegenerative disorders.

Serotonin regulation of nerve growth factor synthesis in neonatal and adult astrocytes: comparison to the beta-adrenergic agonist isoproterenol.

Although serotonin regulates synthesis of the neurotrophic factor S-100 beta by astrocytes, its ability to affect nerve growth factor (NGF) synthesis has never been examined. We report here that there is a correlation between the effect of serotonin on cyclic adenosine monophosphate (cAMP) content and on NGF content in neonatal astrocytes but not in adult astrocytes. In neonatal striatal astrocytes, serotonin increases both cAMP and NGF, whereas, in neonatal cerebellar astrocytes, serotonin decreases both. The increase in neonatal cortical astrocyte cAMP appeared to be too small (45%) to increase NGF significantly. The beta-adrenergic agonist isoproterenol increased cAMP and NGF in both cortical and striatal astrocytes derived from neonatal rats. In contrast, there was a dissociation between cAMP changes and NGF changes in astrocytes derived from adult rats. Both serotonin and isoproterenol increased cAMP in adult cortical astrocytes, without any effect on NGF content. However, adult striatal astrocytes responded to serotonin with an elevation of both cAMP and NGF, whereas isoproterenol could only enhance cAMP, without affecting NGF. Thus, in neonatal astrocytes, a change of sufficient magnitude in cAMP was correlated with a comparable change in NGF, in response to activation of either serotonergic or beta-adrenergic receptors; in cerebellar astrocytes, the decrease in cAMP was accompanied by a decrease in NGF. In contrast, adult astrocytes were not responsive: Although cAMP changes were large, NGF synthesis was increased only in striatal astrocytes and only in response to serotonin. J. Neurosci. Res. 64:261-267, 2001. Published 2001 Wiley-Liss, Inc.

Perinatal opioids reduce striatal nerve growth factor content in rat striatum.

Both human and animal models indicate that perinatal methadone exposure produces a variety of short- and long-term neurobehavioral consequences, including disruption of normal development of striatal cholinergic neurons. Despite this, methadone maintenance is a standard method of managing pregnant heroin addicts, and the opioid receptor partial agonist buprenorphine is under evaluation for the same use. We now report that perinatal administration of either methadone or buprenorphine reduces the content of the neurotrophic factor nerve growth factor (NGF) in rat striatum, which may explain the behavioral deficits observed. Furthermore, although NGF content is reduced, there are no corresponding reductions in striatal NGF mRNA.

PSA-NCAM distinguishes reactive astrocytes in 6-OHDA-lesioned substantia nigra from those in the striatal terminal fields.

6-hydroxydopamine (6-OHDA) lesion of the substantia nigra (SN) causes the appearance of reactive astrocytes not only in the SN but also in the striatal terminal fields, as measured by increased size of the cells and their processes, as well as enhanced expression of glial fibrillary acidic protein (GFAP) and an epitope recognized by monoclonal antibody 19D1. We now demonstrate that polysialylated neural cell adhesion molecule (PSA-NCAM) is induced on reactive astrocytes, as well as on large neurons, on the ipsilateral side of the 6-OHDA-lesioned SN. Colocalization of GFAP and PSA-NCAM was confirmed for reactive astrocytes using a confocal laser scanning microscope. Negligible amounts of PSA-NCAM reactivity were detected contralaterally, although colocalization was noted on astrocytes with sparse, significantly thinner processes. In contrast to the increase of GFAP in the lesioned striatum, few striatal astrocytes expressed PSA-NCAM. In agreement with these results, PSA-NCAM was detected on cultured reactive astrocytes from SN but not reactive striatal astrocytes. Double immunohistochemistry for proliferating cell nuclear antigen (PCNA), a marker of dividing cells, and GFAP demonstrated that reactive astrocytes in lesioned SN were PCNA-positive whereas those in striatum were not. Although NG2 chondroitin sulfate proteoglycan expression also increased in the lesioned SN, NG2 was not colocalized with PSA-NCAM, was not expressed on astrocytes, and labeled only oligodendrocyte precursor cells. Our results suggest that PSA-NCAM can act as a marker for reactive astrocytes only at the site of the lesion and not in the terminal fields, probably because it is reexpressed only when astrocytes divide.

Glial somatostatin-14 expression in the rat pituitary intermediate lobe: a possible neurotrophic function during development?

