Production of membrane proteins in yeast

Background Yeast is an important and versatile organism for studying membrane proteins. It is easy to cultivate and can perform higher eukaryote-like post-translational modifications. S. cerevisiae has a fully-sequenced genome and there are several collections of deletion strains available, whilst P. pastoris can produce very high cell densities (230 g/l). Results We have used both S. cerevisiae and P. pastoris to over-produce the following His6 and His10 carboxyl terminal fused membrane proteins. CD81 ��� 26 kDa tetraspanin protein (TAPA-1) that may play an important role in the regulation of lymphoma cell growth and may also act as the viral receptor for Hepatitis C-Virus. CD82 ��� 30 kDa tetraspanin protein that associates with CD4 or CD8 cells and delivers co-stimulatory signals for the TCR/CD3 pathway. MC4R ��� 37 kDa seven transmembrane G-protein coupled receptor, present on neurons in the hypothalamus region of the brain and predicted to have a role in the feast or fast signalling pathway. Adt2p ��� 34 kDa six transmembrane protein that catalyses the exchange of ADP and ATP across the yeast mitochondrial inner membrane. Conclusion We show that yeasts are flexible production organisms for a range of different membrane proteins. The yields are such that future structure-activity relationship studies can be initiated via reconstitution, crystallization for X-ray diffraction or NMR experiments.

Immunological protein-protein interactions:a critical reflection

Life, and the biochemistry of which it is ultimately comprised, is built from the interactions of proteins, and the study of protein-protein interactions is fast becoming a central feature of molecular bioscience. This is as true of immunobiology as it is of other areas of the wider biological milieu. Protein-protein interactions within an immunological setting comprise both the kind familiar from other areas of biology and instantiations of protein-protein interactions special to the immune arena. Of the generic kind of protein-protein interaction, co-stimulatory receptors, such as CD28, and the interaction of accessory proteins, such as CD4 or CD8, are amongst the most prevalent and apposite of examples. The key examples of special immunological instantiations of protein-protein interactions are the binding of antigens by antibodies and the formation of peptide-MHC-TCR complexes; both prime examples of vital molecular recognition events mediated by protein-protein interactions. In this brief review, and within the context of this burgeoning field, we examine immunological protein-protein interactions, focussing on the problematic nature of defining such interactions. �� 2011 by Nova Science Publishers, Inc. All rights reserved.

IL-1�� and inflammasome-independent IL-1�� promote neutrophil infiltration following alum vaccination

<p>Despite its long record of successful use in human vaccines, the mechanisms underlying the immunomodulatory effects of alum are not fully understood. Alum is a potent inducer of interleukin-1 (IL-1) secretion in vitro in dendritic cells and macrophages via Nucleotide-binding domain and leucine-rich repeat-containing (NLR) family, pyrin domain-containing 3 (NLRP3) inflammasome activation. However, the contribution of IL-1 to alum-induced innate and adaptive immune responses is controversial and the role of IL-1�� following alum injection has not been addressed. This study shows that IL-1 is dispensable for alum-induced antibody and CD8 T cell responses to ovalbumin. However, IL-1 is essential for neutrophil infiltration into the injection site, while recruitment of inflammatory monocytes and eosinophils is IL-1 independent. Both IL-1�� and IL-1�� are released at the site of injection and contribute to the neutrophil response. Surprisingly, these effects are NLRP3-inflammasome independent as is the infiltration of other cell populations. However, while NLRP3 and caspase 1 were dispensable, alum-induced IL-1�� at the injection site was dependent on the cysteine protease cathepsin S. Overall, these data demonstrate a previously unreported role for cathepsin S in IL-1�� secretion, show that inflammasome formation is dispensable for alum-induced innate immunity and reveal that IL-1�� and IL-1�� are both necessary for alum-induced neutrophil influx in vivo.</p>

