Recombinant Subgroup B Human Respiratory Syncytial Virus Expressing Enhanced Green Fluorescent Protein Efficiently Replicates in Primary Human Cells and Is Virulent in Cotton Rats

Human respiratory syncytial virus (HRSV) is the most important viral cause of severe respiratory tract disease in infants. Two subgroups (A and B) have been identified, which cocirculate during, or alternate between, yearly epidemics and cause indistinguishable disease. Existing in vitro and in vivo models of HRSV focus almost exclusively on subgroup A viruses. Here, a recombinant (r) subgroup B virus (rHRSV(B05)) was generated based on a consensus genome sequence obtained directly from an unpassaged clinical specimen from a hospitalized infant. An additional transcription unit containing the gene encoding enhanced green fluorescent protein (EGFP) was introduced between the phosphoprotein and matrix genes (position 5) of the genome to generate rHRSV(B05)EGFP(5). The recombinant viruses replicated efficiently in both HEp-2 cells and in well-differentiated normal human bronchial cells grown at air-liquid interface. Intranasal infection of cotton rats (Sigmodon hispidus) resulted in high numbers of EGFP(+) cells in epithelia of the nasal septum and conchae. When administered in a relatively large inoculum volume, the virus also replicated efficiently in bronchiolar epithelial cells and spread extensively in both the upper and lower respiratory tracts. Virus replication was not observed in ciliated epithelial cells of the trachea. This is the first virulent rHRSV strain with the genetic composition of a currently circulating wild-type virus. In vivo tracking of infected cells by means of EGFP fluorescence in the absence of cytopathic changes increases the sensitivity of virus detection in HRSV pathogenesis studies.

IMPORTANCE

Virology as a discipline has depended on monitoring cytopathic effects following virus culture in vitro. However, wild-type viruses isolated from patients often do not cause significant changes to infected cells, necessitating blind passage. This can lead to genetic and phenotypic changes and the generation of high-titer, laboratory-adapted viruses with diminished virulence in animal models of disease. To address this, we determined the genome sequence of an unpassaged human respiratory syncytial virus from a sample obtained directly from an infected infant, assembled a molecular clone, and recovered a wild-type recombinant virus. Addition of a gene encoding enhanced green fluorescent protein allowed this wild-type virus to be tracked in primary human cells and living animals in the absence of significant cytopathic effects. Imaging of fluorescent cells proved to be a highly valuable tool for monitoring the spread of virus and may help improve assays for evaluating novel intervention strategies.

The Evaluation and Quantitation of Dihydrogen Metabolism Using Deuterium Isotope in Rats

Purpose: Despite the significant interest in molecular hydrogen as an antioxidant in the last eight years, its quantitative metabolic parameters in vivo are still lacking, as is an appropriate method for determination of hydrogen effectivity in the mammalian organism under various conditions.

Basic Procedures: Intraperitoneally-applied deuterium gas was used as a metabolic tracer and deuterium enrichment was determined in the body water pool. Also, in vitro experiments were performed using bovine heart submitochondrial particles to evaluate superoxide formation in Complex I of the respiratory chain.

Main Findings: A significant oxidation of about 10% of the applied dose was found under physiological conditions in rats, proving its antioxidant properties. Hypoxia or endotoxin application did not exert any effect, whilst pure oxygen inhalation reduced deuterium oxidation. During in vitro experiments, a significant reduction of superoxide formation by Complex I of the respiratory chain was found under the influence of hydrogen. The possible molecular mechanisms of the beneficial effects of hydrogen are discussed, with an emphasis on the role of iron sulphur clusters in reactive oxygen species generation and on iron species-dihydrogen interaction.

Principal Conclusions: According to our findings, hydrogen may be an efficient, non-toxic, highly bioavailable and low-cost antioxidant supplement for patients with pathological conditions involving ROS-induced oxidative stress.

Kaempferol Exhibits Progestogenic Effects in Ovariectomized Rats

OBJECTIVE: Progesterone (P4) plays a central role in women's health. Synthetic progestins are used clinically in hormone replacement therapy (HRT), oral contraceptives, and for the treatment of endometriosis and infertility. Unfortunately, synthetic progestins are associated with side effects, including cardiovascular disease and breast cancer. Botanical dietary supplements are widely consumed for the alleviation of a variety of gynecological issues, but very few studies have characterized natural compounds in terms of their ability to bind to and activate progesterone receptors (PR). Kaempferol is a flavonoid that functions as a non-steroidal selective progesterone receptor modulator (SPRM) in vitro. This study investigated the molecular and physiological effects of kaempferol in the ovariectomized rat uteri.

