Cortex, cognition and the cell: New insights into the pyramidal neuron and prefrontal function

Arguably the most complex conical functions are seated in human cognition, the how and why of which have been debated for centuries by theologians, philosophers and scientists alike. In his best-selling book, An Astonishing Hypothesis: A Scientific Search for the Soul, Francis Crick refined the view that these qualities are determined solely by cortical cells and circuitry. Put simply, cognition is nothing more, or less, than a biological function. Accepting this to be the case, it should be possible to identify the mechanisms that subserve cognitive processing. Since the pioneering studies of Lorent de No and Hebb, and the more recent studies of Fuster, Miller and Goldman-Rakic, to mention but a few, much attention has been focused on the role of persistent neural activity in cognitive processes. Application of modern technologies and modelling techniques has led to new hypotheses about the mechanisms of persistent activity. Here I focus on how regional variations in the pyramidal cell phenotype may determine the complexity of cortical circuitry and, in turn, influence neural activity. Data obtained from thousands of individually injected pyramidal cells in sensory, motor, association and executive cortex reveal marked differences in the numbers of putative excitatory inputs received by these cells. Pyramidal cells in prefrontal cortex have, on average, up to 23 times more dendritic spines than those in the primary visual area. I propose that without these specializations in the structure of pyramidal cells, and the circuits they form, human cognitive processing would not have evolved to its present state. I also present data from both New World and Old World monkeys that show varying degrees of complexity in the pyramidal cell phenotype in their prefrontal cortices, suggesting that cortical circuitry and, thus, cognitive styles are evolving independently in different species.

Pyramidal cell heterogeneity in the visual cortex of the nocturnal new world owl monkey (aotus trivirgatus)

Recent studies have revealed marked variation in pyramidal cell structure in the visual cortex of macaque and marmoset monkeys. In particular, there is a systematic increase in the size of, and number of spines in, the arbours of pyramidal cells with progression through occipitotemporal (OT) visual areas. In the present study we extend the basis for comparison by investigating pyramidal cell structure in visual areas of the nocturnal owl monkey. As in the diurnal macaque and marmoset monkeys, pyramidal cells became progressively larger and more spinous with anterior progression through OT visual areas. These data suggest that: 1. the trend for more complex pyramidal cells with anterior progression through OT visual areas is a fundamental organizational principle in primate cortex; 2. areal specialization of the pyramidal cell phenotype provides an anatomical substrate for the reconstruction of the visual scene in OT areas; 3. evolutionary specialization of different aspects of visual processing may determine the extent of interareal variation in the pyramidal cell phenotype in different species; and 4. pyramidal cell structure is not necessarily related to brain size. Crown Copyright (C) 2003 Published by Elsevier Science Ltd on behalf of IBRO. All rights reserved.

Expression of hNP22 is altered in the frontal cortex and hippocampus of the alcoholic human brain

Background: Human neuronal protein (hNP22) is a gene with elevated messenger RNA expression in the prefrontal cortex of the human alcoholic brain. hNP22 has high homology with a rat protein (rNP22). These proteins also share homology with a number of cytoskeleton-interacting proteins. Methods: A rabbit polyclonal antibody to an 18-amino acid epitope was produced for use in Western and immunohistochemical analysis. Samples from the human frontal and motor cortices were used for Western blots (n = 10), whereas a different group of frontal cortex and hippocampal samples were obtained for immunohistochemistry (n = 12). Results: The hNP22 antibody detected a single protein in both rat and human brain. Western blots revealed a significant increase in hNP22 protein levels in the frontal cortex but not the motor cortex of alcoholic cases. Immunohistochemical studies confirmed the increased hNP22 protein expression in all cortical layers. This is consistent with results previously obtained using Northern analysis. Immunohistochemical analysis also revealed a significant increase of hNP22 immunoreactivity in the CA3 and CA4 but not other regions of the hippocampus. Conclusions: It is possible that this protein may play a role in the morphological or plastic changes observed after chronic alcohol exposure and withdrawal, either as a cytoskeleton-interacting protein or as a signaling molecule.

