Paroxetine and bupropion have no in vitro effects on lynphocyte proliferation and viability

OBJECTIVE: Initial studies with tricyclic antidepressants demonstrated that they jeopardize the immune system activity. Recent studies suggested that selective serotonin reuptake inhibitors would have stimulating immunological effects. Here, we explored the in vitro immunological effects of two antidepressants used in clinical practice, paroxetine (selective serotonin reuptake inhibitor) and bupropion (norepinephrine and dopamine reuptake inhibitor). METHOD: Peripheral blood samples were obtained from 16 healthy volunteers and the peripheral blood mononuclear cells were isolated and cultured in vitro. We evaluated the effects of bupropion and paroxetine on cell viability as well as the ability to suppress phytohemagglutinin-induced lymphocyte proliferation. RESULTS: Both antidepressants produced neither significant effect on cell viability nor on T-cell proliferation. CONCLUSIONS: This could be of valuable information for the clinical practice when these drugs are administered. These results indicate a more favorable effect of such psychopharmacological drugs when compared to reported immunological effects associated with tryciclic antidepressants.

Selective serotonin-reuptake inhibitor and norepinephrine dopamine reuptake inhibitor antidepressants do not affect natural killer cell activity in vitro

OBJECTIVE: This study aims to evaluate the citotoxic activity of two commonly used anti-depressants: paroxetine and bupropion. We also evaluated the in vitro natural killer activity (NKA) after incubating the blood samples with the antidepressants. METHODS: Peripheral blood samples from 15 healthy volunteers were collected and the mononuclear cells (PBMCs) were isolated and incubated for 24h with (or without = control cells) paroxetine and bupropion, in concentrations of 30, 100 and 1000 ng/ml. After the incubation period in both groups, the amount of dead cells was calculated using trypam blue technique. NKA was evaluated using the classic51Cr release assay. CONCLUSIONS: PBMCs dead cells occurred in both groups and in proportion to all pharmacological concentrations. Nevertheless, the NKA was not affected, even with the reduction in the number of effective cells.

The role of IFNγ in higher brain function: in health and under chronic stress

Tese de Doutoramento em Ciências da Saúde

Characterization of an in vitro fed-batch model to obtain cells released from S. epidermidis biofilms

Both dynamic and fed-batch systems have been used for the study of biofilms. Dynamic systems, whose hallmark is the presence of continuous flow, have been considered the most appropriate for the study of the last stage of the biofilm lifecycle: biofilm disassembly. However, fed-batch is still the most used system in the biofilm research field. Hence, we have used a fed-batch system to collect cells released from Staphylococcus epidermidis biofilms, one of the most important etiological agents of medical device-associated biofilm infections. Herein, we showed that using this model it was possible to collect cells released from biofilms formed by 12 different S. epidermidis clinical and commensal isolates. In addition, our data indicated that biofilm disassembly occurred by both passive and active mechanisms, although the last occurred to a lesser extent. Moreover, it was observed that S. epidermidis biofilm-released cells presented higher tolerance to vancomycin and tetracycline, as well as a particular gene expression phenotype when compared with either biofilm or planktonic cells. Using this model, biofilm-released cells phenotype and their interaction with the host immune system could be studied in more detail, which could help providing significant insights into the pathophysiology of biofilm-related infections.

Evaluation of zearalenone in vitro removal by Saccharomyces cerevisiae strains isolated from bovine forage

Zearalenone (ZEN) is a mycotoxin that has relatively low acute toxicity. However, it is a potent oestrogen, interfering with the reproductive tract of animals. Among other effects, ZEN decreases animals fertility, and induces fibrosis in the uterus, breast cancer and endometrial carcinoma (Zinedine et al., 2007). Anti-mycotoxin additives (AMA) are defined as a group of products that, when added to animal feed, are capable of adsorbing, inactivating, or neutralizing mycotoxins in the gastrointestinal tract of animals. One example of these products are adsorbents based on yeast cell walls, a safe and beneficial animal feed additive (Abreu et al., 2008). When based on active cells, yeast based products also act as a probiotic, contributing to improve the general animal health because it stimulates their immune system and promotes the integrity of intestinal mucosa (Albino et al., 2006). Strains of Saccharomyces cerevisiae isolated from silage were tested for their ZEN removal capability. Their effect on - and b-zearalenol (-ZOL and b-ZOL) was also tested. Strains were grown on YPD separately supplemented with ZEN, -ZOL and b-ZOL, and their elimination from culture media was quantified over time by HPLC-FL.