METHODS: Since genistein is a phytoestrogen that was previously demonstrated to increase uterine weight and proliferation, the ability of kaempferol to block genistein action in the uterus was investigated. Analyses of proliferation, steroid receptor expression, and induction of well-established PR-regulated targets Areg and Hand2 were completed using histological analysis and qPCR gene induction experiments. In addition, kaempferol in silico binding analysis was completed for PR. The activation of estrogen and androgen receptor signalling was determined in vitro.

RESULTS: Molecular docking analysis confirmed that kaempferol adopts poses that are consistent with occupying the ligand-binding pocket of PRA. Kaempferol induced expression of PR regulated transcriptional targets in the ovariectomized rat uteri, including Hand2 and Areg. Consistent with progesterone-l ke activity, kaempferol attenuated genistein-induced uterine luminal epithelial proliferation without increasing uterine weight. Kaempferol signalled without down regulating PR expression in vitro and in vivo and without activating estrogen and androgen receptors.

CONCLUSION: Taken together, these data suggest that kaempferol is a unique natural PR modulator that activates PR signaling in vitro and in vivo without triggering PR degradation.

MRZ-99030 – a novel modulator of Aβ aggregation: II – reversal of Aβ oligomer-induced deficits in long-term potentiation (LTP) and cognitive performance in rats and mice.

β-amyloid1-42 (Aβ1-42) is a major endogenous pathogen underlying the aetiology of Alzheimer's disease (AD). Recent evidence indicates that soluble Aβ oligomers, rather than plaques, are the major cause of synaptic dysfunction and neurodegeneration. Small molecules that suppress Aβ aggregation, reduce oligomer stability or promote off-pathway non-toxic oligomerization represent a promising alternative strategy for neuroprotection in AD. MRZ-99030 was recently identified as a dipeptide that modulates Aβ1-42 aggregation by triggering a non-amyloidogenic aggregation pathway, thereby reducing the amount of intermediate toxic soluble oligomeric Aβ species. The present study evaluated the relevance of these promising results with MRZ-99030 under pathophysiological conditions i.e. against the synaptotoxic effects of Aβ oligomers on hippocampal long term potentiation (LTP) and two different memory tasks. Aβ1-42 interferes with the glutamatergic system and with neuronal Ca2+ signalling and abolishes the induction of LTP. Here we demonstrate that MRZ-99030 (100–500 nM) at a 10:1 stoichiometric excess to Aβ clearly reversed the synaptotoxic effects of Aβ1-42 oligomers on CA1-LTP in murine hippocampal slices. Co-application of MRZ-99030 also prevented the two-fold increase in resting Ca2+ levels in pyramidal neuron dendrites and spines triggered by Aβ1-42 oligomers. In anaesthetized rats, pre-administration of MRZ-99030 (50 mg/kg s.c.) protected against deficits in hippocampal LTP following i.c.v. injection of oligomeric Aβ1-42. Furthermore, similar treatment significantly ameliorated cognitive deficits in an object recognition task and under an alternating lever cyclic ratio schedule after the i.c.v. application of Aβ1-42 and 7PA2 conditioned medium, respectively. Altogether, these results demonstrate the potential therapeutic benefit of MRZ-99030 in AD.

Analgesic exposure in pregnant rats affects fetal germ cell development with inter-generational reproductive consequences

Analgesics which affect prostaglandin (PG) pathways are used by most pregnant women. As germ cells (GC) undergo developmental and epigenetic changes in fetal life and are PG targets, we investigated if exposure of pregnant rats to analgesics (indomethacin or acetaminophen) affected GC development and reproductive function in resulting offspring (F1) or in the F2 generation. Exposure to either analgesic reduced F1 fetal GC number in both sexes and altered the tempo of fetal GC development sex-dependently, with delayed meiotic entry in oogonia but accelerated GC differentiation in males. These effects persisted in adult F1 females as reduced ovarian and litter size, whereas F1 males recovered normal GC numbers and fertility by adulthood. F2 offspring deriving from an analgesic-exposed F1 parent also exhibited sex-specific changes. F2 males exhibited normal reproductive development whereas F2 females had smaller ovaries and reduced follicle numbers during puberty/adulthood; as similar changes were found for F2 offspring of analgesic-exposed F1 fathers or mothers, we interpret this as potentially indicating an analgesic-induced change to GC in F1. Assuming our results are translatable to humans, they raise concerns that analgesic use in pregnancy could potentially affect fertility of resulting daughters and grand-daughters.

