NK Cells of Kidney Transplant Recipients Display an Activated Phenotype that Is Influenced by Immunosuppression and Pathological Staging

To explore phenotype and function of NK cells in kidney transplant recipients, we investigated the peripheral NK cell repertoire, capacity to respond to various stimuli and impact of immunosuppressive drugs on NK cell activity in kidney transplant recipients. CD56(dim) NK cells of kidney transplanted patients displayed an activated phenotype characterized by significantly decreased surface expression of CD16 (p=0.0003), CD226 (p<0.0001), CD161 (p=0.0139) and simultaneously increased expression of activation markers like HLA-DR (p=0.0011) and CD25 (p=0.0015). Upon in vitro stimulation via Ca++-dependent signals, down-modulation of CD16 was associated with induction of interferon (IFN)-gamma expression. CD16 modulation and secretion of NFAT-dependent cytokines such as IFN-gamma, TNF-alpha, IL-10 and IL-31 were significantly suppressed by treatment of isolated NK cells with calcineurin inhibitors but not with mTOR inhibitors. In kidney transplant recipients, IFN-gamma production was retained in response to HLA class I-negative target cells and to non-specific stimuli, respectively. However, secretion of other cytokines like IL-13, IL-17, IL-22 and IL-31 was significantly reduced compared to healthy donors. In contrast to suppression of cytokine expression at the transcriptional level, cytotoxin release, i.e. perforin, granzyme A/B, was not affected by immunosuppression in vitro and in vivo in patients as well as in healthy donors. Thus, immunosuppressive treatment affects NK cell function at the level of NFAT-dependent gene expression whereby calcineurin inhibitors primarily impair cytokine secretion while mTOR inhibitors have only marginal effects. Taken together, NK cells may serve as indicators for immunosuppression and may facilitate a personalized adjustment of immunosuppressive medication in kidney transplant recipients.

Developmental programming of polycystic ovary syndrome (PCOS): prenatal androgens establish pancreatic islet α/β cell ratio and subsequent insulin secretion

Exogenous androgenic steroids applied to pregnant sheep programmes a PCOS-like phenotype in female offspring. Via ultrasound guidance we applied steroids directly to ovine fetuses at d62 and d82 of gestation, and examined fetal (day 90 gestation) and postnatal (11 months old) pancreatic structure and function. Of three classes of steroid agonists applied (androgen - Testosterone propionate (TP), estrogen - Diethystilbesterol (DES) and glucocorticoid - Dexamethasone (DEX)), only androgens (TP) caused altered pancreatic development. Beta cell numbers were significantly elevated in prenatally androgenised female fetuses (P=0.03) (to approximately the higher numbers found in male fetuses), whereas alpha cell counts were unaffected, precipitating decreased alpha:beta cell ratios in the developing fetal pancreas (P=0.001), sustained into adolescence (P=0.0004). In adolescence basal insulin secretion was significantly higher in female offspring from androgen-excess pregnancies (P=0.045), and an exaggerated, hyperinsulinaemic response to glucose challenge (P=0.0007) observed, whereas prenatal DES or DEX treatment had no effects upon insulin secretion. Postnatal insulin secretion correlated with beta cell numbers (P=0.03). We conclude that the pancreas is a primary locus of androgenic stimulation during development, giving rise to postnatal offspring whose pancreas secreted excess insulin due to excess beta cells in the presence of a normal number of alpha cells.

