Literatura académica sobre el tema "Cluster Differentiation of Antigen 4"

Cluster of differentiation antigen 4 (CD4), the T lymphocyte antigen receptor component and human immunodeficiency virus coreceptor, is down-modulated when cells are activated by antigen or phorbol esters. During down-modulation CD4 dissociates from p56 lck , undergoes endocytosis through clathrin-coated pits, and is then sorted in early endosomes to late endocytic organelles where it is degraded. Previous studies have suggested that phosphorylation and a dileucine sequence are required for down-modulation. Using transfected HeLa cells, in which CD4 endocytosis can be studied in the absence of p56 lck , we show that the dileucine sequence in the cytoplasmic domain is essential for clathrin-mediated CD4 endocytosis. However, this sequence is only functional as an endocytosis signal when neighboring serine residues are phosphorylated. Phosphoserine is required for rapid endocytosis because CD4 molecules in which the cytoplasmic domain serine residues are substituted with glutamic acid residues are not internalized efficiently. Using surface plasmon resonance, we show that CD4 peptides containing the dileucine sequence bind weakly to clathrin adaptor protein complexes 2 and 1. The affinity of this interaction is increased 350- to 700-fold when the peptides also contain phosphoserine residues.

The autoptical observations commonly ascribed to sepsis deal with unspecific general and local signs of inflammation or ischemia, such as myocardial inflammation, pulmonary edema and infiltration, cerebral swallowing, and tubular necrosis in the kidney. In the two last decades, some studies have been carried out to implement immunohistochemical markers for post-mortem diagnosis. All of these target molecules are specifically up-regulated or down-regulated during systemic inflammatory responses, especially for infective causes. Among these, we found some antigens expressed on leukocyte surfaces (very late antigen-4 (VLA-4), cluster differentiation-15 (CD15)), enzyme contained in neutrophils granules (lysozyme (LZ), lactoferrin (LF)), endothelial markers and junctions (E-selectin, vascular endothelial cadherin (VE-cadherin)), and soluble factors (vascular endothelial growth factor (VEGF), tumor necrosis factor alpha (TNFα), procalcitonin (PCT), soluble triggering receptor expressed on myeloid cells-1 (s-TREM-1)). All of these showed potential reliability in differentiating sepsis cases from controls. Further studies are needed to provide a concrete validation for a combination of markers on specific organ samples in order to reach a post-mortem diagnosis of sepsis also in the absence of clinical records.

