Stable expression of a human-like sialylated recombinant thyrotropin in a chinese hamster ovary cell line expressing 'alfa'2,6-sialyltransferase

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The Influence of Age, Cardiorespiratory Fitness, Exercise and Sedentary Behaviours on Circulating Angiogenic Cells and Cell Surface Receptor Expression.

Cardiovascular disease (CVD) is the biggest killer of people in western civilisation. Age is a significant risk factor for the development for CVD, and treatments and therapies to address this increased risk are crucial to quality of life and longevity. Exercise is one such intervention which has been shown to reduce CVD risk. Age is also associated with endothelial dysfunction, reduced angiogenic capabilities, and reduced ability to repair the vessel wall. Circulating angiogenic cells (CACs) are a subset of circulating cells which assist in the repair and growth of the vasculature and in the maintenance of endothelial function. Reductions in these cells are observed in those with vascular disease compared to age-matched healthy controls. Exercise may reduce CVD risk by improvements in number and/or function of these CACs. Data was collected from human volunteers of various ages, cardiorespiratory fitness (CRF) levels and latent viral infection history status to investigate the effects of chronological age, CRF, viral serology and other lifestyle factors, such as sedentary behaviours and exercise on CACs. The levels of CACs in these volunteers were measured using four colour flow cytometry using various monoclonal antibodies specific to cell surface markers that are used to identify specific subsets of these CACs. In addition, the response to acute exercise of a specific subset of these CACs, termed ‘angiogenic T-cells’ (TANG) were investigated, in a group of well-trained males aged 20-40 years, using a strenuous submaximal exercise bout. Advancing age was associated with a decline in various subsets of CACs, including bone marrow-derived CD34+ progenitors, putative endothelial progenitor cells (EPCs) and also TANG cells. Individuals with a higher CRF were more likely to have higher circulating numbers of TANG cells, particularly in the CD4+ subset. CRF did not appear to modulate CD34+ progenitors or EPC subsets. Increasing sitting time was associated with reduction in TANG cells, but after correcting for the effects of fitness, sitting time no longer negatively affected the circulating number of these cells. Acute exercise was a powerful stimulus for increasing the number of TANG cells (140% increase), potentially through an SDF-1:CXCR4-dependent mechanism, but more studies are required to investigate this. Latent CMV infection was associated with higher number of TANG cells (CD8+), but only in 18-40 year old individuals, and not in an older age group (41-65 year old). The significance of this has yet to be understood. In conclusion, advancing age may contribute to increased CVD risk partly due to the observed reductions in angiogenic cells circulating in the peripheral compartment. Maintaining a high CRF may attenuate this CVD reduction by modulating TANG cell number, but potentially not CD34+ progenitor or EPC subsets. Acute exercise may offer a short window for vascular adaptation through the mobilisation of TANG cells into the circulation.

Chemokine Receptor Expression on Normal Blood CD56(+) NK-Cells Elucidates Cell Partners That Comigrate during the Innate and Adaptive Immune Responses and Identifies a Transitional NK-Cell Population

Studies of chemokine receptors (CKR) in natural killer- (NK-) cells have already been published, but only a few gave detailed information on its differential expression on blood NK-cell subsets. We report on the expression of the inflammatory and homeostatic CKR on normal blood CD56(+low) CD16(+) and CD56(+high)  CD16(-/+low) NK-cells. Conventional CD56(+low) and CD56(+high) NK-cells present in the normal PB do express CKR for inflammatory cytokines, although with different patterns CD56(+low) NK-cells are mainly CXCR1/CXCR2(+) and CXCR3/CCR5(-/+), whereas mostly CD56(+high) NK-cells are CXCR1/CXCR2(-) and CXCR3/CCR5(+). Both NK-cell subsets have variable CXCR4 expression and are CCR4(-) and CCR6(-). The CKR repertoire of the CD56(+low) NK-cells approaches to that of neutrophils, whereas the CKR repertoire of the CD56(+high) NK-cells mimics that of Th1(+) T cells, suggesting that these cells are prepared to migrate into inflamed tissues at different phases of the immune response. In addition, we describe a subpopulation of NK-cells with intermediate levels of CD56 expression, which we named CD56(+int) NK-cells. These NK-cells are CXCR3/CCR5(+), they have intermediate levels of expression of CD16, CD62L, CD94, and CD122, and they are CD57(-) and CD158a(-). In view of their phenotypic features, we hypothesize that they correspond to a transitional stage, between the well-known CD56(+high) and CD56(+low) NK-cells populations.