THE ROLE OF THE MECHANICAL ENVIRONMENT ON CANCER CELL TRANSMIGRATION AND MRNA LOCALIZATION

Most cancer-related deaths are due to metastasis formation, the ability of cancer cells to break away from the primary tumor site, transmigrate through the endothelium, and form secondary tumors in distant areas. Many studies have identified links between the mechanical properties of the cellular microenvironment and the behavior of cancer cells. Cells may experience heterogeneous microenvironments of varying stiffness during tumor progression, transmigration, and invasion into the basement membrane. In addition to mechanical factors, the localization of RNAs to lamellipodial regions has been proposed to play an important part in metastasis. This dissertation provides a quantitative evaluation of the biophysical effects on cancer cell transmigration and RNA localization. In the first part of this dissertation, we sought to compare cancer cell and leukocyte transmigration and investigate the impact of matrix stiffness on transmigration process. We found that cancer cell transmigration includes an additional step, ‘incorporation’, into the endothelial cell (EC) monolayer. During this phase, cancer cells physically displace ECs and spread into the monolayer. Furthermore, the effects of subendothelial matrix stiffness and endothelial activation on cancer cell incorporation are cell-specific, a notable difference from the process by which leukocytes transmigrate. Collectively, our results provide mechanistic insights into tumor cell extravasation and demonstrate that incorporation into the endothelium is one of the earliest steps. In the next part of this work, we investigated how matrix stiffness impacts RNA localization and its relevance to cancer metastasis. In migrating cells, the tumor suppressor protein, adenomatous polyposis coli (APC) targets RNAs to cellular protrusions. We observed that increasing stiffness promotes the peripheral localization of these APC-dependent RNAs and that cellular contractility plays a role in regulating this pathway. We next investigated the mechanism underlying the effect of substrate stiffness and cellular contractility. We found that contractility drives localization of RNAs to protrusions through modulation of detyrosinated microtubules, a network of modified microtubules that associate with, and are required for localization of APC-dependent RNAs. These results raise the possibility that as the matrix environment becomes stiffer during tumor progression, it promotes the localization of RNAs and ultimately induces a metastatic phenotype.

Cell surface Glut1 levels distinguish human CD4 and CD8 T lymphocyte subsets with distinct effector functions

International audience

Metastability and dynamics of stem cells: from direct observations to inference at the single cell level

Organismal development, homeostasis, and pathology are rooted in inherently probabilistic events. From gene expression to cellular differentiation, rates and likelihoods shape the form and function of biology. Processes ranging from growth to cancer homeostasis to reprogramming of stem cells all require transitions between distinct phenotypic states, and these occur at defined rates. Therefore, measuring the fidelity and dynamics with which such transitions occur is central to understanding natural biological phenomena and is critical for therapeutic interventions.

While these processes may produce robust population-level behaviors, decisions are made by individual cells. In certain circumstances, these minuscule computing units effectively roll dice to determine their fate. And while the 'omics' era has provided vast amounts of data on what these populations are doing en masse, the behaviors of the underlying units of these processes get washed out in averages.

Therefore, in order to understand the behavior of a sample of cells, it is critical to reveal how its underlying components, or mixture of cells in distinct states, each contribute to the overall phenotype. As such, we must first define what states exist in the population, determine what controls the stability of these states, and measure in high dimensionality the dynamics with which these cells transition between states.

To address a specific example of this general problem, we investigate the heterogeneity and dynamics of mouse embryonic stem cells (mESCs). While a number of reports have identified particular genes in ES cells that switch between 'high' and 'low' metastable expression states in culture, it remains unclear how levels of many of these regulators combine to form states in transcriptional space. Using a method called single molecule mRNA fluorescent in situ hybridization (smFISH), we quantitatively measure and fit distributions of core pluripotency regulators in single cells, identifying a wide range of variabilities between genes, but each explained by a simple model of bursty transcription. From this data, we also observed that strongly bimodal genes appear to be co-expressed, effectively limiting the occupancy of transcriptional space to two primary states across genes studied here. However, these states also appear punctuated by the conditional expression of the most highly variable genes, potentially defining smaller substates of pluripotency.

Having defined the transcriptional states, we next asked what might control their stability or persistence. Surprisingly, we found that DNA methylation, a mark normally associated with irreversible developmental progression, was itself differentially regulated between these two primary states. Furthermore, both acute or chronic inhibition of DNA methyltransferase activity led to reduced heterogeneity among the population, suggesting that metastability can be modulated by this strong epigenetic mark.

Finally, because understanding the dynamics of state transitions is fundamental to a variety of biological problems, we sought to develop a high-throughput method for the identification of cellular trajectories without the need for cell-line engineering. We achieved this by combining cell-lineage information gathered from time-lapse microscopy with endpoint smFISH for measurements of final expression states. Applying a simple mathematical framework to these lineage-tree associated expression states enables the inference of dynamic transitions. We apply our novel approach in order to infer temporal sequences of events, quantitative switching rates, and network topology among a set of ESC states.

Taken together, we identify distinct expression states in ES cells, gain fundamental insight into how a strong epigenetic modifier enforces the stability of these states, and develop and apply a new method for the identification of cellular trajectories using scalable in situ readouts of cellular state.