Effects of early life permethrin exposure on spatial working memory and on monoamine levels in different brain areas of pre-senescent rats

Pesticide exposure during brain development could represent an important risk factor for the onset of neurodegenerative diseases. Previous studies investigated the effect of permethrin (PERM) administered at 34 mg/kg, a dose close to the no observable adverse effect level (NOAEL) from post natal day (PND) 6 to PND 21 in rats. Despite the PERM dose did not elicited overt signs of toxicity (i.e. normal body weight gain curve), it was able to induce striatal neurodegeneration (dopamine and Nurr1 reduction, and lipid peroxidation increase). The present study was designed to characterize the cognitive deficits in the current animal model. When during late adulthood PERM treated rats were tested for spatial working memory performances in a T-maze-rewarded alternation task they took longer to choose for the correct arm in comparison to age matched controls. No differences between groups were found in anxiety-like state, locomotor activity, feeding behavior and spatial orientation task. Our findings showing a selective effect of PERM treatment on the T-maze task point to an involvement of frontal cortico-striatal circuitry rather than to a role for the hippocampus. The predominant disturbances concern the dopamine (DA) depletion in the striatum and, the serotonin (5-HT) and noradrenaline (NE) unbalance together with a hypometabolic state in the medial prefrontal cortex area. In the hippocampus, an increase of NE and a decrease of DA were observed in PERM treated rats as compared to controls. The concentration of the most representative marker for pyrethroid exposure (3-phenoxybenzoic acid) measured in the urine of rodents 12 h after the last treatment was 41.50 µ/L and it was completely eliminated after 96 h.

Characterization of the Toxicological Effects of Aminocarb on Rats: Hematological, Biochemical, and Histological Analyses

Aminocarb is a widely applied carbamate insecticide with action of controlling pests such as Lepidoptera and Coleoptera. In this study, subchronic effects on Wistar rats were investigated using hematological, biochemical, and histological techniques. Rats were exposed orally at sublethal levels of 10, 20, or 40 mg/kg body weight (groups A, B, and C, respectively) for 14 d. Hematological results revealed no statistical differences after 1 d of exposure but significant reduction in white blood cells detected after 7 d of exposure in group C, as well as, in all treated groups after 14 d of exposure. Biochemical data showed a decrease of acetylcholinesterase activity in all groups after 1 d of exposure with a return to normal after 7 and 14 d. Significant increase in alkaline phosphatase activity of rats exposed to aminocarb was noted after 7 d of treatment. The levels of triglycerides were also significantly decreased. The present investigation also showed a significant increase in content of serum urea and creatinine in animals from group A (14 d), and from groups B and C (7 and 14 d). Histological results demonstrated hemorrhagic focus on hepatic and renal parenchyma in all exposed groups. Taken together, the attained results were dose dependent and indicated adverse effects of aminocarb on hepatic and renal functions, as well as on immune responsiveness at sublethal tested doses.

Determination of permethrin and its metabolite in brains and urines of rats by GC-ECD and GC-MS

Synthetic pyrethroids are pesticides derived from naturally occurring pyrethrins, taken from pyrethrum of dried Chrysanthemum flowers. Pyrethroid insecticides are one of the most commonly used residential and agricultural insecticides. Many pyrethroids can significantly harm the nervous system. Permethrin could be one of the factors involved in the onset of neurodegenerative diseases. The present study aims to evaluate in brain, the effect that can induce the exposure to permethrin, during early life of female rats (from 6 to 21 days of life). Therefore, have been examined the concentrations of permethrin and its main metabolite (3-PBA) in the brain and urine in female rats sacrificed the day after and 14 days after treating. The different concentrations of permethrin and 3-PBA (after 24h and after 14 days in the end of treatment) were obtained using two different methods. The evaluation of permethrin by liquid-liquid extraction and GC-ECD was performed. The levels of the 3-PBA (in urine and brains) were obtained by SPE procedure and GC-MS using the 2- PBA as internal standard.