BackgroundRadiotherapy (RT) in combination with CTLA-4 inhibition (CTLA4i) can expand and activate T-cells to reject tumors in both mice and some patients with tumors unresponsive to CTLA4i alone.1 2 However, only a subset of patients achieves long-term control of metastatic disease. Similar responses to RT+CTLA4i are seen in the 4T1 mouse model of triple negative breast cancer (TNBC), making it an ideal model to interrogate the interaction between RT and CTLA4i, and identify barriers to its effectiveness.MethodsMice were inoculated in one or both flanks with 4T1 cells. In some experiments one tumor was removed for analysis before start of treatment with RT (3 × 8 Gy) and/or anti-CTLA-4 antibody (9H10, 3 × 200 ?g i.p.). The intratumoral T cell response was assessed using bulk and single cell RNA/TCR sequencing. The METABRIC dataset3 was used to associate gene expression signatures with patient survival. In some experiments, RT+CTLA4i was combined with PD-1, LAG-3, or CD40 Abs.ResultsRT, alone and with CTLA4i, increased the TCR repertoire clonality and activated T cells density in the tumors (figure 1A-G). In untreated tumors, Gzmb+Prf1+Lag3+Pd1+Cd8+ T cells (cluster 0) were most common. CTLA4i ‘unlocked’ Ifng+Cd40lg+ Cd4+ T cells (cluster 2) while RT favored expansion/persistence of Cd8+ T cell clusters. In tumors of mice treated with RT+CTLA4i activated Treg cells (cluster 1) were decreased and Ifng+Cd40lg+Cd4+ T cells (cluster 2) increased. Relatively among CD8+ T cells, Ifng+Tnf+Cd8+ (cluster 4) was expanded at the expense of cluster 0 (figure 2A-F). Gene signatures defining clusters 0, 2, and 4 were associated with improved survival in the METABRIC TNBC patient cohort using a multivariate model (figure 2G-H). In mice, AH1-tumor antigen-specific CD8+ T cells occupied different transcriptional states, with a shift to cluster 4 in mice treated with RT+CTLA4i (figure 2I), suggesting that multiple functional T cell states are required for tumor rejection. Based on the T cell phenotypes expanded by RT+CTLA4i, antibodies to PD-1, LAG-3, and CD40 were tested for the ability to enhance RT+CTLA4i therapy. Only CD40-agonist improved significantly tumor control (figure 3A-B).Abstract 465 Figure 1RT, alone and with CTLA4i, increased the TCR repertoire clonality and density of activated T cells in the tumors. (A) Design of the experiment enabling collection of pre- and post-treatment (pre-tx and post-tx) 4T1 tumor tissue that was analyzed using RNA- and TCR-sequencing. (B) Tumor growth curves. Statistical significance in tumor volume growth between groups was determined using 2-way repeated measures ANOVA between day 15–21 and t-test at day 21. (C) Shannon clonality of paired pre- and post-tx TCR repertoires. Pairwise and paired t-tests were used to evaluate statistical significance of differences between and within groups, respectively. (D) RNA-seq based gene expression heatmap of selected canonical T cell markers in post-tx tumors. (E) Linear regression between Cd3e and Cd4 or Cd8 gene expression in post-tx tumors. R2 and p indicate R-square and p-value for the models, respectively (F) Ingenuity Pathway Analysis Canonical Pathway and (G) Upstream Regulation analysis. Z-scores indicate predicted activation (> 2) or inhibition (< -2) of pathways and upstream regulators. (all panels) *, **, and ***, and #, ## and ### indicate p-values of pairwise and paired statistical tests, respectively. Tukey’s and Holm’s method for adjusting p-values corrected for multiple comparison was used for the ANOVA and t-tests, respectively. (Abbreviations) tx, treatment; RT, radiation therapy; CTLA4, CTLA-4 Ab therapy; TCR, T cell receptorAbstract 465 Figure 2RT+CTLA-4i increased tumor infiltration by Gzmb+Prf1+Lag3+Pd1+Cd8+, Ifng+Cd40lg+Cd4+, and Ifng+Tnf+Cd8+ T cells in 4T1 tumors. (A) Design of experiment enabling single cell analysis of T cells infiltrating 4T1 tumors. (B) Based on gene expression levels, the T cells were divided into 17 clusters (indicated by colors) and visualized in 2D using UMAP dimensionality reduction algorithm. (C) Gene expression levels of selected high-level T cell markers. (D) Table with main phenotype, key genes representative for each cluster, and the distribution of T cells from each condition falling into the different clusters. (E) Proportion of Cd4+ and Cd8+ T cells for the different treatment groups. (F) The expression of cluster-specific gene signatures in bulk 4T1 tumors for clusters 0, 2, and 4. (G) Survival curves and (H) multivariate analysis of the association between survival and enrichment of the gene signatures of clusters 0, 2, and 4. (I) The positioning of the all AH1-dextramer+ Cd8+T cell clones within the UMAP plot. Color annotate cluster. (Abbreviations) tx, treatment; RT, radiation therapy; Untr., untreated; CTLA4, CTLA-4 Ab; TCR CDR3, T cell receptor complementary determining region 3; UMAP, Uniform Manifold Approximation and Projection for dimension reduction; AH1, tumor antigen in 4T1 tumors; SPSYVYHQF peptide derived from gp70 and restricted to H2-LdAbstract 465 Figure 3Agonistic CD40 treatment improves RT+CTLA-4 therapy. Individual tumor growth curves for untreated, RT+CTLA-4, or RT+CTLA-4+CD40 treated mice. Color annotate group. *, **, ***, and **** indicate p-values < 0.05, 0.01, 0.001, and 0.0001, respectively, calculated using a linear mixed-effects model. (A) and (B) represent two individual experiments. (Abbreviations) RT, radiation therapy; CTLA4, CTLA-4 Ab; CD40, anti-CD40 Ab; mm3, cubic millimeter; d, daysConclusionsAltogether, these results revealed that RT and CTLA4i have complementary effects and besides driving T cells into tumors shape CD4 and CD8 T cell functional differentiation towards subsets that are associated with improved survival in patients. Unexpectedly, inhibition of checkpoint receptors expressed by a large CD8 T cells cluster did not further improve responses to RT+CTLA4i, whereas agonistic CD40 therapy did, suggesting new therapeutic strategies.AcknowledgementsGrant support: R01CA198533ReferencesK, Ferrari de Andrade L, Wucherpfennig KW, Heguy A, Imai N, Gnjatic S, Emerson RO, Zhou XK, Zhang T, Chachoua A, Demaria S. Radiotherapy induces responses of lung cancer to CTLA-4 blockade. Nat Med 2018;24(12):1845–51.Rudqvist NP, Pilones KA, Lhuillier C, Wennerberg E, Sidhom JW, Emerson RO, Robins HS, Schneck J, Formenti SC, Demaria S. Radiotherapy and CTLA-4 Blockade Shape the TCR Repertoire of Tumor-Infiltrating T Cells. Cancer Immunol Res 2018;6(2):139–50.Curtis C, Shah SP, Chin SF, Turashvili G, Rueda OM, Dunning MJ, Speed D, Lynch AG, Samarajiwa S, Yuan Y, Graf S, Ha G, Haffari G, Bashashati A, Russell R, McKinney S, Group M, Langerod A, Green A, Provenzano E, Wishart G, Pinder S, Watson P, Markowetz F, Murphy L, Ellis I, Purushotham A, Borresen-Dale AL, Brenton JD, Tavare S, Caldas C, Aparicio S. The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. Nature 2012;486(7403):346–52.

Toll-like receptor 4 (TLR4), the receptor of bacterial endotoxins in mammalians, plays a pivotal role in the induction of innate immunity and inflammation. TLR4 activation by bacterial lipopolysaccharide (LPS) is achieved by the coordinate and sequential action of three other proteins, the lipopolysaccharide binding protein (LBP), the cluster differentiation antigen CD14, and the myeloid differentiation protein (MD-2) receptors, that bind LPS and present it in a monomeric form to TLR4 by forming the activated [TLR4·MD-2·LPS]2 complex. Small molecules and nanoparticles active in modulating the TLR4 signal by targeting directly the MD-2·TLR4 complex or by interfering in other points of the TLR4 signaling are presented in this paper. These compounds have great pharmacological interest as vaccine adjuvants, immunotherapeutics, anti-sepsis, and anti-inflammatory agents.