Investigating the Role of PDE4D1/2 in Vascular Smooth Muscle Cell Desensitization

Thesis (Master, Biochemistry) -- Queen's University, 2016-10-14 02:44:01.604

Lymphotoxin-beta receptor and RANK signaling in TEL-JAK2-induced T-cell leukemia

Tese de doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015

Functional relationship of colorectal cancer-associated glycans with malignant phenotype and transcriptome changes

Glycosyltransferases ST6GAL1 and B4GALNT2 (and their cognate antigens Sia6LacNAc and Sda, respectively) are associated with colorectal cancer (CRC) but it is not fully clear their biological and clinical significance. We explored the clinical relevance of both glycosyltransferases by interrogating The Cancer Genome Atlas (TCGA) database while the phenotypic/transcriptomic effects of ST6GAL1/B4GALNT2 overexpression were studied in genetically modified CRC cell lines. Transcriptomic data from CRC patients in TCGA database suggested a moderate impact of ST6GAL1 on CRC progression, although it was not possible to define a clear role for this glycosyltransferase. Transcriptomic analysis of ST6GAL1-transduced cell lines revealed a much deeper effect of ST6GAL1 on gene expression in SW948 than in SW48. The overexpression of ST6GAL1 induced opposite effects on soft agar growth and wound healing in both cell lines. These results indicate that the impact of a cancer-associated glycosyltransferase change on phenotype/transcriptome can be extremely variable, depending on the molecular context of the tumor cell. On the contrary, transcriptomic analysis of B4GALNT2-modified cell lines together with TCGA database survey demonstrated a strong impact of B4GALNT2 on the transcriptional activity of CRC cells, in particular its association with a better prognosis. We suggest an anti-tumoral role of B4GALNT2 in CRC. We also investigated the glycan changes related to ST6GAL1/B4GALNT2 expression in a small cohort of tissues/plasma as well as the N-glycomic profile of CRC, normal and polyp tissues. We found an increase of ST6GAL1 activity in CRC and inflammatory bowel disease plasma samples comparing with plasma from healthy donors. A different Sda protein carrier pattern was observed between healthy donors and CRC plasma samples. β-arrestin 1 is a possible candidate as Sda carrier protein in plasma samples although future validation studies are needed. The alterations found in the N-glycan pattern highlight the importance of N-glycome as a molecular signature in cancer.

Genotype-phenotype correlation in trisomy 21

Down syndrome (DS) or trisomy 21 (T21) is the most common genetic cause of intellectual disability (ID). Subjects with DS are characterized by complex and variable clinical features including intellectual disability (ID) and craniofacial dysmorphisms. The aim of the thesis is to uncover genotype-phenotype relationships in DS possibly useful to devise therapies based on molecular and cellular mechanisms. In this work, we have investigated different aspects of DS: - we have collected clinical data of children with DS and we have evaluated the cognitive impairment through specific cognitive tests - we have analysed genomics of DS through the study of partial trisomy (PT21) cases. We have described new PT21 cases confirming the hypothesis of the highly restricted DS critical region (HR-DSCR) recently identified as the minimal region whose duplication is shared by all PT21 subjects diagnosed with DS, while it is absent in all PT21 non-DS subjects. Moreover, we have characterized new transcripts included in the HR-DSCR; - we have studied gene expression through RNAseq in blood cells of children with DS; -metabolic alterations in plasma of children with DS were identified through different methods: Nuclear Magnetic resonance, routine blood exams performed during the follow up of the subjects and enzyme-linked immunosorbent assay (ELISA); - to test possible correlations between specific Hsa21 regions and alterations in transcriptomics and metabolomics, we have used trisomic iPSCs and differentiated them into neuronal derivatives. Significant alterations in gene expression and metabolic profiles have been identified, as well as significant correlations with clinical and cognitive aspects. Specific genes and the HR-DSCR may play a role in these alterations: cell models need to be developed to investigate this role. Neural derivatives from trisomic iPSCs are a promising model to better understand genotype-phenotype correlations in DS.