Perfil de mem��ria e ativa����o de linf��citos T na leishmaniose visceral

Visceral leishmaniasis (VL) in Brazil is a disease caused by Leishmania infantum chagasi (L.i.chagasi). The clinical evolution post-infection depends on the vertebrate host immune response, which is genetically mediated. This study aimed to evaluate the immune response of individuals living in endemic area for VL in the state of the Rio Grande do Norte, considering individuals with VL under treatment (n = 9), recovered VL <1 year post treatment (n = 10), > 10 years posttreatment (n = 9), uninfected individuals living in endemic areas (n = 7), individuals that lost DTH response (n=6) and asymptomatic individuals for VL (n=9). Peripheral blood cells were evaluated in the presence and absence of soluble Leishmania antigens (SLA) and ex vivo, to determine activation, presence of regulatory cells and memory cells. The Leishmania parasitemia and anti-Leishmania antibodies were determined respectively by qPCR and ELISA. Cells from individuals with VL under treatment showed less cell activation after stimulation with SLA for the markers CD4/CD69, CD8/CD69 and CD8/CD25 compared with VL post treatment treatment (p <0.001). Apparently uninfected individuals have a higher cell activation than symptomatic VL (p <0.001), with the exception of CD8/CD25 marker (p = 0.6662). On the other hand, in the ex-vivo group, significant differences were observed for CD4/CD69, CD8/CD69 and CD8/CD25 between the 4 groups due to increased cell activation present in cells of individuals symptomatic LV (p <0.001). VL cells under treatment, ex vivo, have a lower percentage of memory cells (CD4/CD45RO and CD8/CD45RO) than individuals VL post-treatment or control group (p = <0.01). Likewise, individuals with symptomatic VL have fewer regulatory cells when stimulated by SLA [CD4/CD25 (p = 0.0022) and CD4/FOXP3 (p = 0.0016)] and in the ex-vivo group (p = 0.0017). Finally, DNA isolated from recovered VL contained Leishmania DNA, supporting the hypothesis of non-sterile clinical cure for Leishmania infection. Recovered VL, even 10 years after treatment have high levels of memory cells, which may be due to the presence of stimulation, either by reexposure to Leishmania or non-sterile cure

O papel modulat��rio do ferro sobre a resposta imune na Leishmaniose Visceral

Iron is an essential element for many cellular functions, including the immune response against intracellular pathogens. In this study, we aimed evaluate the effect of iron on IRP2, IFN-��, TNF-��, IL-6, IL-10, MIG and IP10 expression in PBMC and assess the effect of the spleen parasite load on the expression of these genes in the spleen of L. infantum naturally infected dogs. Blood sample from 7 DTH+ donor was collected and PBMC was obtained. The cells were cultivated in absence (iron chelator desferroximane, DFO 10 ��M supplemented media) or in presence of iron (hemin 6 mM) for 1 h, followed by stimulation with Leishmania infatum antigen for 4 h. 44 dog spleen samples were obtained and parasite load in this organ was determinate by qPCR. Gene expression was analyzed by qPCR and cytokine production quantified by flow cytometry. In antigen stimulated cells, genes involved in immune response are significantly more expressed in presence of iron. T CD4+ and TCD8+ lymphocytes produces IFN-��, TNF-�� and IL-10 possibly in iron dependent pathway. Monocytes antigen stimulated reduced TNF-��, IL-6 and IL-10 production in presence of iron. We found spleen of infected dogs IRP2 expression increases according to parasite load in that organ, while an inverse profile was found for IFN-��, TNF-�� e IL-10 expression. These results suggest that T lymphocytes depends on iron to produce IFN-��, TNF-�� and IL-10, while iron seems to inhibit cytokine production in monocytes. So, we propose an immunoregulatory mechanism carried out by iron during L. infantum infection in humans and dogs

Efecto de la terapia aut��loga con linfocitos T CD8+ estimulados con ant��geno tumoral total E IL-21 en caninos con tumor ven��reo transmisible