Psychosocial adjustment and physical health in children of divorce

Objective: To review the literature on the effects of parental divorce over the psychological maladjustment and physical health problems in children of divorced parents, thus contributing to the integration of existing scientific knowledge based on the biopsychosocial model of the impact of divorce on children’s physical health as proposed by Troxel and Matthews (2004). Sources: Review of the literature using MEDLINE and PsycInfo (1980-2007) databases, selecting the most representative articles on the subject. Special attention was paid to contributions by internationally renowned investigators on the subject. Summary of the findings: Divorce may be responsible for a decline of physical and psychological health in children. The developmental maladjustment of children is not triggered by divorce itself, but rather by other risk factors associated with it, such as interparental conflict, parental psychopathology, decline in socio-economic level, inconsistency in parenting styles, a parallel and conflicting co-parenting relationship between parents and low levels of social support. Such risk factors trigger maladjusted developmental pathways, marked by psychopathological symptoms, poor academic performance, worst levels of physical health, risk behavior, exacerbated psychophysiological responses to stress and weakening of the immune system. Conclusions: Clear links were observed between experiencing parental divorce and facing problems of physical and psychological maladjustment in children. Divorce is a stressor that should be considered by health professionals as potentially responsible for maladjusted neuropsychobiological responses and for decline in children’s physical health.

Avaliação da expressão de mediadores imunitários em amostras de cetáceos capturados acidentalmente em Portugal Continental

Dissertação de Mestrado Integrado em Medicina Veterinária

Endogenous and exogenously-induced immunomodulation of tumour-host responsiveness

In spite of the availability of multiple effector mechanisms of the immune system to combat tumour growth and metastases, their impairment frequently accompanies the appearance of cancer. Factors contributing to this impairment may be related to properties of the host and/or the tumour itself and may be with respect to their origin -endogenous or exogenour. Based on the unique biological behavior of prostate cancer (PCa), and its apparent escape from immune surveillance in the presence of tumour immuno genicity, continuing investigation of endogenous and exogenous factors thought to be relevant to its pathogenesis have been made. For this purpose further studies of the suggested role of human seminal plasma (SePl) and the synthetic oestrogen, diethylstiboestrol (DES), as representative endogenous and exogenous immunomodulatory factors (IMF) of tumour-host responsiveness, together with evaluation of human prostatic tissue extracts and leuprolide (the luteinizing-hormone-releasing-hormone proposed as an alternate to DES therapy) have been made by evaluating their effect on the lytic activity of natural killer (NK) cells. SePl and prostate extracts significantly suppressed NK cell lysis. Physicochemical studies suggest SePl and prostate IMF to be associated with high and low molecular weight macromolecules; and implicate the participation of transglutaminase and prostaglandins. Comparative study of therapeutic levels of DES vs. leuprolide on NK cell lysis demonstrated significant suppression by DES vs. a negligible effect of leuprolide. Metastases are highly prevalent in PCa, and contribute significantly to its morbidity and mortality. Further knowledge of the range of effects of endogenous and exogenous IMF on effector mechanisms of tumour-host responsiveness, to include suppression of NK cells, and elucidation of their nature, may contribute toward our understanding of the unique biological behavior of tumours of the prostate, in addition to improvement in their clinical management.

Effects of irradiation and tunicamycin on the surface glycoproteins Schistosoma mansoni

The cercarial glycocalyx and schistosomulum surface contains a number of glycoproteins which are expressed in very variable amounts within a parasite population. Tunicamycin inhibits glycoprotein synthesis of schistosomula if the parasites are incubated for 24hr with the drug (10µg ml[raised to the power of -1]). An unexpected increase in lectin binding to the parasite surface was observed but no other changes were detected. Schistosomula treated in this way did not develop in the host past the lung stage. Ultraviolet irradiation (400µW min cm[raised to the power of-2]) also inhibited glycoprotein synthesis. Synthesis of other proteins, and in particular heat shock proteins, were also inhibited. Sera from mice (NIH strain) infected with irradiated cercariae contained antibodies which bound to normal schistosomula with lower affinity than to irradiated parasites. This is evidence that irradiation modifies the surface and secreted glycoproteins of schistosomula, so they are processed in a different way to normal glycoproteins by the host's immune system. The effects of irradiation on heat shock protein synthesis may allow the parasite to release a variety of proteins and glycoproteins in abnormal conformations. This may explain the enhanced immunogenicity of irradiated cercariae.