Urine-derived Renal Epithelial Cells from kidney transplanted patients: phenotype, immunomodulatory properties, and effect of the exposition to NGAL

During kidney transplant procedure transplanted organs can undergo ischaemia reperfusion phenomena, often associated with the onset of acute kidney damage, loss of kidney function and rejection. These events promote cell turnover to replace damaged cells and preserve kidney function, thus cells deriving from nephrons structures are highly voided in urine. Urine derived cells represents a promising cell source since they can be easily isolated and cultured. The aim of this project was to characterise Urine-derived Renal Epithelial Cells (URECs) from transplanted kidney and to evaluate how these cells react to the co-culture with immune cells. URECs expressed typical markers of kidney tubule epithelial cells (Cytokeratin and CD13), and a subpopulation of these cells expressed CD24 and CD133, which are markers of kidney epithelial progenitor cells. The expression of immunosuppressive molecules as HLA-G and CD73 was also observed. As matter of fact, during the co-culture with PBMCs, UREC suppressed the proliferation of CD4 and CD8 Lymphocytes and reduce the T helper 1 subset, while increasing the T regulatory counterpart. Also, preliminary data observed in this study indicated that the exposition to kidney damage associated molecule, such as NGAL, could significantly affect UREC viability and immunomodulatory capacity. These results add new information about the phenotype of urine cells obtained after kidney transplant and reveal that these cells show promising immunomodulatory properties, suggesting their potential application in personalized cell therapy approaches.

Egfr Activating Mutations And Their Association With Response To Platinum-doublet Chemotherapy In Brazilian Non-small Cell Lung Cancer Patients.

The aim of the study was to analyze the frequency of epidermal growth factor receptor (EGFR) mutations in Brazilian non-small cell lung cancer patients and to correlate these mutations with response to benefit of platinum-based chemotherapy in non-small cell lung cancer (NSCLC). Our cohort consisted of prospective patients with NSCLCs who received chemotherapy (platinum derivates plus paclitaxel) at the [UNICAMP], Brazil. EGFR exons 18-21 were analyzed in tumor-derived DNA. Fifty patients were included in the study (25 with adenocarcinoma). EGFR mutations were identified in 6/50 (12 %) NSCLCs and in 6/25 (24 %) adenocarcinomas; representing the frequency of EGFR mutations in a mostly self-reported White (82.0 %) southeastern Brazilian population of NSCLCs. Patients with NSCLCs harboring EGFR exon 19 deletions or the exon 21 L858R mutation were found to have a higher chance of response to platinum-paclitaxel (OR 9.67 [95 % CI 1.03-90.41], p = 0.047). We report the frequency of EGFR activating mutations in a typical southeastern Brazilian population with NSCLC, which are similar to that of other countries with Western European ethnicity. EGFR mutations seem to be predictive of a response to platinum-paclitaxel, and additional studies are needed to confirm or refute this relationship.

An Efficient Protocol For The Generation Of Monocyte Derived Dendritic Cells Using Serum-free Media For Clinical Applications In Post Remission Aml Patients.

Protocols for the generation of dendritic cells (DCs) using serum as a supplementation of culture media leads to reactions due to animal proteins and disease transmissions. Several types of serum-free media (SFM), based on good manufacture practices (GMP), have recently been used and seem to be a viable option. The aim of this study was to evaluate the results of the differentiation, maturation, and function of DCs from Acute Myeloid Leukemia patients (AML), generated in SFM and medium supplemented with autologous serum (AS). DCs were analyzed by phenotype characteristics, viability, and functionality. The results showed the possibility of generating viable DCs in all the conditions tested. In patients, the X-VIVO 15 medium was more efficient than the other media tested in the generation of DCs producing IL-12p70 (p=0.05). Moreover, the presence of AS led to a significant increase of IL-10 by DCs as compared with CellGro (p=0.05) and X-Vivo15 (p=0.05) media, both in patients and donors. We concluded that SFM was efficient in the production of DCs for immunotherapy in AML patients. However, the use of AS appears to interfere with the functional capacity of the generated DCs.

Solid Lipid Nanoparticles For Hydrophilic Biotech Drugs: Optimization And Cell Viability Studies (caco-2 & Hepg-2 Cell Lines).