Somatostatin-14 was first detected on gestational day 17 in radially-oriented, bipolar cells spanning the width of the intermediate lobe of the rat pituitary. Cells were prominent, and constituted approximately 50% of the lobe area. The presence of vimentin, the cellular shape, and the localization identified these cells as glia. At postnatal day 6, somatostatin-14 and vimentin staining appeared in stellate-shaped cells. This is in agreement with the change from bipolar to stellate shape these glia undergo after the onset of innervation ([13] Gary et al. Int. J. Devl. Neurosci. 13, 555-565, 1995). Glia were more abundant, relative to melanotropes, throughout embryonic and early postnatal development compared to adulthood. Reverse transcription-polymerase chain reaction data showed a high level of prosomatostatin mRNA in the intermediate lobe, compared to the anterior and neural lobes from postnatal day 2 animals, and a significant drop in intermediate lobe content in the adult. The correlation between the number of glia and high expression of somatostatin in neonatal relative to adult tissue, together with the close apposition of incoming axons to the abundant, radially oriented glia during innervation of the lobe, support a neurotrophic function of glia-derived somatostatin.

Recombinant forms of the neurotrophic factor pigment epithelium-derived factor activate cellular metabolism and inhibit proliferation of the RAW macrophage cell line.

Recombinant forms of the neurotrophic factor pigment epithelium-derived factor (PEDF) activate metabolism of RAW macrophage cells while simultaneously inhibiting their proliferation. The recombinant forms (rPEDF) acted with EC(50)s of 0.1-1 nM while full-length native bovine PEDF was inactive. Urea, which is the buffer used to extract recombinant PEDF, stimulated RAW cell proliferation, the first report of an effect of urea on non-kidney cells. PEDF acted within 12 h and its effects persisted up to 72 h with continuous exposure. Although rPEDF had no direct action on glioma cell lines, it increased the amount of a soluble factor released by RAW cells which was capable of blocking glioma cell division. Thus PEDF may function as a neuroimmune modulator, affecting both neural and immune system cells.

CNS interleukin-3 (IL-3) expression and neurological syndrome in antisense-IL-3 transgenic mice.

Interleukin-3 (IL-3) is an important mediator of physiological and pathophysiological processes affecting the central nervous system (CNS). It stimulates the proliferation and activation of microglia and can enhance differentiation of cholinergic and sensory neurons. To examine the role of IL-3 in the CNS, we utilized transgenic mice expressing a murine antisense IL-3 (AS-IL-3) RNA under the control of the T cell B19 promoter so that expression is limited to hematopoietic cells. The AS-IL-3 transgenic mice develop either a progressive neurologic dysfunction, which includes ataxia, bradykinesia, and paralysis, or a lymphoproliferative syndrome. Histopathology demonstrated accumulations of reactive astrocytes in the cerebellum, brain stem, and spinal cord, accompanied by activated microglia. Partial loss of cerebellar nuclei neurons as well as neurons in the cranial nerve nuclei and spinal cord motor neurons is seen. Despite depletion of IL-3 peripherally, expression of IL-3 mRNA and protein is turned on in the CNS of the transgenic mice. Astrocytes cultured from the AS-IL-3 mice contain IL-3 mRNA and may thus be responsible for the activation of the microglia. This model should provide important insights into the role of cytokines in neurological disorders.

Differential effects of chemical sympathectomy on expression and activity of tyrosine hydroxylase and levels of catecholamines and DOPA in peripheral tissues of rats.

Tyrosine hydroxylase (TH) mRNA and activity and concentrations of 3,4-dihydroxyphenylalanine (DOPA) and catecholamines were examined as markers of sympathetic innervation and catecholamine synthesis in peripheral tissues of sympathectomized and intact rats. Chemical sympathectomy with 6-hydroxydopamine (6-OHDA) markedly decreased norepinephrine and to a generally lesser extent TH activities and dopamine in most peripheral tissues (stomach, lung, testis, duodenum, pancreas, salivary gland, spleen, heart, kidney, thymus). Superior cervical ganglia, adrenals and descending aorta were unaffected and vas deferens showed a large 92% decrease in norepinephrine, but only a small 38% decrease in TH activity after 6-OHDA. Presence of chromaffin cells or neuronal cell bodies in these latter tissues, indicated by consistent expression of TH mRNA, explained the relative resistance of these tissues to 6-OHDA. Stomach also showed consistent expression of TH mRNA before, but not after 6-OHDA, suggesting that catecholamine synthesizing cells in gastric tissue are sensitive to the toxic effects of 6-OHDA. Tissue concentrations of DOPA were mainly unaffected by 6-OHDA, indicating that much of the DOPA in peripheral tissues is synthesized independently of local TH or sympathetic innervation. The differential effects of chemical sympathectomy on tissue catecholamines, DOPA, TH mRNA and TH activity demonstrate that these variables are not simple markers of sympathetic innervation or catecholamine synthesis. Other factors, including presence of neuronal cell bodies, parenchymal chromaffin cells, non-neuronal sites of catecholamine synthesis and alternative sources of tissue DOPA, must also be considered when tissue catecholamines, DOPA and TH are examined as markers of sympathetic innervation and local catecholamine synthesis.