The pyramidal neuron in occipital, temporal and prefrontal cortex of the owl monkey (Aotus trivirgatus): regional specialization in cell structure

Recent studies have revealed marked regional variation in pyramidal cell morphology in primate cortex. In particular, pyramidal cells in human and macaque prefrontal cortex (PFC) are considerably more spinous than those in other cortical regions. PFC pyramidal cells in the New World marmoset monkey, however, are less spinous than those in man and macaques. Taken together, these data suggest that the pyramidal cell has become more branched and more spinous during the evolution of PFC in only some primate lineages. This specialization may be of fundamental importance in determining the cognitive styles of the different species. However, these data are preliminary, with only one New World and two Old World species having been studied. Moreover, the marmoset data were obtained from different cases. In the present study we investigated PFC pyramidal cells in another New World monkey, the owl monkey, to extend the basis for comparison. As in the New World marmoset monkey, prefrontal pyramidal cells in owl monkeys have relatively few spines. These species differences appear to reflect variation in the extent to which PFC circuitry has become specialized during evolution. Highly complex pyramidal cells in PFC appear not to have been a feature of a common prosimian ancestor, but have evolved with the dramatic expansion of PFC in some anthropoid lineages.

Avaliação imunológica nas pregas vestibulares de pacientes autopsiados com a Síndrome da Imunodeficiência Adquirida

Em pacientes com Síndrome da Imunodeficiência Adquirida há uma diminuição das células envolvidas na resposta imune, o que influencia na população celular dos folículos linfóides encontrados nas pregas vestibulares, favorecendo o aparecimento de infecções nas vias aéreas destes pacientes. Estas infecções são a principal causa de mortalidade e morbidade nestes pacientes. OBJETIVO: Caracterizar a população de células nos folículos linfóides localizados nas pregas vestibulares de adultos autopsiados com Síndrome da Imunodeficiência Adquirida, com e sem infecções respiratórias associadas. MATERIAIS E MÉTODOS: Foi realizado um estudo retrospectivo transversal em 64 laringes de adultos coletadas na rotina das autopsias. Para a imunohistoquímica foram utilizados os anticorpos: Anti-B cells, Anti-CD3, Anti-CD68 e Anti-follicular dendritic cells. RESULTADOS: 46 (71,87%) dos pacientes estudados tinham diagnóstico de Síndrome da Imunodeficiência Adquirida. Nestes pacientes, a celularidade dos folículos linfóides foi estatisticamente menor em relação ao grupo controle em todos os fenótipos estudados. Nos pacientes imunodeprimidos com infecção respiratória associada, o número de células estava diminuído, sendo significante no caso dos linfócitos T (p=0,024). CONCLUSÃO: Em nosso estudo demonstramos que os folículos linfóides das pregas vestibulares são afetados pela infecção viral e representam com fidedignidade o estado imunológico de imunodepressão destes pacientes.

A new cloning system based on the OprI lipoprotein for the production of recombinant bacterial cell wall-derived immunogenic formulations

The conjugation of antigens with ligands of pattern recognition receptors (PRR) is emerging as a promising strategy for the modulation of specific immunity. Here, we describe a new Escherichia coli system for the cloning and expression of heterologous antigens in fusion with the OprI lipoprotein, a TLR ligand from the Pseudomonas aeruginosa outer membrane (OM). Analysis of the OprI expressed by this system reveals a triacylated lipid moiety mainly composed by palmitic acid residues. By offering a tight regulation of expression and allowing for antigen purification by metal affinity chromatography, the new system circumvents the major drawbacks of former versions. In addition, the anchoring of OprI to the OM of the host cell is further explored for the production of novel recombinant bacterial cell wall-derived formulations (OM fragments and OM vesicles) with distinct potential for PRR activation. As an example, the African swine fever virus ORF A104R was cloned and the recombinant antigen was obtained in the three formulations. Overall, our results validate a new system suitable for the production of immunogenic formulations that can be used for the development of experimental vaccines and for studies on the modulation of acquired immunity.

Electrodeposition and characterization of nickel–copper metallic foams for application as electrodes for supercapacitors

Nickel-copper metallic foams were electrodeposited from an acidic electrolyte, using hydrogen bubble evolution as a dynamic template. Their morphology and chemical composition was studied by scanning electron microscopy and related to the deposition parameters (applied current density and deposition time). For high currents densities (above 1 A cm(-2)) the nickel-copper deposits have a three-dimensional foam-like morphology with randomly distributed nearly-circular pores whose walls present an open dendritic structure. The nickel-copper foams are crystalline and composed of pure nickel and a copper-rich phase containing nickel in solid solution. The electrochemical behaviour of the material was studied by cyclic voltammetry and chronopotentiometry (charge-discharge curves) aiming at its application as a positive electrode for supercapacitors. Cyclic voltammograms showed that the Ni-Cu foams have a pseudocapacitive behaviour. The specific capacitance was calculated from charge-discharge data and the best value (105 F g(-1) at 1 mA cm(-2)) was obtained for nickel-copper foams deposited at 1.8 A cm(-2) for 180 s. Cycling stability of these foams was also assessed and they present a 90 % capacitance retention after 10,000 cycles at 10 mA cm(-2).