La Organizaci��n Mundial de la Salud declaro que el c��ncer es una de las principales causas de muerte en el mundo, para el 2012 se present�� un total de 8.2 millones de defunciones. A pesar de la gran cantidad de recursos invertidos en investigaci��n, la tasa de mortalidad no ha sido disminuida, por lo tanto es necesario el desarrollo de nuevas terapias efectivas contra el c��ncer. Desde sus inicios, en el campo de la inmunoterapia se han realizado numerosos ensayos en humanos con c��ncer y en modelos animales, obteniendo algunos casos de regresi��n tumoral completa. En el presente trabajo se evalu�� el efecto de la terapia aut��loga en perros inoculados experimentalmente con Tumor Ven��reo Transmisible (TVT). Se inocul��12 hembras de raza mixta con TVT (1x108 c��lulas) intragenital, una vez que el tumor alcanzo un volumen de 10cm 3 , se colect�� una biopsia con la cual se extrajo el ant��geno tumoral total (300 ��g/mL) el cual fue agregado (30��g/mL) en cultivos de c��lulas dendr��ticas inmaduras. Linfocitos totales obtenidos del mismo paciente y CPAs (CD80+ 80.3%, CD83+ 76.4%, DLA II 86.5%) cargadas con ant��geno tumoral fueron co-cultivados en presencia de est��mulos con IL-21 (50ng/mL) o IL-2 (20ng/mL), posterior a la activaci��n de los linfocitos (746.88 pg/mL IFN��), las c��lulas T CD8+ espec��ficos de tumor fueron separadas por selecci��n negativa (Dynabeads Untouched) con un 82.6% de pureza para finalmente ser expandidas con OKT3. Se llev�� a cabo un ensayo de in vitro de la citotoxicidad de los Linfocitos T CD8+ espec��ficos de tumor contra las c��lulas de TVT, obteniendo 100% de lisis de la c��lula tumoral cultivada en presencia de linfocitos CD8+ espec��ficos de tumor estimulados con IL-21 y un 90% de citotoxicidad tumoral cuando los CD8+ fueron espec��ficos pero estimulados con IL-2, cuando los CD8+ no fueron espec��ficos de tumor el porcentaje de citotoxicidad fue muy poco (10%). In vivo el grupo 1 fue tratado con terapia aut��loga, linfocitos T CD8+ (5x107 c��lulaspor ciclo) espec��ficos de tumor estimulados con IL-21 y aplicados en 3 ciclos en intervalos de 2 semanas. Como grupos control se utilizaron perros con tumor sin tratamiento (grupo 2), perros con tumor tratados con suero glucosado (grupo 3) y perros con tumor tratados con transfusi��n sangu��nea aut��loga (grupo 4). Despu��s de la terapia aut��loga la inmunidad celular (CD4+ y CD8+ ) e IFN�� fue incrementada observando regresi��n tumoral en los perros del grupo 1. Estos resultados indican que la terapia aut��loga es eficaz en la destrucci��n del TVT.

Development of water-soluble polyanionic carbosilane dendrimers as novel and highly potent topical anti-HIV-2 microbicides

This is the author���s version of a work that was accepted for publication in Nanoscale.

Profile of cortisol, glycaemia, and blood parameters of American Bullfrog tadpoles Lithobates catesbeianus exposed to density and hypoxia stressors

O objetivo deste estudo foi avaliar a resposta fisiol��gica (cortisol, glicemia e par��metros sanguineos) de girinos de r��-touro (Lithobates catesbeianus) em diferentes densidades e ap��s exposi����o a��rea. Os animais utilizados no experimento estavam entre os est��gios 31 a 39, na fase de pr��-metamorfose sendo testados 1 girino/L (Tratamento 1), 5 girinos/L (Tratamento 2) e 10 girinos/L (Tratamento 3), conduzidos em 3 r��plicas simult��neas durante 12 dias. O sangue foi retirado por rompimento do vaso caudal na condi����o de Norm��xia - N (tempo zero) e Hip��xia - H (tempo de 15 minutos de exposi����o ao ar). Foi observado um aumento nos valores de cortisol, aos 4 e 8 dias de exposi����o a��rea retornando aos valores basais ao final do experimento, apesar de n��o haver diferen��as significativas. A glicemia n��o apresentou diferen��as significativas quanto aos estressores aplicados. Os par��metros hematol��gicos da s��rie branca, principalmente, o n��mero de linf��citos, neutr��filos e eosin��filos mostraram diferen��a significativa aos 12 dias de experimenta����o quando comparados com o momento zero; concluindo-se que, em condi����es controladas, o adensamento de at�� 10 girinos/litro e a exposi����o a��rea por 15 minutos n��o apresentou danos aos girinos de r��-touro durante o per��odo experimental. O padr��o de resposta a estes est��mulos talvez seja expresso em outro n��vel hormonal (corticosterona).

Alveolar macrophages as accessory cells for human gamma delta T cells activated by Mycobacterium tuberculosis.

Alveolar macrophages form the first line of defense against inhaled droplets containing Mycobacterium tuberculosis by controlling mycobacterial growth and regulating T cell responses. CD4+ and gamma delta T cells, two major T cell subsets activated by M. tuberculosis, require accessory cells for activation. However, the ability of alveolar macrophages to function as accessory cells for T cell activation remains controversial. We sought to determine the ability of alveolar macrophages to serve as accessory cells for resting (HLA-DR-, IL-2R-) and activated (HLA-DR+, IL-2R+) gamma delta T cells in response to M. tuberculosis and its Ag, and to compare accessory cell function for gamma delta T cells of alveolar macrophages and blood monocytes obtained from the same donor. Alveolar macrophages were found to serve as accessory cells for both resting and activated gamma delta T cells in response to M. tuberculosis Ag. At high alveolar macrophage to T cell ratios (> 3:1), however, expansion of resting gamma delta T cells was inhibited by alveolar macrophages. The inhibition of resting gamma delta T cells by alveolar macrophages was dose-dependent, required their presence during the first 24 h, and was partially overcome by IL-2. Alveolar macrophages did not inhibit activated gamma delta T cells even at high accessory cell to T cell ratios, and alveolar macrophages functioned as well as monocytes as accessory cells. Monocytes were not inhibitory for either resting or activated gamma delta T cells. These findings support the following model. In the normal alveolus the alveolar macrophage to T cell ratio is > or = 9:1, and therefore the threshold for resting gamma delta T cell activation is likely to be high. Once a nonspecific inflammatory response occurs, such as after invasion by M. tuberculosis, this ratio is altered, favoring gamma delta T cell activation by alveolar macrophages.

