Biological response to phorbol ester determined by alternative G1 pathways.

A plethora of extracellular signals is known to induce a common set of immediate early genes. The immediate early response, therefore, must not be sufficient to determine the biological outcome. An example of this is found with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). A potent activator of protein kinase C, TPA can either stimulate or inhibit cell proliferation, depending on the cell type. This cell context-dependent response to TPA is observed with two subclones of NIH 3T3 cells, the P- and the N-3T3 clones. TPA is a mitogen for the P-3T3 but an antimitogen for the N-3T3 cells. The immediate early pathway is activated by TPA in both cell types, indicating that this pathway alone does not activate DNA synthesis. The delayed induction of cyclin D1 expression by TPA is observed only in the P-3T3 cells, correlating with mitogenesis. N-Acetylcysteine does not affect the immediate early pathway but can inhibit the TPA-mediated induction of cyclin D1 and DNA synthesis. In the N-3T3 cells, TPA causes an inhibition of the cyclin E-associated kinase at the G1/S transition, correlating with growth inhibition. The growth-inhibitory activity of TPA is not affected by N-acetylcysteine. Thus, the two TPA-regulated G1 pathways can be distinguished by their sensitivity to N-acetylcysteine. These results demonstrate that TPA can activate alternative G1 pathways. Moreover, the selection of the alternative G1 pathways is determined by the cell context, which, in turn, dictates the biological response to TPA.

Decrements in auditory responses to a repeated conspecific song are long-lasting and require two periods of protein synthesis in the songbird forebrain.

Earlier work showed that playbacks of conspecific song induce expression of the immediate early gene ZENK in the caudo-medial neostriatum (NCM) of awake male zebra finches and that this response disappears with repeated presentations of the same stimulus. In the present study, we investigated whether repetitions of a song stimulus also elicited a decrement in the electrophysiological responses in the NCM neurons of these birds. Multiunit auditory responses in NCM were initially vigorous, but their amplitude decreased (habituated) rapidly to repeated stimulation, declining to about 40% of the initial response during the first 50 iterations. A similar time course of change was seen at the single unit level. This habituation occurred specifically for each song presented but did not occur when pure tones were used as a stimulus. Habituation to conspecific, but not heterospecific, song was retained for 20 h or longer. Injections of inhibitors of protein or RNA synthesis at the recording site did not affect the initial habituation to a novel stimulus, but these drugs blocked the long-term habituation when injected at 0.5-3 h and at 5.5-7 h after the first exposure to the stimulus. Thus, at least two waves of gene induction appear to be necessary for long-lasting habituation to a particular song.

