The immune system as drug target

The immune system is perhaps the largest yet most diffuse and distributed somatic system in vertebrates. It plays vital roles in fighting infection and in the homeostatic control of chronic disease. As such, the immune system in both pathological and healthy states is a prime target for therapeutic interventions by drugs-both small-molecule and biologic. Comprising both the innate and adaptive immune systems, human immunity is awash with potential unexploited molecular targets. Key examples include the pattern recognition receptors of the innate immune system and the major histocompatibility complex of the adaptive immune system. Moreover, the immune system is also the source of many current and, hopefully, future drugs, of which the prime example is the monoclonal antibody, the most exciting and profitable type of present-day drug moiety. This brief review explores the identity and synergies of the hierarchy of drug targets represented by the human immune system, with particular emphasis on the emerging paradigm of systems pharmacology. © the authors, publisher and licensee Libertas Academica Limited.

Specific binding of the mammalian high mobility group protein AT-hook 2 to the minor groove of AT -rich DNAs: Thermodynamic and specificity studies

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a small transcriptional factor involved in cell development and oncogenesis. It contains three "AT-hook" DNA binding domains, which specifically recognize the minor groove of AT-rich DNA sequences. It also has an acidic C-terminal motif. Previous studies showed that HMGA2 mediates all its biological effects through interactions with AT-rich DNA sequences in the promoter regions. In this dissertation, I used a variety of biochemical and biophysical methods to examine the physical properties of HMGA2 and to further investigate HMGA2's interactions with AT-rich DNA sequences. The following are three avenues perused in this study: (1) due to the asymmetrical charge distribution of HMGA2, I have developed a rapid procedure to purify HMGA2 in the milligram range. Preparation of large amounts of HMGA2 makes biophysical studies possible; (2) Since HMGA2 binds to different AT-rich sequences in the promoter regions, I used a combination of isothermal titration calorimetry (ITC) and DNA UV melting experiment to characterize interactions of HMGA2 with poly(dA-dT) 2 and poly(dA)poly(dT). My results demonstrated that (i) each HMGA2 molecule binds to 15 AT bp; (ii) HMGA2 binds to both AT DNAs with very high affinity. However, the binding reaction of HMGA2 to poly(dA-dT) 2 is enthalpy-driven and the binding reaction of HMGA2 with poly(dA)poly(dT) is entropy-driven; (iii) the binding reactions are strongly depended on salt concentrations; (3) Previous studies showed that HMGA2 may have sequence specificity. In this study, I used a PCR-based SELEX procedure to examine the DNA binding specificity of HMGA2. Two consensus sequences for HMGA2 have been identified: 5'-ATATTCGCGAWWATT-3' and 5'-ATATTGCGCAWWATT-3', where W represents A or T. These consensus sequences have a unique feature: the first five base pairs are AT-rich, the middle four to five base pairs are GC-rich, and the last five to six base pairs are AT-rich. All three segments are critical for high affinity binding. Replacing either one of the AT-rich sequences to a non-AT-rich sequence causes at least 100-fold decrease in the binding affinity. Intriguingly, if the GC-segment is substituted by an AT-rich segment, the binding affinity of HMGA2 is reduced approximately 5-fold. Identification of the consensus sequences for HMGA2 represents an important step towards finding its binding sites within the genome.

Regulatory factors controlling muscle mass : competition between innate immune function and anabolic signals in regulation of atrogin-1 in Atlantic salmon

The research was supported by an industrial PhD studentship between University of Aberdeen and by BioMar Ltd., for Z. Heidari.

Regulatory factors controlling muscle mass : competition between innate immune function and anabolic signals in regulation of atrogin-1 in Atlantic salmon

The research was supported by an industrial PhD studentship between University of Aberdeen and by BioMar Ltd., for Z. Heidari.

Regulatory factors controlling muscle mass : competition between innate immune function and anabolic signals in regulation of atrogin-1 in Atlantic salmon

The research was supported by an industrial PhD studentship between University of Aberdeen and by BioMar Ltd., for Z. Heidari.