Evaluation of antihypertensive drugs in patients with obstructive sleep apnea and in rats submitted to chronic intermittent hypoxia

RESUMO: A hipertensão arterial (HA) é uma patologia altamente prevalente, embora claramente subdiagnosticada, em doentes com síndrome de apneia obstrutiva do sono (SAOS). Estas duas patologias apresentam uma estreita relação e a monitorização ambulatória da pressão arterial (MAPA), por um período de 24 horas, parece ser o método mais preciso para o diagnóstico de hipertensão em doentes com SAOS. No entanto, esta ferramenta de diagnóstico para além de ser dispendiosa e envolver um número acrescido de meios técnicos e humanos, é mais morosa e, por conseguinte, não é utilizada por rotina no contexto do diagnóstico da SAOS. Por outro lado, apesar da aplicação de pressão positiva contínua nas vias aéreas (CPAP – Continous Positive Airway Pressure) ser considerada a terapêutica de eleição para os doentes com SAOS, o seu efeito no abaixamento da pressão arterial (PA) parece ser modesto, exigindo, por conseguinte, a implementação concomitante de terapêutica anti-hipertensora. Acontece que são escassos os dados relativos aos regimes de fármacos anti-hipertensores utilizados em doentes com SAOS e, acresce ainda que, as guidelines terapêuticas para o tratamento farmacológico da HA, neste grupo particular de doentes, permanecem, até ao momento, inexistentes. A utilização de modelos animais de hipóxia crónica intermitente (CIH), que mimetizam a HA observada em doentes com SAOS, revela-se extremamente importante, uma vez que se torna imperativo identificar fármacos que promovam um controle adequado da PA neste grupo de doentes. No entanto, estudos concebidos com o intuito de investigar o efeito anti-hipertensor dos fármacos neste modelo animal revelam-se insuficientes e, por outro lado, os escassos estudos que testaram fármacos anti-hipertensores neste modelo não foram desenhados para responder a questões de natureza farmacológica. Acresce ainda que se torna imprescindível garantir a escolha de um método para administração destes fármacos que seja não invasivo e que minimize o stress do animal. Embora a gavagem seja uma técnica indiscutivelmente eficaz e amplamente utilizada para a administração diária de fármacos a animais de laboratório, ela compreende uma sequência de procedimentos geradores de stress para os animais e, que podem por conseguinte, constituir um viés na interpretação dos resultados obtidos. O objectivo global da presente investigação translacional foi contribuir para a identificação de fármacos anti-hipertensores mais efectivos para o tratamento da HT nos indivíduos com SAOS e investigar mecanismos subjacentes aos efeitos sistémicos associadas à SAOS bem como a sua modulação por fármacos anti-hipertensores. Os objectivos específicos foram: em primeiro lugar,encontrar novos critérios, baseados nas medidas antropométricas, que permitam a identificação de doentes com suspeita de SAOS, que erroneamente se auto-classifiquem como nãohipertensos, e desta forma promover um uso mais criterioso do MAPA; em segundo lugar, investigar a existência de uma hipotética associação entre os esquemas de fármacos antihipertensores e o controle da PA (antes e após a adaptação de CPAP) em doentes com SAOS em terceiro lugar, avaliar a eficácia do carvedilol (CVD), um fármaco bloqueador β-adrenérgico não selectivo com actividade antagonista α1 intrínseca e propriedades anti-oxidantes num modelo animal de hipertensão induzida pela CIH; em quarto lugar, explorar os efeitos da CIH sobre o perfil farmacocinético do CVD; e, em quinto lugar, investigar um método alternativo à gavagem para a administração crónica de fármacos anti-hipertensores a animais de laboratório. Com este intuito, na primeira fase deste projecto, fizemos uso de uma amostra com um número apreciável de doentes com SAOS (n=369), que acorreram, pela primeira vez, à consulta de Patologia do Sono do CHLN e que foram submetidos a um estudo polissonográfico do sono, à MAPA e que preencheram um questionário que contemplava a obtenção de informação relativa ao perfil da medicação anti-hipertensora em curso. Numa segunda fase, utilizámos um modelo experimental de HT no rato induzida por um paradigma de CIH. Do nosso trabalho resultaram os seguintes resultados principais: em primeiro lugar, o índice de massa corporal (IMC) e o perímetro do pescoço (PP) foram identificados como preditores independentes de “auto-classificação errónea” da HA em doentes com suspeita de SAOS; em segundo lugar, não encontramos qualquer associação com significado estatístico entre os vários esquemas de fármacos anti-hipertensores bem como o número de fármacos incluídos nesse esquemas, e o controle da PA (antes e depois da adaptação do CPAP); em terceiro lugar, apesar das doses de 10, 30 e 50 mg/kg de carvedilol terem promovido uma redução significativa da frequência cardíaca, não foi observado qualquer decréscimo na PA no nosso modelo animal; em quarto lugar, as razões S/(R+S) dos enantiómeros do CVD nos animais expostos à CIH e a condições de normóxia revelaram-se diferentes; e, em quinto lugar, a administração oral voluntária mostrou ser um método eficaz para a administração diária controlada de fármacos anti-hipertensores e que é independente da manipulação e contenção do animal. Em conclusão, os resultados obtidos através do estudo clínico revelaram que o controle da PA, antes e após a adaptação do CPAP, em doentes com SAOS é independente, quer do esquema de fármacos anti-hipertensores, quer do número de fármacos incluídos num determinado esquema. Os nossos resultados salientam ainda a falta de validade da chamada self-reported hypertension e sugerem que em todos os doentes com suspeita de SAOS, com HA não diagnosticada e com um IMC e um PP acima de 27 kg/m2 e 39 cm, respectivamente, a confirmação do diagnóstico de HA deverá ser realizada através da MAPA, ao invés de outros métodos que com maior frequência são utilizados com este propósito. Os resultados obtidos no modelo animal de HA induzida pela CIH sugerem que o bloqueio do sistema nervoso simpático, juntamente com os supostos efeitos pleiotrópicos do CVD, não parece ser a estratégia mais adequada para reverter este tipo particular de hipertensão e indicam que as alterações farmacocinéticas induzidas pela CIH no ratio S/(R+S) não justificam a falta de eficácia anti-hipertensora do CVD observada neste modelo animal. Por último, os resultados do presente trabalho suportam ainda a viabilidade da utilização da administração oral voluntária, em alternativa à gavagem, para a administração crónica de uma dose fixa de fármacos anti-hipertensores.