Endometrial response toward bovine herpesvirus 4 infection

Metriti ed endometriti sono le patologie maggiormente responsabili delle perdite economiche negli allevamenti bovini da latte, specialmente nel periodo successivo al parto. Mentre le metriti coinvolgono e si sviluppano in tutto l’utero e sono caratterizzate dalla presenza di sintomi sistemici, le endometriti consistono in una infiammazione che riguarda il solo endometrio, con la presenza di perdite purulente, distruzione della superficie epiteliale, congestione vascolare, edema stromale ed accumulo di linfociti e plasmacellule. Queste patologie, inoltre, possono causare, disfunzione ovarica, con conseguente infertilità e riduzione sia dell’efficienza riproduttiva della vacca sia della produzione stessa di latte. Nonostante queste malattie siano, nella maggior parte dei casi, correlate all’instaurarsi di infezioni batteriche, che possono subentrare nell’utero direttamente durante il parto, il ruolo di alcuni virus nello sviluppo di queste patologie è stato recentemente approfondito e la correlazione tra l’ Herpesvirus Bovino 4 e l’insorgere di metriti ed endometriti è stata dimostrata. L’ Herpesvirus Bovino 4 (BoHV-4) è un gamma-herpesvirus ed il suo genoma è costituito da una molecola lineare di DNA a doppio filamento con una struttura genomica di tipo B, caratterizzata dalla presenza di un’unica lunga sequenza centrale (LUR) fiancheggiata da multiple sequenze poli-ripetute (prDNA). BoHV-4 è stato isolato sia da animali sani sia da animali con differenti patologie, tra cui malattie oculari e respiratorie, ma soprattutto da casi di metriti, endometriti, vaginiti o aborti. Generalmente, il ruolo svolto dal virus in questo tipo di patologie è associato alla compresenza di altri tipi di patogeni, che possono essere virus, come nel caso del Virus Della Diarrea Virale Bovina (BVDV), o più frequentemente batteri. Usualmente, l’iniziale difesa dell’endometrio bovino nei confronti dei microbi si fonda sul sistema immunitario innato e l’attivazione di specifici recettori cellulari determina la sintesi e la produzione di citochine e chemochine pro infiammatorie, che possono essere in grado di modulare la replicazione di BoHV-4. Il genoma di BoHV-4 possiede due principali trascritti per i geni Immediate Early (IE), trai quali ORF50/IE2 è il più importante ed il suo prodotto, Rta/ORF50, è fortemente conservato tra tutti gli Herpesvirus. Esso è responsabile della diretta trans-attivazione di numerosi geni virali e cellulari e può essere modulato da differenti stimoli extracellulari. Precedentemente è stato dimostrato come il TNF-, prodotto dalle cellule stromali e dai macrofagi all’interno dell’endometrio, in conseguenza ad infezione batterica, sia in grado di aumentare la replicazione di BoHV-4 attraverso l’attivazione del pathway di NFkB e direttamente agendo sul promotore di IE2. Per queste ragioni, è risultato di forte interesse investigare quali potessero essere, invece, i fattori limitanti la replicazione di BoHV-4. In questo lavoro è stata studiata la relazione tra cellule endometriali stromali bovine infettate con l’Herpesvirus Bovino 4 e l’interferon gamma (IFN-) ed è stata dimostrata la capacità di questa molecola di restringere la replicazione di BoHV-4 in maniera IDO1 indipendente ed IE2 dipendente. Inoltre, la presenza di alcuni elementi in grado di interagire con l’ IFN-γ, all’interno del promotore di IE2 di BoHV-4, ha confermato questa ipotesi. Basandoci su questi dati, abbiamo potuto supporre l’esistenza di uno stretto vincolo tra l’attivazione dell’asse dell’interferon gamma e la ridotta replicazione di BoHV-4, andando a porre le basi per una nuova efficiente cura e prevenzione per le patologie uterine. Poiché il meccanismo corretto attraverso il quale BoHV-4 infetta l’endometrio bovino non è ancora ben compreso, è stato interessante approfondire in maniera più accurata l’interazione presente tra il virus ed il substrato endometriale, analizzando le differenze esistenti tra cellule infettate e non, in termini di espressione genica. Basandoci su dati preliminari ottenuti attraverso analisi con RNA sequencing (RNAseq), abbiamo visto come numerosi geni risultino over-espressi in seguito ad infezione con BoHV-4 e come, tra questi, la Metalloproteasi 1 sia uno dei più interessanti, a causa delle sue possibili implicazioni nello sviluppo delle patologie dell’endometrio uterino bovino. Successive analisi, effettuate tramite westernblotting e real time PCR, sono state in grado di confermare tale dato, sottolineando l’efficacia di un nuovo approccio sperimentale, basato sul RNAseq, per lo studio dell’insorgenza delle patologie.

Assessment of fight outcome is needed to activate socially driven transcriptional changes in the zebrafish brain

Group living animals must be able to express different behavior profiles depending on their social status. Therefore, the same genotype may translate into different behavioral phenotypes through socially driven differential gene expression. However, how social information is translated into a neurogenomic response and what are the specific cues in a social interaction that signal a change in social status are questions that have remained unanswered. Here, we show for the first time, to our knowledge, that the switch between status-specific neurogenomic states relies on the assessment of fight outcome rather than just on self- or opponent-only assessment of fighting ability. For this purpose, we manipulated the perception of fight outcome in male zebrafish and measured its impact on the brain transcriptome using a zebrafish whole genome gene chip. Males fought either a real opponent, and a winner and a loser were identified, or their own image on a mirror, in which case, despite expressing aggressive behavior, males did not experience either a victory or a defeat. Massive changes in the brain transcriptome were observed in real opponent fighters, with losers displaying both a higher number of differentially expressed genes and of coexpressed gene modules than winners. In contrast, mirror fighters expressed a neurogenomic state similar to that of noninteracting fish. The genes that responded to fight outcome included immediate early genes and genes involved in neuroplasticity and epigenetic modifications. These results indicate that, even in cognitively simple organisms such as zebrafish, neurogenomic responses underlying changes in social status rely on mutual assessment of fighting ability.