Helicobacter pylori is a gram-negative, intracellular, microaerophilic bacteria which causing Peptic ulcer. This bacterium can change its shape which helps the bacteria to survive in the host gastric microenvironment. The Peptic ulcer caused by this bacterium stimulates the humoral and cellular immune response in individuals. The current study was carried out to access the role of interleukin-2, interleukin-4, and cluster differentiation-22 as immune markers in the identification of H. pylori infection. The presence of H. pylori has been diagnosed by feces test (antigen rapid test). In this study, the presence of three immunological markers viz., IL-2, IL-4, and CD22 were measured in the serum of 60 individuals infected with H. pylori and 30 healthy individuals by the Enzyme-Linked Immune-sorbent Assay method. Results of this study indicated a significant increase (P-value=0.0307*) in the concentration of IL-2 (294.27ng/ml), IL-4(151.28ng/ml), and CD22 (492.73ng/ml) in the serum of individuals infected with H. pylori while these concentrations were reported 235.98ng/ml, 116.14ng/ml and 369.33ng/ml respectively in the healthy individuals. Results of the study can be concluded that H.pylori infection stimulates the Cellular and humoral immune response which resulted in the increased production of IL-2, IL-4, and CD22.

Inducible T cell costimulator (ICOS, cluster of differentiation (CD278)) is an activating costimulatory immune checkpoint expressed on activated T cells. Its ligand, ICOSL is expressed on antigen-presenting cells and somatic cells, including tumour cells in the tumour microenvironment. ICOS and ICOSL expression is linked to the release of soluble factors (cytokines), induced by activation of the immune response. ICOS and ICOSL binding generates various activities among the diversity of T cell subpopulations, including T cell activation and effector functions and when sustained also suppressive activities mediated by regulatory T cells. This dual role in both antitumour and protumour activities makes targeting the ICOS/ICOSL pathway attractive for enhancement of antitumour immune responses. This review summarises the biological background and rationale for targeting ICOS/ICOSL in cancer together with an overview of the principal ongoing clinical trials that are testing it in combination with anti-cytotoxic T lymphocyte antigen-4 and anti-programmed cell death-1 or anti-programmed cell death ligand-1 based immune checkpoint blockade.

Endothelial cell vascular cell adhesion molecule-1 (VCAM-1) activates adherent monocytes by clustering their very late antigen-4 (VLA-4) receptors, resulting in the modulation of the inwardly rectifying ( I ir) and delayed rectifying ( I dr) K+ currents, hyperpolarization of the cells, and enhanced Ca2+ influx (Colden-Stanfield M and Gallin EK. Am J Physiol Cell Physiol 275: C267–C277, 1998; Colden-Stanfield M and Scanlon M. Am J Physiol Cell Physiol 279: C488–C494, 2000). The present study was undertaken to test the hypothesis that monoclonal antibodies (MAbs) against VLA-4 (MAbVLA-4) mimic VCAM-1 to cluster VLA-4 integrins, which play a key role in signaling an increase in the secretion of the proinflammatory cytokine interleukin-8 (IL-8). Whole cell ionic currents and IL-8 secretion from THP-1 monocytes that were incubated on polystyrene, VCAM-1-immobilized MAbVLA-4 or an isotype-matched MAb against CD45 (MAbCD45) were measured. Clustering of VLA-4 integrins with a cross-linked MAbVLA-4, but not a monovalent MAbVLA-4, modulated the K+ currents in an identical manner to incubation of cells on VCAM-1. Similarly, cross-linked MAbVLA-4 or VCAM-1 augmented Ca2+-mediated IL-8 secretion from THP-1 monocytes and was completely abolished by exposure to CsCl, an I ir blocker. Thus VLA-4 integrin clustering by cross-linked MAbVLA-4 mimics VCAM-1/VLA-4 interactions sufficiently to be associated with events leading to monocyte differentiation, enhanced Ca2+-mediated macrophage function, and possibly atherosclerotic plaque formation.

Microcapsules that release antigen-capturing nanoparticles (AC-NPs) with macrophage inflammatory protein-3 alpha (MIP-3[Formula: see text]) and anti-programmed death-1 (PD-1) antibody are developed, and these microcapsules have the ability to enhance immunoresponses through cross-priming of cluster of differentiation 8+ (CD8+) T cells by dendritic cells (DCs) in vivo in BALB/c mice. Lipid protamine hyaluronic acid nanoparticles containing AC-NPs generated via nanoprecipitation of 4 mg/mL of polylactic-co-glycolic acid (PLGA), 1,000 ng/mL of MIP-3[Formula: see text] and 400 [Formula: see text]g of anti-PD-1 were mixed with 1 mL of 4.0% alginate and 3.0% of hyaluronate and then sprayed with 0.5 mM of ferrous chloride. These capsules were injected subcutaneously around LM17 tumor in the left hind legs of BALB/c mice. The tumors were exposed to a radiation dose of 10 or 20 Gy from 100 keV soft X-ray radiation. PLGA AC-NPs and MIP-3[Formula: see text] were released in response to the radiation dose. PLGA AC-NPs captured tumor-derived protein antigens are released by exposure to radiation, and these antigens were transported to DCs that were recruited and activated by MIP-3[Formula: see text], intensifying the DC-associated cross-priming of CD8+ T cells. These treatments resulted in increased antitumor effect and reduced metastasis by abscopal effect. Our targeted immunotherapy may lead to better tumor therapy.