Dual regulation of P-glycoprotein expression by Trichostatin A in cancer cell lines

Background. It has been reported that the histone deacetylase inhibitor (iHDAc) trichostatin A (TSA) induces an increase in MDR1 gene transcription (ABCB1). This result would compromise the use of iHDACs in combination with other cytotoxic agents that are substrates of P-glycoprotein (Pgp). It has also been reported the use of alternative promoters by the ABCB1 gene and the existence of a traslational control of Pgp protein. Finally, the ABCB1 gene is located in a genetic locus with the nested gene RUNDC3B in the complementary DNA strand, raising the possibility that RUNDC3B expression could interfere with ABCB1 alternative promoter regulation. Methods. A combination of RT-PCR, real time RT-PCR, Western blot and drug accumulation assays by flow cytometry have been used in this study. Results. The iHDACs-induced increase in MDR1 mRNA levels is not followed by a subsequent increase in Pgp protein levels or activity in several pancreatic and colon carcinoma cell lines, suggesting a traslational control of Pgp in these cell lines. In addition, the MDR1 mRNA produced in these cell lines is shorter in its 5' end that the Pgp mRNA produced in cell lines expressing Pgp protein. The different size of the Pgp mRNA is due to the use of alternative promoters. We also demonstrate that these promoters are differentially regulated by TSA. The translational blockade of Pgp mRNA in the pancreatic carcinoma cell lines could be related to alterations in the 5' end of the MDR1 mRNA in the Pgp protein expressing cell lines. In addition, we demonstrate that the ABCB1 nested gene RUNDC3B expression although upregulated by TSA is independent of the ABCB1 alternative promoter used. Conclusions. The results show that the increase in MDR1 mRNA expression after iHDACs treatment is clinically irrelevant since this mRNA does not render an active Pgp protein, at least in colon and pancreatic cancer cell lines. Furthermore, we have demonstrated that TSA in fact, differentially regulates both ABCB1 promoters, downregulating the upstream promoter that is responsible for active P-glycoprotein expression. These results suggest that iHDACs such as TSA may in fact potentiate the effects of antitumoral drugs that are substrates of Pgp. Finally, we have also demonstrate that TSA upregulates RUNDC3B mRNA independently of the ABCB1 promoter in use.

Mir-190b negatively contributes to the Trypanosoma cruzi -infected cell survival by repressing PTEN protein expression

Chagas disease, which is caused by the intracellular protozoan Trypanosoma cruzi , is a serious health problem in Latin America. The heart is one of the major organs affected by this parasitic infection. The pathogenesis of tissue remodelling, particularly regarding cardiomyocyte behaviour after parasite infection, and the molecular mechanisms that occur immediately following parasite entry into host cells are not yet completely understood. Previous studies have reported that the establishment of parasitism is connected to the activation of the phosphatidylinositol- 3 kinase (PI3K), which controls important steps in cellular metabolism by regulating the production of the second messenger phosphatidylinositol-3,4,5-trisphosphate. Particularly, the tumour suppressor PTEN is a negative regulator of PI3K signalling. However, mechanistic details of the modulatory activity of PTEN on Chagas disease have not been elucidated. To address this question, H9c2 cells were infected with T. cruzi Berenice 62 strain and the expression of a specific set of microRNAs (miRNAs) were investigated. Our cellular model demonstrated that miRNA-190b is correlated to the decrease of cellular viability rates by negatively modulating PTEN protein expression in T. cruzi-infected cells.

Expression of TIMP-2 in HPV-16 infected oral squamous cell carcinoma in patients in Iraq

Aim: To determine the expression of tissue inhibitors of metalloproteinases (TIMP-2) in oral squamous cell carcinoma (OSCC) and the difference in its expression level between positive and negative HPV-16 (human papilloma virus- 16) OSCC patients. Methods: This study was conducted on 33 biopsies obtained from patients with OSCC and 10 normal oral mucosa as controls. In situ hybridization (ISH) was used to investigate the presence of HPV-16, while immunohistochemistry (IHC) was used to estimate the expression level of TIMP-2. Results: The TIMP-2 was expressed in 27 (81.8%) of OSCC sections with no significant difference between its expression level in HPV-16 positive and HPV-16 negative OSCC cases (p=0.058). TIMP-2 was found to be highly expressed in OSCC sections, and the presence of HPV was not related to its overexpression. Conclusions: The percentage of samples that appeared to accommodate detectable HPV-16 was high, but no significant difference was observed in relation to TIMP-2 expression level. Future studies with a larger number of patients are highly recommended to address the possible association between TIMp-2 and OSCC positive HPV-16.