(Table 1, page 126) Concentration of elements in Fe and Mn rich pisoliths (nodules) from Lake Storsjoen, South Norway

A detailed description of the ores of Lake Storsjoen was given by Vogt J. H. L., 1915 who pointed out that the ores may be divided into two principal types; first, iron ore with 2% or less of manganese (ex: Ertemalm), and, second, ores with manganese contents of up to 30% (ex: Korinter). The iron-rich ore sometimes occurs as a conglomerate embedded in manganese-rich ores, clearly demonstrating that two distinctly different precipitates are involved. In the iron-rich ore, a concentric structure is common of which light brown layers of loose, almost dusty material alternate with hard and brittle black layers, the thickness of each being 0.5 mm or less. The analyses presented in this paper seem to demonstrate that the composition of the sedimentary ores of Lake Storsjden could result from fluctuations in the composition of ground waters feeding the lake.

(Table 1, page 1169) Chemical composition of land (fossil) manganese deposits rich in todorokite

Chemical, x-ray and other data are given for todorokite, (Mn, Mg, Ca, Ba, Na, K)2.Mn5O12.3H2O, from Charco Redondo, Cuba, Farragudo, Portugal, and Hüttenberg, Austria. Additional localities at Romanèche, France, Saipan Island, Bahia, Brazil and Sterling Hill, New Jersey, are noted. Delatorreite of Simon and Straczek (1958) is identical with todorokite.

Extracellular cathepsin S and intracellular caspase 1 activation are surrogate biomarkers of particulate-induced lysosomal disruption in macrophages

BACKGROUND: Particulate matter has been shown to stimulate the innate immune system and induce acute inflammation. Therefore, while nanotechnology has the potential to provide therapeutic formulations with improved efficacy, there are concerns such pharmaceutical preparations could induce unwanted inflammatory side effects. Accordingly, we aim to examine the utility of using the proteolytic activity signatures of cysteine proteases, caspase 1 and cathepsin S (CTSS), as biomarkers to assess particulate-induced inflammation.

METHODS: Primary peritoneal macrophages and bone marrow-derived macrophages from C57BL/6 mice and ctss(-/-) mice were exposed to micro- and nanoparticulates and also the lysosomotropic agent, L-leucyl-L-leucine methyl ester (LLOME). ELISA and immunoblot analyses were used to measure the IL-1β response in cells, generated by lysosomal rupture. Affinity-binding probes (ABPs), which irreversibly bind to the active site thiol of cysteine proteases, were then used to detect active caspase 1 and CTSS following lysosomal rupture. Reporter substrates were also used to quantify the proteolytic activity of these enzymes, as measured by substrate turnover.

RESULTS: We demonstrate that exposure to silica, alum and polystyrene particulates induces IL-1β release from macrophages, through lysosomal destabilization. IL-1β secretion positively correlated with an increase in the proteolytic activity signatures of intracellular caspase 1 and extracellular CTSS, which were detected using ABPs and reporter substrates. Interestingly IL-1β release was significantly reduced in primary macrophages from ctss(-/-) mice.

CONCLUSIONS: This study supports the emerging significance of CTSS as a regulator of the innate immune response, highlighting its role in regulating IL-1β release. Crucially, the results demonstrate the utility of intracellular caspase 1 and extracellular CTSS proteolytic activities as surrogate biomarkers of lysosomal rupture and acute inflammation. In the future, activity-based detection of these enzymes may prove useful for the real-time assessment of particle-induced inflammation and toxicity assessment during the development of nanotherapeutics.