---------------------------- ABSTRACT: Hypertension (HT) is a highly prevalent condition, although under diagnosed, in patients with obstructive sleep apnea (OSA). These conditions are closely related and 24-hour ambulatory blood pressure monitoring (ABPM) seems to be the most accurate measurement for diagnosing hypertension in OSA. However, this diagnostic tool is expensive and time-consuming and, therefore, not routinely used. On the other hand, although continuous positive airway pressure (CPAP) is considered the gold standard treatment for symptomatic OSA, its lowering effect on blood pressure (BP) seems to be modest and, therefore, concomitant antihypertensive therapy is still required. Data on antihypertensive drug regimens in patients with OSA are scarce and specific therapeutic guidelines for the pharmacological treatment of hypertension in these patients remain absent. The use of animal models of CIH, which mimic the HT observed in patients with OSA, is extremely important since it is imperative to identify preferred compounds for an adequate BP control in this group of patients. However, studies aimed at investigating the antihypertensive effect of antihypertensive drugs in this animal model are insufficient, and most reports on CIH animal models in which drugs have been tested were not designed to respond to pharmacological issues. Moreover, when testing antihypertensive drugs (AHDs) it becomes crucial to ensure the selection of a non-invasive and stress-free method for drug delivery. Although gavage is effective and a widely performed technique for daily dosing in laboratory rodents, it comprises a sequence of potentially stressful procedures for laboratory animals that may constitute bias for the experimental results. The overall goal of the present translational research was to contribute to identify more effective AHDs for the treatment of hypertension in patients with OSA and investigate underlying mechanisms of systemic effects associated with OSA, as well as its modulation by AHDs. The specific aims were: first, to find new predictors based on anthropometric measures to identify patients that misclassify themselves as non-hypertensive, and thereby promote the selective use of ABPM; second, to investigate a hypothetical association between ongoing antihypertensive regimens and BP control rates in patients with OSA, before and after CPAP adaptation; third, to determine, in a rat model of CIH-induced hypertension, the efficacy of carvedilol (CVD), a nonselective beta-blocker with intrinsic anti-α1-adrenergic activity and antioxidant properties; fourth, to explore the effects of CIH on the pharmacokinetics profile of CVD and fifth, to investigate an alternative method to gavage, for chronic administration of AHDs to laboratory rats. For that, in the first phase of this project, we used a sizeable sample of patients with OSA (n=369), that attended a first visit at Centro Hospitalar Lisboa Norte, EPE Sleep Unit, and underwent overnight polysomnography, 24-h ABPM and filled a questionnaire that included ongoing antihypertensive medication profile registration. In the second phase, a rat experimental model of HT induced by a paradigm of CIH that simulates OSA was used. The main findings of this work were: first, body mass index (BMI) and neck circumference (NC) were identified as independent predictors of hypertension misclassification in patients suspected of OSA; second, in patients with OSA, BP control is independent of both the antihypertensive regimen and the number of antihypertensive drugs, either before or after CPAP adaptation; third, although the doses of 10, 30 and 50 mg/Kg of CVD promoted a significant reduction in heart rate, no decrease in mean arterial pressure was observed; fourth, the S/(R+S) ratios of CVD enantiomers, between rats exposed to CIH and normoxic conditions, were different and fifth, voluntary ingestion proved to be an effective method for a controlled daily dose administration, with a define timetable, that is independent of handling and restraint procedures. In conclusion, the clinical study showed that BP control in OSA patients is independent of both the antihypertensive regimen and the number of antihypertensive drugs. Additionally, our results highlight the lack of validity of self-reported hypertension and suggest that all patients suspected of OSA with undiagnosed hypertension and with a BMI and NC above 27 Kg/m2 and 39 cm should be screened for hypertension, through ABPM. The results attained in the rat model of HT related to CIH suggest that the blockade of the sympathetic nervous system together with the putative pleiotropic effects of carvedilol is not able to revert hypertension induced by CIH and point out that the pharmacokinetic changes induced by CIH on S/(R+S) ratio are not apparently responsible for the lack of efficacy of carvedilol in reversing this particular type of hypertension. Finally, the results here presented support the use of voluntary oral administration as a viable alternative to gavage for chronic administration of a fixed dose of AHDs.