Eligibility for antiretroviral therapy (ART) in HIV-infected patients is defined either by a cluster of differentiation antigen 4 (CD4) count of less than 200cells/mm3 or clinical diagnosis of WHO stage III and IV. Therefore, the decision to start ART becomes difficult when CD4 cell count is not available. With limited laboratory infrastructure, the decision to start ART is usually made based on clinical symptoms leading to late commencement of ART. This calls for alternative criteria to see if nail discoloration (ND) correlates with low CD4 count among untreated HIV infected patients. This will serve as a complementary screening tool for identifying asymptomatic ARV naive HIV patients with a CD4 cell count of less than 200cells/mm3 which signifies 
severe immunosuppression. Study Design and Setting: This was a quantitative cross-sectional descriptive and analytical study involving adult ART naï
ve HIV infected patients in WHO stage I and II. Systematic sampling was used to select the participants from all adult ART naï
ve HIV infected patients attending APIN clinic, located at the Jos University Teaching Hospital (JUTH), Jos, Nigeria. Data Collection: Face-to-face interviews, physical examination and relevant laboratory investigations with selected participants were conducted using a questionnaire guide. Questions on socio-demographic characteristics, clinical data, general physical examinations including finger nail examination and photographing with subsequent laboratory investigations including CD4 count and western blot were employed. Data Analysis: Variables were categorized and data analyzed using descriptive statistics including the frequency, percentage frequency
mean and standard deviation of continuous variables. Association between CD4 count of &le
200cells/mm3 and ND was tested using the chisquare test with an alpha level of 0.05. Prevalence of ND, sensitivity, specificity, positive predictive and negative predictive values and accuracy of the screening test of ND was calculated. Results: 394 patients had their fingernails photographed and assessed. It was shown that distal banded and grey nails were the common types of ND seen with a prevalence of 38%. There was an association between CD4 count &le
200cells/mm3 and ND (p<
0.0001). CD4 count &le
200cells/mm3 was a risk factor for developing ND (RR=2.3[1.8-3.6]). The association has a sensitivity of 78%, specificity of 55%, positive predictive value of 50%, and negative predictive value of 80% and accuracy of test 63%. Conclusion: With a significant association (p<
0.0001) and a sensitivity of 78%, ND can be a useful clinical indicator of immune dysfunction mediated by HIV among patients in WHO stage I or II. ND can either be a clinical sign or a symptom in HIV patients with a CD4 of &le
200cells/mm3 as seen in the study as the specificity and sensitivity of ND compared favourably with other WHO stage III diagnosis. Recommendations: Nail discoloration should complement CD4 count as an additional staging sign to help identify patients likely to benefit from ART especially in resource-limited settings. Finally, all patients with grey or distal banded should be on co-trimoxaxole prophylaxis in line with WHO /national guideline on the use of co-trimoxaxole for all HIV positive patients with a CD4 cell count of &le
350cells/mm3.

Philosophiae Doctor - PhD
AIDS is considered a pandemic causing millions of deaths worldwide and a cure for this disease is still not available. Failure to implement early treatments due to the poor diagnostic methods and ineffective therapeutic regimens to treat HIV patients to achieve complete viral eradication from the human body has encouraged the escalation of this disease at an exponential rate. Though the current treatment regimens (High Active Antiretroviral Therapy) have aided in increasing the lifespan of HIV patients, it still suffers from some shortcomings such as adverse side effects and non-eradication of the virus. Thus, there is a need for a non-toxic therapeutic regimen to stop further infection of HIV-infected patients. Antimicrobial Peptides (AMPs) are naturally occurring peptides which are components of the first line of defence of many organisms against infections and have been proven to be promising therapeutic agents against HIV. The use of AMPs as anti-microbial agents is due to the fact that most AMPs have a net positive charge and are mostly hydrophobic molecules. These features allow AMPs to be site directed electro-statistically to the mostly negatively charged pathogens. In a previous study, a number of novel anti-HIV AMPs was identified using a predictive algorithm Profile Hidden Markov Models (HMMER). The AMP's threedimensional structures were predicted using an in-silico modelling tool I-TASSER and an insilico protein-peptide interaction study of the AMPs to HIV protein gp120 was performed using PatchDock. Five AMPs were identified to bind gp120, at the site where gp120 interacts with CD4 to prevent HIV invasion and HIV replication. Therefore, the aims of this research were to perform in-silico site-directed mutation on the parental anti-HIV AMPs to increase their binding affinity to the gp120 protein, validate the anti-HIV activity of these peptides and confirm the exclusivity of this activity by testing possible anti-bacterial and anti-cancer activities of the AMPs. Firstly, the five parental anti-HIV AMPs were used to generate mutated AMPs through insilico site-directed mutagenesis. The AMPs 3-D structures were determined using I-TASSER and the modelled AMPs were docked against the HIV protein gp120 using PatchDock. Secondly, an "in house" Lateral Flow Device (LFD) tool developed by our industrial partner, Medical Diagnostech (Pty) Ltd, was utilised to confirm the in-silico docking results. Furthermore, the ability of these AMPs to inhibit HIV-1 replication was demonstrated and additional biological activities of the peptides were shown on bacteria and cancer cell lines. In an effort to identify AMPs with increased binding affinity, the in-silico results showed that two mutated AMPs Molecule 1.1 and Molecule 8.1 bind gp120 with high affinity, at the point where gp120 bind with CD4. The molecular binding however showed that only Molecule 3 and Molecule 7 could prevent the interaction of gp120 protein and CD4 surface protein of human cells, in a competitive binding assay. Additionally, the testing of the anti-HIV activity of the AMPs showed that Molecule 7, Molecule 8 and Molecule 8.1 could inhibit HIV-1 NL4-3 with maximal effective concentration (EC₅₀) values of 37.5 μg/ml and 93.75 μg/ml respectively. The EC₅₀ of Molecule 8.1 was determined to be around 12.5 μg/ml. This result looks promising since 150 μg/ml of the AMPs could not achieve 80% toxicity of the human T cells, thus high Therapeutics Index (TI) might be obtained if 50% cytotoxic concentration (CC₅₀) is established. Further biological activity demonstrates that Molecule 3 and Molecule 7 inhibited P. aeruginosa completely after 24 hours treatment with peptide concentrations ranging from 0.5 mg/ml to 0.03125 mg/ml. Nevertheless, moderate inhibition was observed when CHO, HeLa, MCF-7 and HT-29 were treated with these peptides at peptides concentration of 100 μg/ml. The ability of these AMPs to block the entrance of HIV via the binding to CD4 of the host cells is a good concept since they pave the way for the design of anti-HIV peptide-based drugs Entry Inhibitors (FIs) or can be exploited in the production microbicide gels/films to suppress the propagation of the virus.
DST-NIC/Mintek