Autoimmune Regulator (AIRE) in the Maintenance of Immunological Tolerance

Autoimmune regulator (AIRE) is the gene mutated in the human polyglandular autoimmune disease called Autoimmune polyendocrinopathy, candidiasis, and ectodermal dystrophy (APECED) that belongs to the Finnish disease heritage. Murine Aire has been shown to be important in the generation of the T cell central tolerance in the thymus by promoting the expression of ectopic tissue-specific antigens in the thymic medulla. Aire is also involved in the thymus tissue organization during organogenesis. In addition to the thymus, AIRE/Aire is expressed in the secondary lymphoid organs. Accordingly, a role for AIRE/Aire in the maintenance of peripheral tolerance has been suggested. Peripheral tolerance involves mechanisms that suppress immune responses in secondary lymphoid organs. Regulatory T cells (Tregs) are an important suppressive T cell population mediating the peripheral tolerance. Tregs are generated in the thymus but also in the peripheral immune system T cells can acquire the Treg-phenotype. The aim of this study was to characterize Tregs in APECED patients and in the APECED mouse model (Aire-deficient mice). In the mouse model, it was possible to separate Aire expression in the thymus and in the secondary lymphoid organs. The relative importance of thymic and peripheral Aire expression in the maintenance of immunological tolerance was studied in an experimental model that was strongly biased towards autoimmunity, i.e. lymphopenia-induced proliferation (LIP) of lymphocytes. This experimental model was also utilised to study the behaviour of T cells with dual-specific T cell receptors (TCR) during the proliferation. The Treg phenotype was studied by flow cytometry and relative gene expression with real-time polymerase chain reaction. TCR repertoires of the Tregs isolated from APECED patients and healthy controls were also compared. The dual-specific TCRs were studied with the TCR repertoire analysis that was followed with sequencing of the chosen TCR genes in order to estimate changes in the dual-specific TCR diversity. The Treg function was tested with an in vitro suppression assay. The APECED patients had normal numbers of Tregs but the phenotype and suppressive functions of the Tregs were impaired. In order to separate Aire functions in the thymus from its yet unknown role in the secondary lymphoid organs, the phenomenon of LIP was utilised. In this setting, the lymphocytes that are adoptively transferred to a lymphopenic recipient proliferate to stimuli from self-originating antigens. This proliferation can result in autoimmunity if peripheral tolerance is not fully functional. When lymphocytes that had matured without Aire in the thymus were transferred to lymphopenic Aire-sufficient recipients, no clinical autoimmunity followed. The Aire-deficient donor-originating lymphocytes hyperproliferated, and other signs of immune dysregulation were also found in the recipients. Overt autoimmunity, however, was prevented by the Aire-deficient donor-originating Tregs that hyperproliferated in the recipients. Aire-deficient lymphopenic mice were used to study whether peripheral loss of Aire had an impact on the maintenance of peripheral tolerance. When normal lymphocytes were transferred to these Aire-deficient lymphopenic recipients, the majority of recipients developed a clinically symptomatic colitis. The colitis was confirmed also by histological analysis of the colon tissue sections. In the Aire-deficient lymphopenic recipients Tregs were proliferating significantly less than in the control group s recipients that had normal Aire expression in their secondary lymphoid organs. This study shows that Aire is not only important in the central tolerance but is also has a significant role in the maintenance of the peripheral tolerance both in mice and men. Aire expressed in the secondary lymphoid organs is involved in the functions of Tregs during an immune response. This peripheral expression appears to be relatively more important in some situations since only those lymphopenic recipients that had lost peripheral expression of Aire developed a symptomatic autoimmune disease. This AIRE-related Treg defect could be clinically important in understanding the pathogenesis of APECED.