Effector ExoU from the type III secretion system is an important modulator of gene expression in lung epithelial cells in response to Pseudomonas aeruginosa infection

Pseudomonas aeruginosa is an important pathogen in immunocompromised patients and secretes a diverse set of virulence factors that aid colonization and influence host cell defenses. An important early step in the establishment of infection is the production of type III-secreted effectors translocated into host cells by the bacteria. We used cDNA microarrays to compare the transcriptomic response of lung epithelial cells to P. aeruginosa mutants defective in type IV pili, the type III secretion apparatus, or in the production of specific type III-secreted effectors. Of the 18,000 cDNA clones analyzed, 55 were induced or repressed after 4 It of infection and could be classified into four different expression patterns. These include (i) host genes that are induced or repressed in a type III secretion-independent manner (32 clones), (ii) host genes induced specifically by ExoU (20 clones), and (iii) host genes induced in an ExoU-independent but type III secretion dependent manner (3 clones). In particular, ExoU was essential for the expression of immediate-early response genes, including the transcription factor c-Fos. ExoU-dependent gene expression was mediated in part by early and transient activation of the AN transcription factor complex. In conclusion, the present study provides a detailed insight into the response of epithelial cells to infection and indicates the significant role played by the type III virulence mechanism in the initial host response.

Differential involvement of rat medial prefrontal cortex dopamine receptors in modulation of hypothalamic-pituitary-adrenal axis responses to different stressors

Recent investigations have implicated the medial prefrontal cortex (mPFC) in modulation of subcortical pathways that contribute to the generation of behavioural, autonomic and endocrine responses to stress. However, little is known of the mechanisms involved. One of the key neurotransmitters involved in mPFC function is dopamine, and we therefore aimed, in this investigation, to examine the role of mPFC dopamine in response to stress in Wistar rats. In this regard, we infused dopamine antagonists SCH23390 or sulpiride into the mPFC via retrodialysis. We then examined changes in numbers of cells expressing the c-fos immediate-early gene protein product, Fos, in subcortical neuronal populations associated with regulation of hypothalamic-pituitary-adrenal (HPA) axis stress responses in response to either of two stressors; systemic injection of interleukin-1beta, or air puff. The D-1 antagonist, SCH23390, and the D-2 antagonist, sulpiride, both attenuated expression of Fos in the medial parvocellular hypothalamic paraventricular nucleus (mpPVN) corticotropin-releasing factor cells at the apex of the HPA axis, as well as in most extra-hypothalamic brain regions examined in response to interleukin-1beta. By contrast, SCH23390 failed to affect Fos expression in response to air puff in any brain region examined, while sulpiride resulted in an attenuation of the air puff-induced response in only the mpPVN and the bed nucleus of the stria terminalis. These results indicate that the mPFC differentially processes the response to different stressors and that the two types of dopamine receptor may have different roles.