Electrodeposition and characterization of nikel-copper metallic foams for application as electrodes for supercapacitors

Nickel-copper metallic foams were electrodeposited from an acidic electrolyte, using hydrogen bubble evolution as a dynamic template. Their morphology and chemical composition was studied by scanning electron microscopy and related to the deposition parameters (applied current density and deposition time). For high currents densities (above 1 A cm(-2)) the nickel-copper deposits have a three-dimensional foam-like morphology with randomly distributed nearly-circular pores whose walls present an open dendritic structure. The nickel-copper foams are crystalline and composed of pure nickel and a copper-rich phase containing nickel in solid solution. The electrochemical behaviour of the material was studied by cyclic voltammetry and chronopotentiometry (charge-discharge curves) aiming at its application as a positive electrode for supercapacitors. Cyclic voltammograms showed that the Ni-Cu foams have a pseudocapacitive behaviour. The specific capacitance was calculated from charge-discharge data and the best value (105 F g(-1) at 1 mA cm(-2)) was obtained for nickel-copper foams deposited at 1.8 A cm(-2) for 180 s. Cycling stability of these foams was also assessed and they present a 90 % capacitance retention after 10,000 cycles at 10 mA cm(-2).

Characterisation and electrochemical behaviour of electrodeposited Cu-Fe foams applied as pseudocapacitor electrodes

Copper iron (Cu-Fe) 3D porous foams for supercapacitor electrodes were electrodeposited in the cathodic regime, on stainless steel current collectors, using hydrogen bubbling dynamic template. The foams were prepared at different current densities and deposition times. The foams were submitted to thermal conditioning at temperatures of 150 and 250 degrees C. The morphology, composition and structure of the formed films were studied by SEM, EDS and XRD, respectively. The electrochemical behaviour was studied by cyclic voltammetry, electrochemical impedance spectroscopy and chronopotentiometry. The morphology of the 3D Cu-Fe foams is sensitive to the electrodeposition current and time. The increase of the current density produces a denser, larger and more ramified dendritic structure. Thermal conditioning at high temperature induces a coarser grain structure and the formation of copper oxides, which affect the electrochemical behaviour. The electrochemical response reveals the presence of various redox peaks assigned to the oxidation and reduction of Cu and Fe oxides and hydroxides in the foams. The specific capacitance of the 3D Cu Fe foams was significantly enhanced by thermal conditioning at 150 degrees C. The highest specific capacitance values attained 297 Fg(-1) which are much above the ones typically observed for single Cu or Fe Oxides and hydroxides. These values highlight a synergistic behaviour resulting from the combination of Cu and Fe in the form of nanostructured metallic foams. Moreover, the capacitance retention observed in an 8000 charge/discharge cycling test was above 66%, stating the good performance of these materials and its enhanced electrochemical response as supercapacitor negative electrodes. (C) 2014 Elsevier B.V. All rights reserved.

Mathematical model for HIV dynamics in HIV-specific helper cells

In this paper we study a delay mathematical model for the dynamics of HIV in HIV-specific CD4 + T helper cells. We modify the model presented by Roy and Wodarz in 2012, where the HIV dynamics is studied, considering a single CD4 + T cell population. Non-specific helper cells are included as alternative target cell population, to account for macrophages and dendritic cells. In this paper, we include two types of delay: (1) a latent period between the time target cells are contacted by the virus particles and the time the virions enter the cells and; (2) virus production period for new virions to be produced within and released from the infected cells. We compute the reproduction number of the model, R0, and the local stability of the disease free equilibrium and of the endemic equilibrium. We find that for values of R0<1, the model approaches asymptotically the disease free equilibrium. For values of R0>1, the model approximates asymptotically the endemic equilibrium. We observe numerically the phenomenon of backward bifurcation for values of R0⪅1. This statement will be proved in future work. We also vary the values of the latent period and the production period of infected cells and free virus. We conclude that increasing these values translates in a decrease of the reproduction number. Thus, a good strategy to control the HIV virus should focus on drugs to prolong the latent period and/or slow down the virus production. These results suggest that the model is mathematically and epidemiologically well-posed.