Masters of Science
The rapidly expanding field of nanotechnology has been the focus of many biologists with regard to drug delivery. The ability of nanoparticles to enter cellular compartments makes it possible to explore specific treatment strategies for life-threatening diseases such as AIDS. Since HIV primarily infects CD4+ cells, we aim to use CD4 as a selectable marker to deliver pro-apoptotic nano-devices to HIV infected cells. The objective is to selectively induce cell death or apoptosis in CD4+ HIV infected cells. Apoptosis is activated through a number of biochemical pathways. The apoptosis promoting protease, caspase-3 is central to the induction of apoptosis. Caspase-3 is produced as an inactive zymogen and is activated by other proteases through proteolytic cleavage. We take advantage of the fact that HIV-infected cells produce HIV-1 protease, which is responsible for the production of infectious virions through proteolytic cleavage of the HIV proteins, Gag and Pol. Our strategy was to generate a mutant form of the caspase-3 protease that is only cleavable by HIV-1 protease.

SUMMARY:

The bacterial lipopolysaccharide (LPS)-sensing machinery with the innate immune system receptor Toll-like receptor 4 (TLR4) at its centre has been the subject of extensive research but while TLR4 and myeloid differentiation factor 2 (MD2) were both shown to be essential, the role of other, so-called "accessory", molecules is much less clear. The co-receptor cluster of differentiation 14 (CD14) has been widely perceived as being a mere facilitator for the capture and transfer of LPS to TLR4, until recent studies suggested it might have a determining influence on which TLR4-dependent signaling cascades are triggered in response to LPS. The TLR4 receptor complex was shown to be specifically activated by diC14 amidine, a cationic lipid originally synthesized for its carrier properties. The lipid's immunostimulatory activity extends to both TLR4-dependent signaling cascades, the MyD88 and TRIF pathways.

The aim of this work was to gain more insight into how diC14 amidine is able to trigger these cascades and to contribute to the general understanding of the TLR4 machinery and its activation by non-LPS ligands. More precisely we were interested in the role of CD14 in the activation of both MyD88 and TRIF pathways by diC14-amidine and in potential consequences of possible divergent requirements of diC14 amidine and LPS for this co receptor.

Our study of the role of the membrane-associated and the soluble form of CD14 in the activation of the TLR4-dependent pathways by diC14 amidine revealed that – unlike LPS – the cationic lipid does not require CD14 to exercise its immunostimulatory activity, although the presence of the co receptor modulates the TLR4 activation and infrared spectroscopy experiments suggest a direct interaction.

In the case of sensing LPS, CD14 is required for the endocytosis of TLR4 and the subsequent activation of the TRIF pathway. By blocking the endocytosis mechanism at different stages we found that diC14-amidine generally enters the cell via endocytosis and that it activates – unlike LPS – both signaling cascades from inside endosomal vesicles, albeit at different stages of the endocytosis process.

Although the eventual immunological responses caused by diC14 amidine and LPS resemble each other or are even identical, our research revealed differences in the actual mechanism of activating TLR4, the receptor responsible for the corresponding innate immune response. These findings illustrate the uniqueness of diC14 amidine and the potential of further exploring its intriguing properties and mechanisms as a tool to decipher the TLR4 signaling machinery and with the perspective of designing new immunomodulators for vaccination and therapy.

RÉSUMÉ:

Le mécanisme de reconnaissance des lipopolysaccharides bactériens (LPS) par le récepteur de l'immunité innée Toll-like receptor 4 (TLR4) a fait l'objet d'une recherche intensive ces dernières années. Alors que TLR4 et son co-récepteur myeloid differentiation factor 2 (MD2) ont été démontrés comme étant essentiels pour la détection du LPS, le rôle des molécules dites "accessoires" est beaucoup moins évident. Le co-récepteur cluster of differentiation 14 (CD14) a largement été considéré comme un simple facilitateur pour la capture et le transfert des LPS à TLR4, mais des études récentes suggèrent qu'il pourrait avoir une influence déterminante sur les cascades de signalisation dépendantes de TLR4 induites en réponse au LPS. La diC14-amidine, un lipide cationique synthétisé initialement pour ses qualités en tant que vecteur de transfection, a révélé récemment une activité immunostimulatrice dépendante du récepteur TLR4, impliquant les deux cascades de signalisation dépendantes de TLR4, les voies MyD88 et TRIF.

Le but de ce travail était de mieux comprendre le mécanisme par lequel la diC14¬ amidine induit ces cascades et de contribuer à la compréhension générale du fonctionnement du complexe récepteur TLR4 et son activation par des ligands non-LPS. Plus précisément nous nous sommes intéressés au rôle de CD14 dans l'activation des voies MyD88 et TRIF par la diC14-amidine et des conséquences potentielles d’éventuelles divergences en termes d’exigence pour ce co-récepteur entre la diC14-amidine et le LPS.