NLRP3 inflammasome plays a key role in the regulation of intestinal homeostasis.

BACKGROUND:: Attenuated innate immune responses to the intestinal microbiota have been linked to the pathogenesis of Crohn's disease (CD). Recent genetic studies have revealed that hypofunctional mutations of NLRP3, a member of the NOD-like receptor (NLR) superfamily, are associated with an increased risk of developing CD. NLRP3 is a key component of the inflammasome, an intracellular danger sensor of the innate immune system. When activated, the inflammasome triggers caspase-1-dependent processing of inflammatory mediators, such as IL-1β and IL-18. METHODS:: In the current study we sought to assess the role of the NLRP3 inflammasome in the maintenance of intestinal homeostasis through its regulation of innate protective processes. To investigate this role, Nlrp3(-/-) and wildtype mice were assessed in the dextran sulfate sodium and 2,4,6-trinitrobenzenesulfonic acid models of experimental colitis. RESULTS:: Nlrp3(-/-) mice were found to be more susceptible to experimental colitis, an observation that was associated with reduced IL-1β, reduced antiinflammatory cytokine IL-10, and reduced protective growth factor TGF-β. Macrophages isolated from Nlrp3(-/-) mice failed to respond to bacterial muramyl dipeptide. Furthermore, Nlrp3-deficient neutrophils exhibited reduced chemotaxis and enhanced spontaneous apoptosis, but no change in oxidative burst. Lastly, Nlrp3(-/-) mice displayed altered colonic β-defensin expression, reduced colonic antimicrobial secretions, and a unique intestinal microbiota. CONCLUSIONS:: Our data confirm an essential role for the NLRP3 inflammasome in the regulation of intestinal homeostasis and provide biological insight into disease mechanisms associated with increased risk of CD in individuals with NLRP3 mutations. (Inflamm Bowel Dis 2010).

Characterisation of heteromers and oligomers in the TNF family of ligands and receptors

TNF family ligands and receptors fulfill a number of functions, mainly in the immune system. For example, the ligands BAFF and APRIL control growth and survival of mature Β cells at various stages of differentiation. TNF family ligands usually form homotrimers, but heteromers have also been described for lymphotoxin α1β2 and for BAFF and APRIL. Interestingly, twenty BAFF homotrimers can assemble into virus-like particles coined BAFF 60-mer, which are superior to BAFF 3-mer regarding their ability to signal in primary Β cells. A screen was performed in 293T cells, by co-transfecting differently tagged ligands, to identify six novel heteromers. The specificity of these novel heteromers, however, did not correspond to that of orphan receptors in the TNFR family. Little is known about heteromers of BAFF and APRIL, in particular their receptor-binding specificity and their ability to signal. A method to produce and purify heteromers of defined stoechiometry was developed, and the resulting reagents were used to demonstrate that BAFF2APRIL, like BAFF, binds to all BAFF receptors - namely BAFFR, TACI and Β CM A -, while APRIL2BAFF and APRIL only binds to TACI and BCMA. Heteromers could signal via their cognate receptors, sometimes as potently and sometimes less potently than homomers, depending on the receptors. A promising system to measure the activity of single-chain homo- and heteromers in vivo was set up: it measures mature Β cell rescue upon administration of single-chain ligands into BAFF-ko mice. To tackle the question of the physiological importance of BAFF 60-mer, a point mutation that prevents assembly of mouse BAFF into 60-mer while retaining its ability to form trimers was identified. This mutation (E247K) was introduced by homologous recombination into mouse embryonic stem cells that are now being used to generate knock-in mice. Results obtained in this work will help to better understand the role of various BAFF and APRIL forms that are elevated in a several autoimmune diseases. - Les ligands et récepteurs de la famille du TNF joue un rôle prédominant dans le système immunitaire. Par exemple, les ligands BAFF et APRIL contrôlent la croissance et la survie des cellules Β matures à différents stades de différenciation. Ces ligands existent souvent sous forme d'homotrimères (3-mer), bien que des héteromères aient été décrits pour la lymphotoxine α1β2 et pour BAFF et APRIL. Dans le cas de BAFF, vingt trimères peuvent, telle une particule virale, s'assembler en 60-mer qui surpasse le 3-mer pour signaler dans des cellules Β primaires. Un crible effectué dans des cellules 293T, par co-transfection de ligands différemment marqués, a permis d'identifier six nouveaux heteromères dont la spécificité n'a, hélas, pas correspondu à celle d'un récepteur orphelin de la famille du TNFR. Les connaissances sur la spécificité de liaison aux récepteurs et la capacité à signaler des heteromères de BAFF et d'APRIL sont fragmentaires. Une méthode pour produire et purifier des heteromères "simple chaîne" de stoechiométrie déterminée a été mise au point, et les réactifs ainsi obtenus utilisés pour démontrer que BAFF2APRIL, comme BAFF, lie tous les récepteurs de BAFF - c'est-à-dire BAFFR, TACI et BCMA -, alors qu'APRIL2BAFF et APRIL ne lient que TACI et BCMA. Les héteromères peuvent transmettre des signaux, parfois aussi bien et parfois plus faiblement que les homomères, selon les récepteurs. Un système prometteur pour mesurer l'activité des ligands simple chaîne in vivo a été mis au point. Il mesure la réapparition de cellules Β matures dans des souris déficientes pour BAFF après administration des ligands. Pour s'attaquer à la question de l'importance physiologique du 60-mer de BAFF, ime mutation empêchant l'assemblage en 60-mer sans affecter la capacité à former des trimères a été identifiée. Cette mutation (E247K) a été introduite par recombinaison homologue dans des cellules souches embryonnaires de souris qui sont utilisées pour obtenir des souris déficientes en BAFF 60-mer. Les résultats de ces travaux contribueront à mieux cerner le rôle des différentes formes de BAFF et d'APRIL produites en excès dans plusieurs maladies auto-immunes.