���-������������������������������������������������������

���������:���������2���������������������������������������(DXR) ������������������������:������������������2������������������������������ ���������������,������������������������������(Y2CVF) ������������������������������,������������������A(CsA) ���������������(CTX) ���������������(M. P) ���������������������������������������C3���C4���������������������������������,������������������������������������������C3���C5b29���IgG���IgM������������ ������������21 ( ICAM21) ���������������������2��(TNF2��) ���������������������(CD68) ���NK������(CD57) ���CD4 + T ���������CD8 + T ������������������ ������:������������������������������8���10���13 ���13 ���,������C3 ������������������������������0 ,������������������������������������������������������ ���������������������������,���������������������2���4 ���������������������������������,������������������������������������������������������������������ C3���C4���C5b29���IgG���IgM ������,���������������������(50 %) ,���������NK������(8 %���10 %) ���CD4 + T ������(15 %) ���CD8 + T ������ (25 %) ������������������������������������������ICAM21 ���������������,������������������������TNF2��������������������������:������������������������ ������������2������DXR ������������������������

Capillary electrophoretic study on UDP-sugars in cells

Glucose is an important regulator of cell growth and metabolism. Uridine diphosphate sugars (UDP-sugars), as the intermediate products of metabolism, play pivotal roles as precursors in the synthesis of complex carbohydrates and glycolipids as well as lectose. It is very important to study their metabolism in cells in clinical biochemistry. A capillary electrophoretic method has been developed for the analysis of UDP-sugars and nucleotides, By using an uncoated capillary (70cm x 50 mu m) and 20 mmol/L borax buffer (pH 9), 4 important UDP-sugars can be analyzed in 15 min at 22 kV with satisfactory precision and sensitivity. The developed method has been applied to analyze UDP-sugars concentrations in lymphocytes, fibroblasts and mesangial cells, and the results show it not only is much better than HPLC method, but also can be used to measure the energy charge of cells.

Selective enhancement of donor hematopoietic cell engraftment by the CXCR4 antagonist AMD3100 in a mouse transplantation model.

The interaction between stromal cell-derived factor-1 (SDF-1) with CXCR4 chemokine receptors plays an important role in hematopoiesis following hematopoietic stem cell transplantation. We examined the efficacy of post transplant administration of a specific CXCR4 antagonist (AMD3100) in improving animal survival and in enhancing donor hematopoietic cell engraftment using a congeneic mouse transplantation model. AMD3100 was administered subcutaneously at 5 mg/kg body weight 3 times a week beginning at day +2 post-transplant. Post-transplant administration of AMD3100 significantly improves animal survival. AMD3100 reduces pro-inflammatory cytokine/chemokine production. Furthermore, post transplant administration of AMD3100 selectively enhances donor cell engraftment and promotes recovery of all donor cell lineages (myeloid cells, T and B lymphocytes, erythrocytes and platelets). This enhancement results from a combined effect of increased marrow niche availability and greater cell division induced by AMD3100. Our studies shed new lights into the biological roles of SDF-1/CXCR4 interaction in hematopoietic stem cell engraftment following transplantation and in transplant-related mortality. Our results indicate that AMD3100 provides a novel approach for enhancing hematological recovery following transplantation, and will likely benefit patients undergoing transplantation.

Expression of the common heat-shock protein receptor CD91 is increased on monocytes of exposed yet HIV-1-seronegative subjects.

The significantly higher surface expression of the surface heat-shock protein receptor CD91 on monocytes of human immunodeficiency virus type-1 (HIV-1)-infected, long-term nonprogressors suggests that HIV-1 antigen uptake and cross-presentation mediated by CD91 may contribute to host anti-HIV-1 defenses and play a role in protection against HIV-1 infection. To investigate this further, we performed phenotypic analysis to compare CD91 surface expression on CD14+ monocytes derived from a cohort of HIV-1-exposed seronegative (ESN) subjects, their seropositive (SP) partners, and healthy HIV-1-unexposed seronegative (USN) subjects. The median fluorescent intensity (MFI) of CD91 on CD14+ monocytes was significantly higher in ESN compared with SP (P=0.028) or USN (P=0.007), as well as in SP compared with USN subjects (P=0.018). CD91 MFI was not normalized in SP subjects on highly active antiretroviral therapy (HAART) despite sustainable, undetectable plasma viraemia. Data in three SP subjects experiencing viral rebounds following interruption of HAART showed low CD91 MFI comparable with levels in USN subjects. There was a significant positive correlation between CD91 MFI and CD8+ T cell counts in HAART-na��ve SP subjects (r=0.7, P=0.015). Increased surface expression of CD91 on CD14+ monocytes is associated with the apparent HIV-1 resistance that is observed in ESN subjects.

Iron homeostasis in immune mononuclear cells : a potential role in a atherogenesis

Tese de doutoramento, Biologia (Biologia-Molecular), Universidade de Lisboa, Faculdade de Ci��ncias, 2015