Notre étude sur le rôle de la forme membranaire ou soluble de CD14 dans l'activation des voies dépendantes de TLR4 par la diC14-amidine a révélé que - contrairement au LPS - le lipide cationique ne nécessite pas de CD14 pour exercer son activité immunostimulatrice. Cependant, la présence du co-récepteur module l'activation de TLR4 et des expériences de spectroscopie infrarouge suggèrent une interaction directe entre le lipide et le CD14.

Dans le cas de la détection de LPS, le CD14 est nécessaire pour l'endocytose de TLR4 et l'activation subséquente de la voie TRIF. En bloquant le mécanisme d'endocytose à différents stades, nous avons montré que la diC14-amidine active - contrairement au LPS - les deux cascades de signalisation depuis l'intérieur des vésicules endosomiales, mais à des stades différents du processus d'endocytose.

En conclusion, bien que les réponses immunologiques causées par la diC14-amidine et le LPS se ressemblent, notre recherche a mis en évidence des différences substantielles dans leurs modes d'action. Ces différences illustrent le caractère unique de la diC14-amidine et son potentiel comme outil pour explorer la complexité du système de signalisation du TLR4 et en tirer des enseignements qui permettront de contribuer à la conception de nouveaux immunomodulateurs pour la vaccination et la thérapie.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished

Protein sequencing has produced overwhelming amount of protein sequences, especially in the last decade. Nevertheless, the majority of the proteins' functional and structural classes are still unknown, and experimental methods currently used to determine these properties are very expensive, laborious and time consuming. Therefore, automated computational methods are urgently required to accurately and reliably predict functional and structural classes of the proteins. Several bioinformatics methods have been developed to determine such properties of the proteins directly from their sequence information. Such methods that involve signal processing methods have recently become popular in the bioinformatics area and been investigated for the analysis of DNA and protein sequences and shown to be useful and generally help better characterise the sequences. However, there are various technical issues that need to be addressed in order to overcome problems associated with the signal processing methods for the analysis of the proteins sequences. Amino acid indices that are used to transform the protein sequences into signals have various applications and can represent diverse features of the protein sequences and amino acids. As the majority of indices have similar features, this project proposes a new set of computationally derived indices that better represent the original group of indices. A study is also carried out that resulted in finding a unique and universal set of best discriminating amino acid indices for the characterisation of allergenic proteins. This analysis extracts features directly from the protein sequences by using Discrete Fourier Transform (DFT) to build a classification model based on Support Vector Machines (SVM) for the allergenic proteins. The proposed predictive model yields a higher and more reliable accuracy than those of the existing methods. A new method is proposed for performing a multiple sequence alignment. For this method, DFT-based method is used to construct a new distance matrix in combination with multiple amino acid indices that were used to encode protein sequences into numerical sequences. Additionally, a new type of substitution matrix is proposed where the physicochemical similarities between any given amino acids is calculated. These similarities were calculated based on the 25 amino acids indices selected, where each one represents a unique biological protein feature. The proposed multiple sequence alignment method yields a better and more reliable alignment than the existing methods. In order to evaluate complex information that is generated as a result of DFT, Complex Informational Spectrum Analysis (CISA) is developed and presented. As the results show, when protein classes present similarities or differences according to the Common Frequency Peak (CFP) in specific amino acid indices, then it is probable that these classes are related to the protein feature that the specific amino acid represents. By using only the absolute spectrum in the analysis of protein sequences using the informational spectrum analysis is proven to be insufficient, as biologically related features can appear individually either in the real or the imaginary spectrum. This is successfully demonstrated over the analysis of influenza neuraminidase protein sequences. Upon identification of a new protein, it is important to single out amino acid responsible for the structural and functional classification of the protein, as well as the amino acids contributing to the protein's specific biological characterisation. In this work, a novel approach is presented to identify and quantify the relationship between individual amino acids and the protein. This is successfully demonstrated over the analysis of influenza neuraminidase protein sequences. Characterisation and identification problem of the Influenza A virus protein sequences is tackled through a Subgroup Discovery (SD) algorithm, which can provide ancillary knowledge to the experts. The main objective of the case study was to derive interpretable knowledge for the influenza A virus problem and to consequently better describe the relationships between subtypes of this virus. Finally, by using DFT-based sequence-driven features a Support Vector Machine (SVM)-based classification model was built and tested, that yields higher predictive accuracy than that of SD. The methods developed and presented in this study yield promising results and can be easily applied to proteomic fields.