Profile of a serial killer: cellular and molecular approaches to study individual cytotoxic T-cells following therapeutic vaccination.

T-cell vaccination may prevent or treat cancer and infectious diseases, but further progress is required to increase clinical efficacy. Step-by-step improvements of T-cell vaccination in phase I/II clinical studies combined with very detailed analysis of T-cell responses at the single cell level are the strategy of choice for the identification of the most promising vaccine candidates for testing in subsequent large-scale phase III clinical trials. Major aims are to fully identify the most efficient T-cells in anticancer therapy, to characterize their TCRs, and to pinpoint the mechanisms of T-cell recruitment and function in well-defined clinical situations. Here we discuss novel strategies for the assessment of human T-cell responses, revealing in part unprecedented insight into T-cell biology and novel structural principles that govern TCR-pMHC recognition. Together, the described approaches advance our knowledge of T-cell mediated-protection from human diseases.

The research on radioprotective agents in Chinese materia medica

A series of studies has been carried out in the field of traditional medicine for searching radio-protective agents. According to the theory of traditional Chinese medicine, may prescriptions were tested with experimental animals. Some of them could raise the survival rate of dogs irradiated with lethal dose of Pi-rays by 30-40%. Some symptoms of radiation sickness could be improved. More than one thousand kinds of Chinese herbs were screened. Some of them have pronounced radioprotectice activities. A series of bioactive components wee isolated from these herbs. The mechanism of radiation protection were studied. Having the capability of hemopoietic system and immune system may be the characteristics of these Chinese herbs.

Revisiting the specificity of the MHC class II transactivator CIITA in vivo.

CIITA is a master regulatory factor for the expression of MHC class II (MHC-II) and accessory genes involved in Ag presentation. It has recently been suggested that CIITA also regulates numerous other genes having diverse functions within and outside the immune system. To determine whether these genes are indeed relevant targets of CIITA in vivo, we studied their expression in CIITA-transgenic and CIITA-deficient mice. In contrast to the decisive control of MHC-II and related genes by CIITA, nine putative non-MHC target genes (Eif3s2, Kpna6, Tap1, Yars, Col1a2, Ctse, Ptprr, Tnfsf6 and Plxna1) were found to be CIITA independent in all cell types examined. Two other target genes, encoding IL-4 and IFN-gamma, were indeed found to be up- and down-regulated, respectively, in CIITA-transgenic CD4(+) T cells. However, there was no correlation between MHC-II expression and this Th2 bias at the level of individual transgenic T cells, indicating an indirect control by CIITA. These results show that MHC-II-restricted Ag presentation, and its indirect influences on T cells, remains the only pathway under direct control by CIITA in vivo. They also imply that precisely regulated MHC-II expression is essential for maintaining a proper Th1-Th2 balance.