Noradrenergic modulatoins of odor learning and odor representation in the rat

How experience alters neuronal ensemble dynamics and how locus coeruleus-mediated norepinephrine release facilitates memory formation in the brain are the topics of this thesis. Here we employed a visualization technique, cellular compartment analysis of temporal activity by fluorescence in situ hybridization (catFISH), to assess activation patterns of neuronal ensembles in the olfactory bulb (OB) and anterior piriform cortex (aPC) to repeated odor inputs. Two associative learning models were used, early odor preference learning in rat pups and adult rat go-no-go odor discrimination learning. With catFISH of an immediate early gene, Arc, we showed that odor representation in the OB and aPC was sparse (~5-10%) and widely distributed. Odor associative learning enhanced the stability of the rewarded odor representation in the OB and aPC. The stable component, indexed by the overlap between the two ensembles activated by the rewarded odor at two time points, increased from ~25% to ~50% (p = 0.004-1.43E⁻4; Chapter 3 and 4). Adult odor discrimination learning promoted pattern separation between rewarded and unrewarded odor representations in the aPC. The overlap between rewarded and unrewarded odor representations reduced from ~25% to ~14% (p = 2.28E⁻⁵). However, learning an odor mixture as a rewarded odor increased the overlap of the component odor representations in the aPC from ~23% to ~44% (p = 0.010; Chapter 4). Blocking both α- and β-adrenoreceptors in the aPC prevented highly similar odor discrimination learning in adult rats, and reduced OB mitral and granule ensemble stability to the rewarded odor. Similar treatment in the OB only slowed odor discrimination learning. However, OB adrenoceptor blockade disrupted pattern separation and ensemble stability in the aPC when the rats demonstrated deficiency in discrimination (Chapter 5). In another project, the role of α₂-adrenoreceptors in the OB during early odor preference learning was studied. OB α2-adrenoceptor activation was necessary for odor learning in rat pups. α₂-adrenoceptor activation was additive with β-adrenoceptor mediated signalling to promote learning (Chapter 2). Together, these experiments suggest that odor representations are highly adaptive at the early stages of odor processing. The OB and aPC work in concert to support odor learning and top-down adrenergic input exerts a powerful modulation on both learning and odor representation.

Asymmetrical processing of olfactory input in the piriform cortex mediates "activation" of the avian navigation circuitry

The role of odors in the long-distance navigation of birds has elicited intense debate for more than half a century. Failure to resolve many of the issues fueling this debate is due at least in part to the absence of controls for a variety of non-specific effects that odors have on the navigational process. The present experiments were carried out to investigate whether the olfactory inputs are involved only in “activation” of neuronal circuitry involved in navigation or are also playing a role in providing directional information. Experienced adult pigeons were exposed to controlled olfactory stimuli during different segments of the journey (release site vs. displacement + release site). Protein levels of IEGs (immediate early genes used to mark synaptic activity) were analyzed in areas within the olfactory/navigation avian circuitry. The results indicate that 1) exposure to natural odors at the release site (and not before) elicit greater activation across brain regions than exposure to filtered air, artificial odors, and natural odors along the entire outward journey (from home to the release site, inclusive); 2) activation of the piriform cortex in terms of odor discrimination is lateralized; 3) activation of the navigation circuitry is achieved by means of lateralized activation of piriform cortex neurons. Altogether, the findings provide the first direct evidence that activation of the avian navigation circuitry is mediated by asymmetrical processing of olfactory input occurring in the right piriform cortex.

Social buffering of fear in zebrafish

Tese de Doutoramento em Biologia Comportamental apresentada ao ISPA - Instituto Universitário

Immediate and delayed effects of subchronic Paraquat exposure during an early differentiation stage in 3D-rat brain cell cultures.

Xenobiotic exposure is a risk factor in the etiology of neurodegenerative disease. It was recently hypothesized that restricted exposure during brain development could predispose for a neurodegenerative disease later in life. As neuroinflammation contributes to progressive neurodegeneration, it is suspected that neurodevelopmental xenobiotic exposure could elicit a neuroinflammatory process, which over time may assume a detrimental character. We investigated the neurotoxic effects of paraquat (PQ) in three-dimensional whole rat brain cell cultures, exposed during an early differentiation stage, comparing immediate effects-directly post exposure-with long-term effects, 20 days after interrupted PQ-administration. Adverse effects and neuroinflammatory responses were assessed by measuring changes in gene- and protein-expression as well as by determining cell morphology changes. Differentiating neural cultures were highly susceptible to PQ and showed neuronal damage and strong astrogliosis. After the 20-day washout period, neurons partially recovered, whereas astrogliosis persisted, and was accompanied by microglial activation of a neurodegenerative phenotype. Our data shows that immediate and long-term effects of subchronic PQ-exposure differ. Also, PQ-exposure during this window of extensive neuronal differentiation led to a delayed microglial activation, of a character that could promote further pro-inflammatory signals that enable prolonged inflammation, thereby fueling further neurodegeneration.