Actuellement le polyéthylènimine (PEI) est l’agent de transfection transitoire le plus utilisé par l’industrie pharmaceutique pour la production de protéines recombinantes à grande échelle par les cellules de mammifères. Il permet la condensation de l’ADN plasmidique (ADNp) en formant spontanément des nanoparticules positives appelées polyplexes, lui procurant la possibilité de s’attacher sur la membrane cellulaire afin d’être internalisé, ainsi qu’une protection face aux nucléases intracellulaires. Cependant, alors que les polyplexes s’attachent sur la quasi-totalité des cellules seulement 5 à 10 % de l’ADNp internalisé atteint leur noyau, ce qui indique que la majorité des polyplexes ne participent pas à l’expression du transgène. Ceci contraste avec l’efficacité des vecteurs viraux où une seule particule virale par cellule peut être suffisante. Les virus ont évolués afin d’exploiter les voies d’internalisation et de routage cellulaire pour exprimer efficacement leur matériel génétique. Nous avons donc supposé que l’exploitation des voies d’internalisation et de routage cellulaire d’un récepteur pourrait, de façon similaire à plusieurs virus, permettre d’optimiser le processus de transfection en réduisant les quantités d’ADNp et d’agent de transfection nécessaires. Une alternative au PEI pour transfecter les cellules de mammifèreest l’utilisation de protéines possédant un domaine de liaison à l’ADNp. Toutefois, leur utilisation reste marginale à cause de la grande quantité requise pour atteindre l’expression du transgène. Dans cette étude, nous avons utilisé le système E/Kcoil afin de cibler un récepteur membranaire dans le but de délivrer l’ADNp dans des cellules de mammifères. Le Ecoil et le Kcoil sont des heptapeptides répétés qui peuvent interagir ensemble avec une grande affinité et spécificité afin de former des structures coiled-coil. Nous avons fusionné le Ecoil avec des protéines capables d’interagir avec l’ADNp et le Kcoil avec un récepteur membranaire que nous avons surexprimé dans les cellules HEK293 de manière stable. Nous avons découvert que la réduction de la sulfatation de la surface cellulaire permettait l’attachement ciblé sur les cellules par l’intermédiaire du système E/Kcoil. Nous démontrons dans cette étude comment utiliser le système E/Kcoil et une protéine interagissant avec l’ADNp pour délivrer un transgène de manière ciblée. Cette nouvelle méthode de transfection permet de réduire les quantités de protéines nécessaires pour l’expression du transgène.
Pharmaceutical industry often employs polyethylenimine (PEI) for large scale protein production processes by transient transfection of mammalian cells. PEI condenses plasmid DNA (pDNA) by spontaneously forming positive nanoparticles known as polyplexes. Condensed pDNA is favoured for cell surface binding, internalization and protection from intracellular nucleases. While most of the cells efficiently uptake polyplexes, only 5 to 10% of captured pDNA reaches the nucleus for transgene expression. This suggests that polyplexes are hampered in their ability to route and to translocate to the nucleus necessitating large amounts of polyplexes to achieve high expression levels. By contrast, many viruses can efficiently transduce cells with only one or a few viral genome copies. Viruses have evolved to exploit cellular internalization and routing properties to express their own genetic material. We hypothesized that less pDNA would be used in an optimized transfection process if we exploited the internalization and routing properties that viruses use. DNA binding proteins could be used as an alternative to PEI to transfect mammalian cells. However, their usage is marginal due to the large protein quantities required to bind pDNA for transgene expression. If less pDNA is used less binding protein is needed. In this study, we used the E/Kcoil system to target a membrane receptor to deliver pDNA in mammalian cells. The Ecoil and Kcoil are two repeated heptapeptides which interact with a high affinity and specificity to form coiled-coil structures. We fused the Ecoil with a recombinant pDNA-binding protein. The Kcoil was fused to a stably-expressed membrane receptor in HEK293 cells. We discovered that low sulfation of the cell surface reduced non-specific binding of the pDNA:protein complex and permitted targeted binding via the E/Kcoil interaction. We demonstrate how to use recombinant pDNA-binding protein and the E/Kcoil system for targeted transgene delivery. This newly developed system provides a new transfection method, with reduced pDNA-binding protein quantities needed to achieve transgene expression.

The hallmark of the human immunodeficiency virus (HIV) patient with a cluster of differentiation 4 (CD4) T lymphocyte count below 200 is the development of opportunistic infections. Although the use of antiretroviral therapy (ART) has decreased the incidence of these infections, they continue to be a major case of morbidity and mortality in the patient with HIV. These infections can be respiratory in nature and present with cough or shortness of breath: Pneumocystis pneumonia (PCP), tuberculosis (TB), aspergillosis, and coccidioidomycosis. Neurological infections, which can present with change in mental status, include toxoplasmosis encephalitis (TE), meningoencephalitis, John Cunningham (JC) virus, and progressive multifocal leukoencephalopathy (PML). Gastrointestinal infections, such as Cryptosporidium, present with abdominal pain and diarrhea. Viral changes can result from cytomegalovirus retinitis. Fever or nonspecific symptoms can result from disseminated Mycobacterium Avium complex disease, histoplasmosis, bartonellosis, and cytomegalovirus.

T cell activation plays a central role in inflammation, autoimmune diseases and cancer. Cancer immunotherapies, such as immune checkpoint inhibitor, bi-specific antibody, chimeric antigen receptor T (CAR T) cell, and adoptive tumor-infiltrating lymphocyte (TIL) therapies require the characterization and monitoring of T cell activation. Here we describe a novel, multiplex immune assay platform based on high-throughput flow cytometry technology and advanced computational algorithms for data analysis. The assay simultaneously measures T cell dynamics including phenotype, time-dependent expression of activation markers, secreted effector cytokines, and proliferation. The assay screened a kinase chemogenomic library and identified 25 kinase inhibitors with distinct inhibition profiles on early (CD69) and late (CD25) activation markers and the cytokines IFNγ and TNFα. We identified 5 kinase inhibitors with dissimilar effects on CD69 and CD25 expression, and a cluster of total 4 MEK1//2 inhibitors with similar activation profiles. The screening revealed 3 kinase inhibitors for PKC, IKK2, and MEK1/2 respectively, all with a phenotypic signature similar to ruxolitinib, a Jak1/2 inhibitor used to treat myelofibrosis disease. These results suggest this multiplexed assay platform, combined with a chemogenomic library screening, may be used as primary screen for phenotypic or target-based drug discovery, target identification, and potential drug repositioning.