Reactions of atomic and molecular ions with acetone, 1,1,1- trifluoroacetone, and hexafluoroacetone: An investigation of the effects of molecular structure on the dynamics and kinetics of ion-molecule reactions

A selected ion flow tube study of the reactions of a series of gas-phase atomic cations (S⁺, Xe⁺, O⁺, Kr⁺, N⁺, Ar⁺ and Ne⁺) and molecular ions (SF _n⁺ (n = 1-5), CF_n⁺ (n = 1-3), CF₂Cl⁺, H₃O⁺, NO⁺, N ₂O⁺, CO₂⁺, CO⁺, and N₂⁺) spanning a large range of recombination energies (6.3-21.6 eV), with acetone, 1,1,1-trifluoroacetone, and hexafluoroacetone has been undertaken with the objective of exploring the nature of the reaction ion chemistry as the methyl groups in acetone are substituted for CF₃. The reaction rate coefficients and product ion branching ratios for all 66 reactions, measured at 298 K, are reported. The experimental reaction rate coefficients are compared to theoretically calculated collisional values. Several distinct reaction processes were observed among the large number of reactions studied, including charge transfer (non-dissociative and dissociative), abstraction, ion-molecule associations and, in the case of the reactions involving the reagent ion H₃O⁺, proton transfer. 

Circle of Friends a sampling of the rich and prolific repertoires of Johannes Brahms and Felix Mendelssohn December 1, 2013

Concert Program

Study and development of UV photodetectors envisaging COMPASS RICH-1 upgrade and applications

COMPASS is an experiment at CERN’s SPS whose goal is to study hadron structure and spectroscopy. The experiment includes a wide acceptance RICH detector, operating since 2001 and subject to a major upgrade of the central region of its photodetectors in 2006. The remaining 75% of the photodetection area are still using MWPCs from the original design, who suffer from limitations in gain due to aging of the photocathodes from ion bombardment and due to ion-induced instabilities. Besides the mentioned limitations, the increased luminosity conditions expected for the upcoming years of the experiment make an upgrade to the remaining detectors pertinent. This upgrade should be accomplished in 2016, using hybrid detectors composed of ThGEMs and MICROMEGAS. This work presents the study, development and characterization of gaseous photon detectors envisaging the foreseen upgrade, and the progress in production and evaluation techniques necessary to reach increasingly larger area detectors with the performances required. It includes reports on the studies performed under particle beam environment of such detectors. MPGD structures can also be used in a variety of other applications, of which nuclear medical imaging is a notorious example. This work includes, additionally, the initial steps in simulating, assembling and characterizing a prototype of a gaseous detector for application as a Compton Camera.

Genome Wide Association Studies of Phagocytosis and the Cellular Immune Response in Drosophila melanogaster.

Phagocytosis of bacteria by specialized blood cells, known as hemocytes, is a vital component of Drosophila cellular immunity. To identify novel genes that mediate the cellular response to bacteria, we conducted three separate genetic screens using the Drosophila Genetic Reference Panel (DGRP). Adult DGRP lines were tested for the ability of their hemocytes to phagocytose the Gram-positive bacteria Staphylococcus aureus or the Gram-negative bacteria Escherichia coli. The DGRP lines were also screened for the ability of their hemocytes to clear S. aureus infection through the process of phagosome maturation. Genome-wide association analyses were performed to identify potentially relevant single nucleotide polymorphisms (SNPs) associated with the cellular immune phenotypes. The S. aureus phagosome maturation screen identified SNPs near or in 528 candidate genes, many of which have no known role in immunity. Three genes, dpr10, fred, and CG42673, were identified whose loss-of-function in blood cells significantly impaired the innate immune response to S. aureus. The DGRP S. aureus screens identified variants in the gene, Ataxin 2 Binding Protein-1 (A2bp1) as important for the cellular immune response to S. aureus. A2bp1 belongs to the highly conserved Fox-1 family of RNA-binding proteins. Genetic studies revealed that A2bp1 transcript levels must be tightly controlled for hemocytes to successfully phagocytose S. aureus. The transcriptome of infected and uninfected hemocytes from wild type and A2bp1 mutant flies was analyzed and it was found that A2bp1 negatively regulates the expression of the Immunoglobulin-superfamily member Down syndrome adhesion molecule 4 (Dscam4). Silencing of A2bp1 and Dscam4 in hemocytes rescues the fly’s immune response to S. aureus indicating that Dscam4 negatively regulates S. aureus phagocytosis. Overall, we present an examination of the cellular immune response to bacteria with the aim of identifying and characterizing roles for novel mediators of innate immunity in Drosophila. By screening panel of lines in which all genetic variants are known, we successfully identified a large set of candidate genes that could provide a basis for future studies of Drosophila cellular immunity. Finally, we describe a novel, immune-specific role for the highly conserved Fox-1 family member, A2bp1.