The costs of parasitism and anti-parasitic defense in the common vole

Résumé : Les relations entre un parasite et son hôte sont avant tout marquées par le coût pour l'hôte que représente la ponction de ressources au profit du parasite et ses conséquences sur les traits d'histoires de vie de l'hôte. Pour contenir la réduction de leur valeur reproductive, les hôtes ont acquis au cours de l'évolution des mécanismes soit de lutte contre les parasites, soit de réallocations des ressources. Curieusement les effets des ectoparasites sur la biologie de mammifères ont été peu étudiés. Dans une première expérience à long terme, nous avons examiné sous un angle intégratif si les puces Nosopsyllus fasciatus affectent certains paramètres physiologiques des campagnols des champs Microtus arvalis. Nous avons également testé si les puces peuvent réduire la longévité et si oui, si ce pourrait être dû à une accélération de la sénescence. Ensuite nous avons testé si la simple activation répétée du système immunitaire comme lors d'une infestation chronique pouvait aussi réduire la longévité. Dans une dernière expérience, nous avons d'abord testé si l'infestation par des puces de jeunes campagnols au stade néonatal (21 jours) pouvait modifier leur développement et leur phénotype adulte. Puis nous avons testé si la modification du phénotype adulte est une réponse prédite et potentiellement adaptative pour minimiser les effets des puces à l'âge adulte. Nos résultats montrent que l'infestation par des puces réduit la croissance subadulte, induit une forte anémie et une immunodépression, et augmente le métabolisme de repos. De plus les puces réduisent la longévité et la taille des testicules, réduisant fortement le succès reproducteur potentiel des individus parasités. La taille finale, c'est-à-dire le développement pré-adulte, détermine en grande part la longévité. La réduction de longévité ne devrait pas être due à l'investissement au profit du système immunitaire car l'activation chronique seule du système immunitaire ne réduit pas la longévité. L'infestation néonatale retarde légèrement le développement mais surtout modifie l'hématocrite et réduit les performances locomotrices des campagnols plus de 3 mois après l'infestation. Les effets immédiats du parasitisme sur la physiologie semblent bien supérieurs comparés aux effets à long terme. Nous n'avons pas d'éléments permettant d'affirmer que le parasitisme néonatal prépare les campagnols à faire face aux puces à l'âge adulte. Au contraire, le parasitisme néonatal interagit sur le parasitisme adulte pour augmenter le métabolisme de repos. Cette thèse offre une vision intégrative des mécanismes par lesquels les puces peuvent affecter la valeur reproductive de leurs hôtes. De façon générale, ces résultats 35 montrent l'importance des puces comme force de sélection chez les campagnols. Il est indispensable de prendre en compte les ectoparasites dans l'étude de l'écologie et des dynamiques de populations chez les mammifères. Summary : The relationship between a parasite and its host is fundamentally marked by the costs for host of the withdrawals of resources by parasite and the subsequent reduction in host life-history traits. Hosts have evolved a number of strategies to reduce these costs, either by fighting against the parasite directly or by reallocating resources to reduce costs on lifetime reproductive value. The effects of ectoparasites on burrowing mammals have been scarcely studied. In a first long-term experiment, we examined how fleas Nosopsyllus fasciatus affect physiological levels of the common vole, Microtus arvalis. We also examined whether fleas reduce longevity and if so, if it is due to an early senescence pattern. Then we tested if experimental activation of the immune system by repeated injections of an antigen could result in a shorter longevity. In the last experiment, we tested if short-lasting neonatal parasitism can have long-term effects on phenotype, and if these effects could induce a predictive response to reduce damages when parasitized at the adult stage. We found that parasitism by flea reduced subadult growth, induced anaemia and immunodepression, and increased energy consumption even when resting. Moreover fleas reduce longevity and testes size associated to splenomegaly, suggesting an overall reduction in fitness but we did not find any pattern of accelerated senescence explaining the early death of parasitized voles compared to non-parasitzed. The cost of mounting an immune response throughout life does not impair longevity, suggesting that it is the cost of parasitism that limits the longevity and not the immune investment. Neonatal infestation by fleas has long-term effects on physiology and reduces motor activity more than 3 months after infestation. The modification of physiology due to long-term effects seems weak compared to the immediate effects of adult infestation. We found no evidence that neonatal parasitism prepares voles to mount a predictive adaptive response in order to reduce effects of fleas on fitness components. On the contrary, neonatal parasitism seems to worsen the effect of adult parasitism. This thesis offers an integrative view of mechanisms by which fleas affect their host at the individual level. Overall, our results demonstrate the importance of fleas as a selective force in voles. These results highlight the importance of ectoparasitism in ecology of micromarnrnals and suggest a role in the dynamic of host populations.