TGFβ influences cell growth and differentiation in a variety of normal and transformed cells and was recently shown to have growth inhibitory activity toward cultured endothelium. The fibrinolytic activity of endothelial cells changes dramatically with their growth state. Experiments were thus performed to determine the effect of TGFβ on the fibrinolytic system of these cells. Confluent BAEs were incubated with increasing concentrations of purified human or porcine TGFβ for various times in the presence or absence of serum. Conditioned medium (CM) and extracellular matrix (ECM) were prepared and assayed for tissue-type plasminogen activator (tPA), urokinase-type PA (uPA) and PA inhibitor-1 (PAI-1) by immunological methods and by fibrin and reverse fibrin autography. TGFβ induced a dose and time dependent increase in PAI-1 antigen and activity. The half-maximal effect was observed at 0.5 ng/ml while the maximal effect was at 2-5 ng/ml and resulted in 100-fold increase of PAI-1 in CM and at least a 20-fold increase in ECM. 35S-methionine labeling experiments indicated that these effects (1) could be detected in CM within 3-4 hrs, (2) resulted from an increased rate of synthesis of PAI-1, (3) were also observed following transient exposure (1-2 h) of the cells to TGFβ, and (4) were relatively specific for PAI-1. TGFβ treated cells had normal amounts of tPA activity but greatly reduced levels of uPA activity. Human serum had a synergistic effect on PAI-1 production by these cells, but neither epidermal growth factor nor platelet derived growth factor altered their response to TGFβ. TGFβ also stimulated PAI-1 production by cultured human umbilical vein endothelial cells. The release of TGFβ by platelets may thus suppress the fibrinolytic system of the vascular wall.

Several serine protease inhibitorsof plasma inhibit the activated coagulation enzymes but only antithrombin III(AT-III)and heparin cofactor II (HC-II) are implicated in the pathogenesisof the familial thrombosis. Since thefirst publication (1965) many thrombophilic families with reduced AT-III synthesis have been investigated. These studies have proved that the disorder is associated with a high risk forvenous thrombosis and the inheritanceis autosomal dominant. The AT-III activity in the plasma of the affected patients is about 50% of the normalvalue.In recent years the heterogeneity of the inherited AT-III deficiency has been verified. The various AT-III abnormalities are not mere interestingrarities but they provide naturally occurring models for the solution of theoretical problems such as the function of AT-III molecule, the physiological significance of heparin, etc. Furthermore, the clinical manifestationof the particular variants greatly differs from symptomless abnormality tosevere thrombotic cases.In the majority of cases, reduced functional activity is accompanied with a parallel decrease of antigen concentration of AT-III. This is the characteristic feature of the quantitative or Type I ("classical")AT-III deficiency. By means of crossed immunoelectrophoresis, electro-focussing and recombinant DNA techniques the heterogeneity of this group has been established. In one subgroup (Type la) AT-III molecules are normal as regards their biochemical characteristics. In Type lb, subnormal AT-IIIquantity is accompanied with decreased heparin affinity. Differentiation of these subgroups has practical consequences: therapeutic concentrations of heparin apparently does not decrease AT-III level in the plasma of patients with Type lb AT-III deficiency.The other main form is the qualitative deficiency of AT-III (Type II) which is characterised by reduced functional activity at normal antigen concentration. In general, two populations of AT-III molecules can be detected in the blood of these patients: a normal and an abnormal one. Up till now at least 24 different abnormalities were found and designatedwith toponymes. These disorders can be classified with relatively simple laboratory methods such as functional anti-IIaXaFirstDepartment of Medicine, Postgraduate Medical University, Budapest, Hungary.arin cofactor activity, crossed immunoelectrophoresiswith and without heparin, heparin-affinity chromatography. Type na is characterised by profound structural changes of the molecule,variably: reflected in reduced inactivation of F Ha and F Xa, abnormal heparin-AT-III reaction and aberrant immunochemical structure Seven different abnormalities fall into this group (Budapest I, Tokyo, MalmÖ, Chicago, Milano, Trento and Northwick Park). The last three abnormalities are very similar.In Type lib an isolated defect of protease inactivation can be detected and an isolated disturbance of the active centre ofthe molecule is assumed.Until now 6 apparently different variants belonging to this group have been described. (Aalborg, Vicenza, Denver, Hvidovre, Charleville, Milano 2.) Type lie abnormality is characterised by an isolated defect of the heparin-AT-III reaction. In these cases a disturbance of the heparin binding site(s) is assumed. Eleven families with this type of abnormality have been recorded (Ann Arbor, Basel, Paris 1 and 2, Toyama* Tours, Padova I and 2, Algers, Fontainebleu and Budapest 2). This subgroup is heterogeneous in respect ofheparin affinity: in the majority of cases the abnormal AT-III molecules have no heparin affinity at all while in rare cases (such as Basel, Budapest 2) they have reduced affinity.TheType lie AT-III deficiency has several distinctive features compared with the other subtypesJClinically, the thromboembolic complications are rare: in 4 families thrombosis has notoccurred at all. Only one member in each of 4 other families had thrombosis. In 3 families homozygous patients suffered severe thrombosis in young age and/or in unusual localisations (intraarterial, intracardiac, etc.) butthe other heterozygous members were free of thrombotic symptoms. No increased intravascular coagulation could be detected in Type lie heterozygous cases incontrast to the "classical" AT-III deficiency.These observations suggest a different mechanism and clinical manifestation of the deficiency of progressiveserine protease inactivation and of heparin cofactor activity. In case of progressive inactivation, reduction of 50% of the activity predisposes mainly to venous thrombosis as a consequence of the hypercoagulability of theblood. The isolated reduction of heparin cofactor activity seems to bringabout thrombosis in any part of the vascular system, but only if this reduction is as severe as that of the coagulant factors in case of coagulopathies.In accordance with this finding, rare cases of HCII deficiency give rise to thrombosis in both the arteries and the veins. Heparin cofactor activities may play an important role in the antithrombotic mechanism along theendothelial surface of the whole vascular system.