Olfactory traces and spatial learning in rats.

We conducted an experiment to assess the use of olfactory traces for spatial orientation in an open environment in rats, Rattus norvegicus. We trained rats to locate a food source at a fixed location from different starting points, in the presence or absence of visual information. A single food source was hidden in an array of 19 petri dishes regularly arranged in an open-field arena. Rats were trained to locate the food source either in white light (with full access to distant visuospatial information) or in darkness (without any visual information). In both cases, the goal was in a fixed location relative to the spatial frame of reference. The results of this experiment revealed that the presence of noncontrolled olfactory traces coherent with the spatial frame of reference enables rats to locate a unique position as accurately in darkness as with full access to visuospatial information. We hypothesize that the olfactory traces complement the use of other orientation mechanisms, such as path integration or the reliance on visuospatial information. This experiment demonstrates that rats can rely on olfactory traces for accurate orientation, and raises questions about the establishment of such traces in the absence of any other orientation mechanism. Copyright 1998 The Association for the Study of Animal Behaviour.

Influence of local environmental olfactory cues on place learning in rats

The aim of the present study was to assess the influence of local environmental olfactory cues on place learning in rats. We developed a new experimental design allowing the comparison of the use of local olfactory and visual cues in spatial and discrimination learning. We compared the effect of both types of cues on the discrimination of a single food source in an open-field arena. The goal was either in a fixed or in a variable location, and could be indicated by local olfactory and/or visual cues. The local cues enhanced the discrimination of the goal dish, whether it was in a fixed or in a variable location. However, we did not observe any overshadowing of the spatial information by the local olfactory or visual cue. Rats relied primarily on distant visuospatial information to locate the goal, neglecting local information when it was in conflict with the spatial information.