Insulin was used as model protein to developed innovative Solid Lipid Nanoparticles (SLNs) for the delivery of hydrophilic biotech drugs, with potential use in medicinal chemistry. SLNs were prepared by double emulsion with the purpose of promoting stability and enhancing the protein bioavailability. Softisan(®)100 was selected as solid lipid matrix. The surfactants (Tween(®)80, Span(®)80 and Lipoid(®)S75) and insulin were chosen applying a 2(2) factorial design with triplicate of central point, evaluating the influence of dependents variables as polydispersity index (PI), mean particle size (z-AVE), zeta potential (ZP) and encapsulation efficiency (EE) by factorial design using the ANOVA test. Therefore, thermodynamic stability, polymorphism and matrix crystallinity were checked by Differential Scanning Calorimetry (DSC) and Wide Angle X-ray Diffraction (WAXD), whereas the effect of toxicity of SLNs was check in HepG2 and Caco-2 cells. Results showed a mean particle size (z-AVE) width between 294.6 nm and 627.0 nm, a PI in the range of 0.425-0.750, ZP about -3 mV, and the EE between 38.39% and 81.20%. After tempering the bulk lipid (mimicking the end process of production), the lipid showed amorphous characteristics, with a melting point of ca. 30 °C. The toxicity of SLNs was evaluated in two distinct cell lines (HEPG-2 and Caco-2), showing to be dependent on the concentration of particles in HEPG-2 cells, while no toxicity in was reported in Caco-2 cells. SLNs were stable for 24 h in in vitro human serum albumin (HSA) solution. The resulting SLNs fabricated by double emulsion may provide a promising approach for administration of protein therapeutics and antigens.

Autologous Platelet Gel: Five Cases Illustrating Use On Sickle Cell Disease Ulcers.

Leg ulcers represent a particularly disabling complication in patients with sickle cell disease (SCD). Platelet gel (PG) is a novel therapeutic strategy used for accelerating wound healing of a wide range of tissues through the continuous release of platelet growth factors. Here, we describe the use of PG preparation according to Anitua's PRGF (preparations rich in growth factors) protocol for treating chronic nonhealing ulcers in patients with SCD. A positive response occurred in 3 patients with an area reduction of 85.7% to 100%, which occurred within 7 to 10 weeks, and a 35.2% and 20.5% of area reduction in 2 other patients, who however, had large ulcers. After calcium chloride addition, the platelet-rich plasmas demonstrated enhanced platelet-derived growth factors-BB (P < .001), transforming growth factor-β1 (P = .015), vascular endothelial growth factors (P = .03), and hepatocyte growth factors (nonsignificant) secretion. Furthermore, calcium chloride addition induced a significant decrease in platelet number (P = .0134) and there was no leukocyte detection in the PG product. These results demonstrate that PG treatment might impact the healing of leg ulcers in sickle cell disease, especially in patients with small ulcers.

Impaired Compensatory Beta-cell Function And Growth In Response To High-fat Diet In Ldl Receptor Knockout Mice.

In this study, we investigated the effect of low density lipoprotein receptor (LDLr) deficiency on gap junctional connexin 36 (Cx36) islet content and on the functional and growth response of pancreatic beta-cells in C57BL/6 mice fed a high-fat (HF) diet. After 60 days on regular or HF diet, the metabolic state and morphometric islet parameters of wild-type (WT) and LDLr-/- mice were assessed. HF diet-fed WT animals became obese and hypercholesterolaemic as well as hyperglycaemic, hyperinsulinaemic, glucose intolerant and insulin resistant, characterizing them as prediabetic. Also they showed a significant decrease in beta-cell secretory response to glucose. Overall, LDLr-/- mice displayed greater susceptibility to HF diet as judged by their marked cholesterolaemia, intolerance to glucose and pronounced decrease in glucose-stimulated insulin secretion. HF diet induced similarly in WT and LDLr-/- mice, a significant decrease in Cx36 beta-cell content as revealed by immunoblotting. Prediabetic WT mice displayed marked increase in beta-cell mass mainly due to beta-cell hypertrophy/replication. Nevertheless, HF diet-fed LDLr-/- mice showed no significant changes in beta-cell mass, but lower islet-duct association (neogenesis) and higher beta-cell apoptosis index were seen as compared to controls. The higher metabolic susceptibility to HF diet of LDLr-/- mice may be explained by a deficiency in insulin secretory response to glucose associated with lack of compensatory beta-cell expansion.