Case for Diagnosis

Blastic plasmacytoid dendritic cell tumor is a rare, highly aggressive systemic neoplasm for which effective therapies have not yet been established. We describe a 73-year-old man with multiple nodules and patches emerging on the trunk and limbs. Lesional skin biopsy revealed a plasmacytoid dendritic cell tumor with dense dermal infiltrate of tumor cells with blastoid features. No apparent systemic involvement was identified in the initial stage. The patient was treated with prednisone daily, with notorious improvement of the skin lesions, although no complete remission was obtained. During the six-month follow-up period, no disease progression was documented, but fatal systemic progression occurred after that period of time.

Expression of TLR2 and TLR4 in lesions of patients with tegumentary american leishmaniasis

OBJECTIVES: The aim of this study was to describe the pattern of expression of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) in skin biopsies of patients with American tegumentary leishmaniasis (ATL) caused by Leishmania braziliensis. METHODS: This prospective study evaluated 12 patients with ATL caused by Leishmania braziliensis confirmed by polymerase chain reaction. Immunohistochemistry was performed to determine the expression of TLR2 and TLR4. The number of NK cells, dendritic cells and macrophages in the tissue were calculated. The cytokine expression was determined using the anti-TNF-α, anti-IFN-Γ, anti-IL-1 and anti-IL-6. Double immunostaining reactions were used to determine the cell expressing TLR2 and TLR4. RESULTS: The numbers of cells expressing TLR2 and TLR4 were 145.48 ± 82.46 cell/mm² and 3.26 ± 4.11 cell/mm² respectively (p < 0.05). There was no correlation of TLR2 and TLR4 with the amount of cytokines and the number of NK cells, dendritic cells or macrophages. The double immunostaining revealed that TLR2 was expressed by macrophages. CONCLUSION: In human cutaneous leishmaniasis caused by Leishmania braziliensis, TLR2 is the most common TLR expressed during active disease, mainly by macrophages although without correlation with the amount of cytokines and number of cells.

Sialyl Tn-Expressing Bladder Cancer Cells Induce a Tolerogenic Phenotype in Innate and Adaptive Immune Cells

Despite the wide acceptance that glycans are centrally implicated in immunity, exactly how they contribute to the tilt immune response remains poorly defined. In this study, we sought to evaluate the impact of the malignant phenotype-associated glycan, sialyl-Tn (STn) in the function of the key orchestrators of the immune response, the dendritic cells (DCs). In high grade bladder cancer tissue, the STn antigen is significantly overexpressed and correlated with the increased expression of ST6GALNAC1 sialyltransferase. Bladder cancer tissue presenting elevated expression of ST6GALNAC1 showed a correlation with increased expression of CD1a, a marker for bladder immature DCs and showed concomitant low levels of Th1-inducing cytokines IL-12 and TNF-α. In vitro, human DCs co-incubated with STn+ bladder cancer cells, had an immature phenotype (MHC-IIlow, CD80low and CD86low) and were unresponsive to further maturation stimuli. When contacting with STn+ cancer cells, DCs expressed significantly less IL-12 and TNF-α. Consistent with a tolerogenic DC profile, T cells that were primed by DCs pulsed with antigens derived from STn+ cancer cells were not activated and showed a FoxP3high IFN-γlow phenotype. Blockade of STn antigens and of STn+ glycoprotein, CD44 and MUC1, in STn+ cancer cells was able to lower the induction of tolerance and DCs become more mature. Overall, our data suggest that STn-expressing cancer cells impair DC maturation and endow DCs with a tolerogenic function, limiting their capacity to trigger protective anti-tumour T cell responses. STn antigens and, in particular, STn+ glycoproteins are potential targets for circumventing tumour-induced tolerogenic mechanisms.