High-titer human immunodeficiency virus type 1-based vector systems for gene delivery into nondividing cells.

Previously we designed novel pseudotyped high-titer replication defective human immunodeficiency virus type 1 (HIV-1) vectors to deliver genes into nondividing cells (J. Reiser, G. Harmison, S. Kluepfel-Stahl, R. O. Brady, S. Karlsson, and M. Schubert, Proc. Natl. Acad. Sci. USA 93:15266-15271, 1996). Since then we have made several improvements with respect to the safety, flexibility, and efficiency of the vector system. A three-plasmid expression system is used to generate pseudotyped HIV-1 particles by transient transfection of human embryonic kidney 293T cells with a defective packaging construct, a plasmid coding for a heterologous envelope (Env) protein, and a vector construct harboring a reporter gene such as neo, ShlacZ (encoding a phleomycin resistance/beta-galactosidase fusion protein), HSA (encoding mouse heat-stable antigen), or EGFP (encoding enhanced green fluorescent protein). The packaging constructs lack functional Vif, Vpr, and Vpu proteins and/or a large portion of the Env coding region as well as the 5' and 3' long terminal repeats, the Nef function, and the presumed packaging signal. Using G418 selection, we routinely obtained vector particles pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G) with titers of up to 8 x 10(7) CFU/microgram of p24, provided that a functional Tat coding region was present in the vector. Vector constructs lacking a functional Tat protein yielded titers of around 4 x 10(6) to 8 x 10(6) CFU/microgram of p24. Packaging constructs with a mutation within the integrase (IN) core domain profoundly affected colony formation and expression of the reporter genes, indicating that a functional IN protein is required for efficient transduction. We explored the abilities of other Env proteins to allow formation of pseudotyped HIV-1 particles. The rabies virus and Mokola virus G proteins yielded high-titer infectious pseudotypes, while the human foamy virus Env protein did not. Using the improved vector system, we successfully transduced contact-inhibited primary human skin fibroblasts and postmitotic rat cerebellar neurons and cardiac myocytes, a process not affected by the lack of the accessory proteins.

IFN-gamma inhibits AP-1 binding activity in human brain-derived cells through a nitric oxide dependent mechanism.

It has been demonstrated that CNS levels of the cytokine IFN-gamma are elevated in association with a number of neuro-inflammatory diseases. In the present study, we have examined the effect of this cytokine on human brain derived cells. We show that prolonged treatment (22 h) of such cells with IFN-gamma inhibits the DNA binding activity of transcription factor AP-1. Furthermore, we show that this effect can be reversed by either N(G)-monomethyl-L-arginine (L-NMMA) or L-N5-(1-iminoethyl)ornithine (L-NIO), competitive inhibitors of nitric oxide synthase activity [Rees et al., 1990]. In addition, we show that treatment of brain-derived cells with the nitric oxide donor 3-morpholinosydnonimine, HCl (SIN-1), or [N-(b-D-glucopyranosyl)-N2-acetyl-S-nitroso-D,L-penicillaminamide] (glyco-SNAP-1), also inhibits the binding activity of AP-1. Together, these results suggest that IFN-gamma can inhibit AP-1 binding activity through a nitric oxide dependent mechanism.

A culture model of reactive astrocytes: increased nerve growth factor synthesis and reexpression of cytokine responsiveness.

Reactive gliosis, which occurs in response to damage to the central nervous system, has been recognized for years but is not yet understood. We describe here a tissue culture model of reactive astrocytes used to characterize their properties. Cultures are prepared 1 week following 6-hydroxydopamine (6-OHDA) lesion of rat substantia nigra and compared with astrocytes cultured from normal adult rats or rats injected with saline only. Astrocytes from the 6-OHDA-lesioned side contained elevated levels of glial fibrillary acidic protein (GFAP) and GFAP mRNA and were intensely immunoreactive for GFAP, vimentin, and two epitopes that in vivo are found only on reactive astrocytes. The basal content of nerve growth factor (NGF) mRNA and NGF in astrocytes from 6-OHDA-lesioned rats was significantly higher relative to control astrocytes. Two inflammatory cytokines, interleukin-1beta and interferon-gamma, increased synthesis of NGF up to 20-fold in the reactive cells, whereas there was no response in the normal adult astrocytes. Astrocytes from postnatal day 2 rats shared many of the properties of the reactive adult astrocytes. These cultures offer the possibility to characterize the cellular and molecular properties of reactive astrocytes and to determine the factors responsible for activation of astrocytes.