Phosphate uptake and phosphate transporter expression during immune cell proliferation in in vitro and ex vivo leukemic murine models

Dissertação de Mestrado, Ciências Biomédicas, Departamento de Medicina e Ciências Biomédicas, Universidade do Algarve, 2015

Mineral stress response of Vitis cell wall transcriptome: identification, characterization and expression of genes from key families involved in wall biosynthesis and modification

Doutoramento em Engenharia Agronómica - Instituto Superior de Agronomia - UL

A syntenic cross species aneuploidy genetic screen links RCAN1 expression to β-cell mitochondrial dysfunction in Type 2 diabetes

Type 2 diabetes (T2D) is a complex metabolic disease associated with obesity, insulin resistance and hypoinsulinemia due to pancreatic β-cell dysfunction. Reduced mitochondrial function is thought to be central to β-cell dysfunction. Mitochondrial dysfunction and reduced insulin secretion are also observed in β-cells of humans with the most common human genetic disorder, Down syndrome (DS, Trisomy 21). To identify regions of chromosome 21 that may be associated with perturbed glucose homeostasis we profiled the glycaemic status of different DS mouse models. The Ts65Dn and Dp16 DS mouse lines were hyperglycemic, while Tc1 and Ts1Rhr mice were not, providing us with a region of chromosome 21 containing genes that cause hyperglycemia. We then examined whether any of these genes were upregulated in a set of ~5,000 gene expression changes we had identified in a large gene expression analysis of human T2D β-cells. This approach produced a single gene, RCAN1, as a candidate gene linking hyperglycemia and functional changes in T2D β-cells. Further investigations demonstrated that RCAN1 methylation is reduced in human T2D islets at multiple sites, correlating with increased expression. RCAN1 protein expression was also increased in db/db mouse islets and in human and mouse islets exposed to high glucose. Mice overexpressing RCAN1 had reduced in vivo glucose-stimulated insulin secretion and their β-cells displayed mitochondrial dysfunction including hyperpolarised membrane potential, reduced oxidative phosphorylation and low ATP production. This lack of β-cell ATP had functional consequences by negatively affecting both glucose-stimulated membrane depolarisation and ATP-dependent insulin granule exocytosis. Thus, from amongst the myriad of gene expression changes occurring in T2D β-cells where we had little knowledge of which changes cause β-cell dysfunction, we applied a trisomy 21 screening approach which linked RCAN1 to β-cell mitochondrial dysfunction in T2D.

Transcriptional analysis of PRRSV-infected porcine dendritic cell response to Streptococcus suis infection reveals up-regulation of inflammatory-related genes expression

The porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important swine pathogens and often serves as an entry door for other viral or bacterial pathogens, of which Streptococcus suis is one of the most common. Pre-infection with PRRSV leads to exacerbated disease caused by S. suis infection. Very few studies have assessed the immunological mechanisms underlying this higher susceptibility. Since antigen presenting cells play a major role in the initiation of the immune response, the in vitro transcriptional response of bone marrow-derived dendritic cells (BMDCs) and monocytes in the context of PRRSV and S. suis co-infection was investigated. BMDCs were found to be more permissive than monocytes to PRRSV infection; S. suis phagocytosis by PRRSV-infected BMDCs was found to be impaired, whereas no effect was found on bacterial intracellular survival. Transcription profile analysis, with a major focus on inflammatory genes, following S. suis infection, with and without pre-infection with PRRSV, was then performed. While PRRSV pre-infection had little effect on monocytes response to S. suis infection, a significant expression of several pro-inflammatory molecules was observed in BMDCs pre-infected with PRRSV after a subsequent infection with S. suis. While an additive effect could be observed for CCL4, CCL14, CCL20, and IL-15, a distinct synergistic up-regulatory effect was observed for IL-6, CCL5 and TNF-α after co-infection. This increased pro-inflammatory response by DCs could participate in the exacerbation of the disease observed during PRRSV and S. suis co-infection.

Transcriptional analysis of PRRSV-infected porcine dendritic cell response to Streptococcus suis infection reveals up-regulation of inflammatory-related genes expression

The porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important swine pathogens and often serves as an entry door for other viral or bacterial pathogens, of which Streptococcus suis is one of the most common. Pre-infection with PRRSV leads to exacerbated disease caused by S. suis infection. Very few studies have assessed the immunological mechanisms underlying this higher susceptibility. Since antigen presenting cells play a major role in the initiation of the immune response, the in vitro transcriptional response of bone marrow-derived dendritic cells (BMDCs) and monocytes in the context of PRRSV and S. suis co-infection was investigated. BMDCs were found to be more permissive than monocytes to PRRSV infection; S. suis phagocytosis by PRRSV-infected BMDCs was found to be impaired, whereas no effect was found on bacterial intracellular survival. Transcription profile analysis, with a major focus on inflammatory genes, following S. suis infection, with and without pre-infection with PRRSV, was then performed. While PRRSV pre-infection had little effect on monocytes response to S. suis infection, a significant expression of several pro-inflammatory molecules was observed in BMDCs pre-infected with PRRSV after a subsequent infection with S. suis. While an additive effect could be observed for CCL4, CCL14, CCL20, and IL-15, a distinct synergistic up-regulatory effect was observed for IL-6, CCL5 and TNF-α after co-infection. This increased pro-inflammatory response by DCs could participate in the exacerbation of the disease observed during PRRSV and S. suis co-infection.