Immediate and early non-occlusal loading of Straumann implants with a chemically modified surface (SLActive) in the posterior mandible and maxilla: interim results from a prospective multicenter randomized-controlled study

OBJECTIVE: Immediate and early loading of dental implants can simplify treatment and increase overall patient satisfaction. The purpose of this 3-year prospective randomized-controlled multicenter study was to assess the differences in survival rates and bone level changes between immediately and early-loaded implants with a new chemically modified surface (SLActive). This investigation shows interim results obtained after 5 months. MATERIAL AND METHODS: Patients > or =18 years of age missing at least one tooth in the posterior maxilla or mandible were enrolled in the study. Following implant placement, patients received a temporary restoration either on the day of surgery (immediate loading) or 28-34 days after surgery (early loading); restorations consisted of single crowns or two to four unit fixed dental prostheses. Permanent restorations were placed 20-23 weeks following surgery. The primary efficacy variable was change in bone level (assessed by standardized radiographs) from baseline to 5 months; secondary variables included implant survival and success rates. RESULTS: A total of 266 patients were enrolled (118 males and 148 females), and a total of 383 implants were placed (197 and 186 in the immediate and early loading groups, respectively). Mean patient age was 46.3+/-12.8 years. After 5 months, implant survival rates were 98% in the immediate group and 97% in the early group. Mean bone level change from baseline was 0.81+/-0.89 mm in the immediate group and 0.56+/-0.73 mm in the early group (P<0.05). Statistical analysis revealed a significant center effect (P<0.0001) and a significant treatment x center interaction (P=0.008). CONCLUSIONS: The results suggested that Straumann implants with an SLActive can be used predictably in time-critical (early or immediate) loading treatment protocols when appropriate patient selection criteria are observed. The mean bone level changes observed from baseline to 5 months (0.56 and 0.81 mm) corresponded to physiological observations from other studies, i.e., were not clinically significant. The presence of a significant center effect and treatment x center interaction indicated that the differences in bone level changes between the two groups were center dependent.

Esthetic outcomes following immediate and early implant placement in the anterior maxilla: a systematic review.

PURPOSE The objectives of this systematic review are (1) to quantitatively estimate the esthetic outcomes of implants placed in postextraction sites, and (2) to evaluate the influence of simultaneous bone augmentation procedures on these outcomes. MATERIALS AND METHODS Electronic and manual searches of the dental literature were performed to collect information on esthetic outcomes based on objective criteria with implants placed after extraction of maxillary anterior and premolar teeth. All levels of evidence were accepted (case series studies required a minimum of 5 cases). RESULTS From 1,686 titles, 114 full-text articles were evaluated and 50 records included for data extraction. The included studies reported on single-tooth implants adjacent to natural teeth, with no studies on multiple missing teeth identified (6 randomized controlled trials, 6 cohort studies, 5 cross-sectional studies, and 33 case series studies). Considerable heterogeneity in study design was found. A meta-analysis of controlled studies was not possible. The available evidence suggests that esthetic outcomes, determined by esthetic indices (predominantly the pink esthetic score) and positional changes of the peri-implant mucosa, may be achieved for single-tooth implants placed after tooth extraction. Immediate (type 1) implant placement, however, is associated with a greater variability in outcomes and a higher frequency of recession of > 1 mm of the midfacial mucosa (eight studies; range 9% to 41% and median 26% of sites, 1 to 3 years after placement) compared to early (type 2 and type 3) implant placement (2 studies; no sites with recession > 1 mm). In two retrospective studies of immediate (type 1) implant placement with bone graft, the facial bone wall was not detectable on cone beam CT in 36% and 57% of sites. These sites had more recession of the midfacial mucosa compared to sites with detectable facial bone. Two studies of early implant placement (types 2 and 3) combined with simultaneous bone augmentation with GBR (contour augmentation) demonstrated a high frequency (above 90%) of facial bone wall visible on CBCT. Recent studies of immediate (type 1) placement imposed specific selection criteria, including thick tissue biotype and an intact facial socket wall, to reduce esthetic risk. There were no specific selection criteria for early (type 2 and type 3) implant placement. CONCLUSIONS Acceptable esthetic outcomes may be achieved with implants placed after extraction of teeth in the maxillary anterior and premolar areas of the dentition. Recession of the midfacial mucosa is a risk with immediate (type 1) placement. Further research is needed to investigate the most suitable biomaterials to reconstruct the facial bone and the relationship between long-term mucosal stability and presence/absence of the facial bone, the thickness of the facial bone, and the position of the facial bone crest.