An Immune Inspired Approach to Anomaly Detection

The immune system provides a rich metaphor for computer security: anomaly detection that works in nature should work for machines. However, early artificial immune system approaches for computer security had only limited success. Arguably, this was due to these artificial systems being based on too simplistic a view of the immune system. We present here a second generation artificial immune system for process anomaly detection. It improves on earlier systems by having different artificial cell types that process information. Following detailed information about how to build such second generation systems, we find that communication between cells types is key to performance. Through realistic testing and validation we show that second generation artificial immune systems are capable of anomaly detection beyond generic system policies. The paper concludes with a discussion and outline of the next steps in this exciting area of computer security.

Diet, microbes and gut immune responses in the pathogenesis of experimental type 1 diabetes

Type 1diabetes (T1D) is an autoimmune disease, which is influenced by a variety of environmental factors including diet and microbes. These factors affect the homeostasis and the immune system of the gut. This thesis explored the altered regulation of the immune system and the development of diabetes in non-obese diabetic (NOD) mice. Inflammation in the entire intestine of diabetes-prone NOD mice was studied using a novel ex-vivo imaging system of reactive oxygen and nitrogen species (RONS), in relation to two feeding regimens. In parallel, gut barrier integrity and intestinal T-cell activation were assessed. Extra-intestinal manifestations of inflammation and decreased barrier integrity were sought for by studying peritoneal leukocytes. In addition, the role of pectin and xylan as dietary factors involved in diabetes development in NOD mice was explored. NOD mice showed expression of RONS especially in the distal small intestine, which coincided with T-cell activation and increased permeability to macromolecules. The introduction of a casein hydrolysate (hydrolysed milk protein) diet reduced these phenomena, altered the gut microbiota and reduced the incidence of T1D. Extra-intestinally, macrophages appeared in large numbers in the peritoneum of NOD mice after weaning. Peritoneal macrophages (PM) expressed high levels of interleukin-1 receptor associated kinase M (IRAK-M), which was indicative of exposure to ligands of toll-like receptor 4 (TLR-4) such as bacterial lipopolysaccharide (LPS). Intraperitoneal LPS injections activated T cells in the pancreatic lymph nodes (PaLN) and thus, therefore potentially could activate islet-specific T cells. Addition of pectin and xylan to an otherwise diabetes-retarding semisynthetic diet affected microbial colonization of newly-weaned NOD mice, disturbed gut homeostasis and promoted diabetes development. These results help us to understand how diet and microbiota impact the regulation of the gut immune system in a way that might promote T1D in NOD mice.

Characterization of the immune response induced by a commercially available inactivated bluetongue virus serotype 1 vaccine in sheep

The protective immune response generated by a commercial monovalent inactivated vaccine against bluetongue virus serotype 1 (BTV1) was studied. Five sheep were vaccinated, boost-vaccinated, and then challenged against BTV1 ALG/2006. RT-PCR did not detect viremia at any time during the experiment. Except a temperature increase observed after the initial and boost vaccinations, no clinical signs or lesions were observed. A specific and protective antibody response checked by ELISA was induced after vaccination and boost vaccination. This specific antibody response was associated with a significant increase in B lymphocytes confirmed by flow cytometry, while significant increases were not observed in T lymphocyte subpopulations (CD4(+), CD8(+), and WC1(+)), CD25(+) regulatory cells, or CD14(+) monocytes. After challenge with BTV1, the antibody response was much higher than during the boost vaccination period, and it was associated with a significant increase in B lymphocytes, CD14(+) monocytes, CD25(+) regulatory cells, and CD8(+) cytotoxic T lymphocytes.