Establishing a cell biology platform: isolation and preservation of human blood products

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Unravelling the role of alpha 2,6 sialic acid on mouse dentritic cells functions

RESUMO: As células dendríticas (CDs) são fundamentais na imunomodulação e iniciação de respostas imunes adaptativas, enquanto os ácidos siálicos (Sias) são potenciais imunomoduladores. Estas células expressam níveis elevados da sialiltransferase ST6Gal-1, que transfere Sias para a posição terminal de oligossacáridos. De facto, a maturação de CDs está associada a uma diminuição da sialilação na sua superfície celular. Apesar de ter função biológica desconhecida, a forma solúvel, extracelular de ST6Gal-1 aumenta em cancros e inflamação. Ainda assim, esta foi recentemente identificada como moduladora da hematopoiese. Considerando o importante papel das CDs na iniciação de respostas anticancerígenas, uma ligação entre a sialilação extrínseca induzida por ST6Gal-1 extracelular e o seu papel na modulação de CDs deve ser identificada. Neste trabalho hipotetizou-se que a sialilação α2,6 extrínseca de CDs diminui o seu perfil de maturação mediante ativação por lipopolissacarídeo (LPS). O objetivo principal foi sialilar extrinsecamente em α2,6 CDs da medula óssea de murganhos, avaliando os seus perfis de maturação e de libertação de citocinas, após estimulação com LPS (por Citometria de Fluxo e ELISA, respetivamente). Ao contrário da hipótese, o perfil celular não foi modulado, usando várias abordagens. Por outro lado, a consequência da falta de α2,6 Sias na maturação de CDs foi avaliada analisando: 1) CDs da medula óssea de murganhos tratadas com sialidase, 2) CDs da medula óssea e 3) CDs das vias aéreas, ambas de murganhos deficientes em ST6Gal-1, comparando com a estirpe selvagem. Estes resultados sugerem que a perta total de α2,6 Sias se relaciona com o aumento da expressão do complexo de histocompatibilidade principal de classe II. Apesar de controverso, é provável existirem mecanismos inerentes à ativação por LPS, reduzindo a eficácia de ST6Gal-1 extracelular. Por outro lado, a modificação no perfil de CDs de murganhos deficientes em ST6Gal-1 poderá relacionar-se com uma predisposição para um estado inflamatório severo. Com isto, o trabalho desenvolvido abriu futuras linhas de investigação, nomeadamente explorar outros fatores envolvidos na (de)sialilação α2,6 de CDs, podendo ter impacto em imunoterapia com uso de CDs.--------------------------ABSTRACT: Dendritic cells (DCs) are vital for immunomodulation and the initiation of adaptive immune responses, whereas sialic acids (Sias) are potential immunomodulators. These cells express high levels of sialyltransferase ST6Gal-1, responsible for transferring Sias to the terminal position of oligosaccharide chains. Indeed, DCs’ maturation is associated with decreased cell surface sialylation. Although its biological significance is unknown, the soluble, extracellular form of ST6Gal-1 increases in cancers and inflammation. However, extracellular ST6Gal-1 was recently identified as modulator of hematopoiesis. Considering that DCs play a crucial role in the initiation of a productive anti-cancer immune response, a link between extrinsic sialylation by the extracellular ST6Gal-1 on DC function needs to be investigated. We hypothesize that extrinsic α2,6 sialylation of DCs diminishes their maturation features upon lipopolysaccharide (LPS) stimulation. The main goal was to extrinsically α2,6 sialylate mice bone marrow derived DCs (BMDCs) and to evaluate their maturation and cytokine profiles upon LPS stimulation (by Flow Cytometry and ELISA, respectively). Unlike the hypothesis, we observed that BMDCs’ profile is not modulated, even using several approaches. In contrast, the consequence of lacking cell surface α2,6 Sias in DC maturation was assessed by analysing: 1) sialidase treated BMDCs, 2) BMDCs from mice lacking ST6Gal-1 and 3) DCs from mice airways, comparing wild type with ST6Gal-1 knockout mice. These results suggest that overall lack in α2,6 Sias is related with increased expression of major histocompatibility class II (MHC-II). Although appearing to be controversial findings, other intracellular mechanisms might be occurring upon LPS-induced BMDC activation, probably reducing extracellular ST6Gal-1 effect. In opposite, the modification observed in DC profile of ST6Gal-1 knockout mice might be related to its predisposition to a more severe inflammatory status. With this, the developed work opened future lines of investigation, namely exploring other factors involved in α2,6 (de)sialylation of DC, which might have influence in immunotherapy using DCs.