Pigment epithelium-derived factor (PEDF) differentially protects immature but not mature cerebellar granule cells against apoptotic cell death.

We have shown previously that pigment epithelium-derived factor (PEDF) acts as a survival factor for cerebellar granule cell neurons in culture, as well as protecting them against glutamate toxicity. In this study we have examined effects of PEDF on apoptotic cell death. We find that the granule cells die of apoptosis throughout the culture period, what we have termed "natural" apoptosis. PEDF prevents this natural apoptosis if added to immature cells, within the first 2 days in vitro (DIV), and the effect is maintained for up to DIV12. However, PEDF has no effect if added to mature cells at DIV5. Similar results are obtained when apoptosis is induced by shifting the cells from a serum- and 25 mM KCl-containing medium to serum-free medium with 5 mM KCl. PEDF most effectively blocks induced apoptosis in immature cells (DIV2) when added 24 hr prior to the change of medium, but still provides some protection when added simultaneously. However, 24 hr pretreatment with PEDF has a minimal effect when apoptosis is induced in mature DIV6 cells; addition at the same time is completely ineffective. Two polypeptide fragments of PEDF, only one of which contains the serine-protease inhibitory site, are equally active, supporting previous results which suggest that the neurotrophic effects of PEDF are not mediated by protease inhibition. We conclude that PEDF protects immature but not mature granule cells against both natural and induced apoptosis.

Involvement of cytokines in normal CNS development and neurological diseases: recent progress and perspectives.

Cytokines have been recognized to play an important role both in normal development of the brain, when they act as neurotrophic factors, as well as following injury. While both the cytokines and their receptors are synthesized and expressed in the brain normally (albeit at low levels), it has become clear that elevated levels are associated with many neurological disorders. In this review, we have chosen to present the data for only a few of the cytokines, including interleukin-1beta, interleukin-3, interleukin-6, interferon-gamma, transforming growth factor-beta, and tumor necrosis factor-alpha. Data are presented that suggest roles they may play in human disorders, including stroke, multiple sclerosis, Alzheimer's disease, and several psychiatric disorders. The results in human disease are compared with results obtained in a variety of transgenic animal models. The mouse models have very different disorders depending on whether a cytokine is overexpressed either peripherally or in either astrocytes or neurons. The potential significance of this to the understanding of human disease is discussed.

Cell culture models for reactive gliosis: new perspectives.

Reactive gliosis, which occurs in response to any damage or disturbance to the central nervous system, has been recognized for many years, but is still not completely understood. The hallmark is the increased expression of glial fibrillary acidic protein (GFAP), yet studies in GFAP knockout mice suggest that GFAP may not be required for an astrocyte to become hypertrophic. In this review, we describe a series of tissue culture models that have been established in order to address: 1) the biochemical phenotype of reactive astrocytes; 2) the factor and/or cell responsible for induction of gliosis; 3) the mechanisms by which one might block the induction. These models range from cultures of astrocytes, both neonatal and adult, to co-cultures of astrocytes with either neurons or microglia, to organ cultures. None is ideal: each addresses a different set of questions, but taken together, they are beginning to provide useful information which should allow a better understanding of the plasticity response of astrocytes to brain injury.

Somatostatin as a neurotrophic factor. Which receptor/second messenger transduction system is involved?

A variety of studies support a trophic role for somatostatin in the developing nervous system, evidenced as stimulation of neurite outgrowth and axonal or neuronal migration in both in vivo and culture models. Cloning experiments have now demonstrated the existence of five subtypes of somatostatin receptor, differentially distributed in the nervous system, differentially linked to specific signal transduction systems and in certain cases differentially expressed during development. The combination of the differential and developmental regulation of expression of both the somatostatin peptides and their receptors thus provides great potential in terms of trophic effects. To substantiate trophic effects of somatostatin, data are presented from two different model systems, cultures of cerebellar granule cells as well as transgenic mice in which somatostatin is expressed under the control of the glial fibrillary acidic protein promoter. Finally, potential receptor subtypes and second messenger systems involved in these trophic effects are addressed.