Immediate reconstruction following breast-conserving surgery: management of the positive surgical margins and influence on secondary reconstruction

The efficacy of breast-conserving surgery for the local control of early breast cancer has been repeatedly evidenced. Although immediate reconstruction following breast-conserving surgery has been described, little information is available regarding surgical management in reoperative settings due to positive margins. We studied the influence of intraoperatively assessed and postoperatively controlled surgical margin status on the type of breast-conserving surgery and report our results regarding complications in a reoperative breast reconstruction scenario. All patients were seen by a multidisciplinary team who recommended breast-conserving surgery. According to the breast volume, ptosis and tumor size/location, the patients were also evaluated by a plastic surgeon, who recommended reconstruction with the appropriate technique. Intraoperative assessment of surgical margins was determined by histological examination of frozen sections. The mean follow-up time was 48months. Two hundred and eighteen patients (88.5 per cent ) underwent breast-conserving surgery and immediate reconstruction. Twelve (5.5 per cent ) patients had a positive tumor margin after review of the permanent section. All patients underwent re-exploration. In 1.3 per cent , a second reconstructive technique was indicated and in 2.2 per cent a skin-sparing mastectomy with total reconstruction was performed. Our findings support the important role of the intraoperative assessment of surgical margins and its interference in the selection of reconstruction techniques and negative margins; however, it will not guarantee complete excision of the tumor. Success depends on coordinated planning with the oncologic surgeon and careful intraoperative management

Effects of age and alcohol exposure during early life on pyramidal cell numbers in the CA1-CA3 region of the rat hippocampus

We have previously shown that exposing rats to a relatively high dose of ethanol during early postnatal life can result in an alteration in spatial learning ability. The hippocampal formation is known to be involved in the control of this ability. The purpose of the present study was to determine whether exposure of rats to ethanol during early postnatal life had either immediate or delayed effects on the numbers of pyramidal cells in the CA1-CA3 subregion of the hippocampus. Wistar rats were exposed to a relatively high daily dose of ethanol at postnatal day 10-15 by placing them for 3 h/day in a chamber containing ethanol vapor. Groups of ethanol-treated (ET), separation control (SC), and mother-reared control (MRC) rats were anesthetized and killed at 16 and 30 days of age by perfusion with phosphate-buffered 2.5% glutaraldehyde. The Cavalieri principle was used to determine the volumes of the CA1 and CA2+CA3 regions. The physical disector method was used to estimate the numerical density of neurons in each of the subdivisions. The total number of pyramidal cells was calculated by multiplying the appropriate estimates of the numerical density by the volume. There were significant age-related reductions in the total numbers of pyramidal cells at 16-30 days of age irrespective of the groups examined. Ethanol treated rats were found to have slightly but significantly fewer pyramidal cell neurons than either the MRC or SC groups. These observations indicate that pyramidal cells in the hippocampus may be vulnerable to a relatively high dose of ethanol exposure during this short period of early postnatal life. (C) 2003 Wiley-Liss, Inc.

Psychometric characteristics of the Rivermead Behavioural Memory Test (RBMT) as an early detection instrument for dementia and mild cognitive impairment in Brazil

Background: The Rivermead Behavioural Memory Test (RBMT) assesses everyday memory by means of tasks which mimic daily challenges. The objective was to examine the validity of the Brazilian version of the RBMT to detect cognitive decline. Methods: 195 older adults were diagnosed as normal controls (NC) or with mild cognitive impairment (MCI) or Alzheimer`s disease (AD) by a multidisciplinary team, after participants completed clinical and neuropsychological protocols. Results: Cronbach`s alpha was high for the total sample for the RBMT profile (PS) and screening scores (SS) (PS=0.91, SS=0.87) and for the AD group (PS=0.84, SS=0.85), and moderate for the MCI (PS=0.62, SS=0.55)and NC (PS=0.62, SS=0.60) groups. RBMT total scores, Appointment, Pictures, Immediate and Delayed Story, Immediate and Delayed Route, Delayed Message and Date contributed to differentiate NC from MCI. ROC curve analyses indicated high accuracy to differentiate NC from AD patients, and, moderate accuracy to differentiate NC from MCI. Conclusions: The Brazilian version of the RBMT seems to be an appropriate instrument to identify memory decline in Brazilian older adults.