Inhibition Of Lysis Research Articles

Inhibition of NKp30- and 2B4-mediated NK cell activation by evolutionary different human and bovine CEACAM1 receptors.

Carcinoembryonicantigen-related cell adhesion molecule 1 (CEACAM1) is a receptor involved in the regulation of NK-cell function. In most species, the CEACAM1 cytoplasmic tail possesses a membrane-proximal ITIM paired with a membrane-distal immunoreceptor tyrosine-based switch motif (ITSM) signaling motif. Human CEACAM1 has phylogenetically relatively recently acquired a second ITIM instead of the ITSM and was shown to inhibit NKG2D-mediated NK-cell activation. Here, we compare the function of bovine and human CEACAM1. We show that in addition to NKG2D, human CEACAM1 can inhibit NK-cell activation via NKp30 or 2B4. Bovine CEACAM1, possessing an ITIM and an ITSM signaling motif, is also inhibitory. However, bovine CEACAM1 inhibition of NKp30-mediated lysis is less pronounced compared with its human counterpart. Bovine CEACAM1 inhibition is dependent on the membrane-proximal ITIM and our data suggest that also the membrane distal ITSM motif contributes to inhibitory signaling. Biochemically, human and bovine CEACAM1 can recruit the phosphatases SHP-1 and SHP-2 after receptor phosphorylation to a similar extend. Bovine CEACAM1 can additionally recruit the adapter molecule Ewing's sarcoma virus-activated transcript-2 (EAT-2), but not SLAM-associated protein (SAP). Taken together, we show that although human and bovine CEACAM1 are differentially equipped with ITIM and ITSM motifs, both receptors can inhibit NKp30 and 2B4 activation of NK cells.

Interferon-γ-induced activation of JAK1 and JAK2 suppresses tumor cell susceptibility to NK cells through upregulation of PD-L1 expression

Inhibition of JAK1 or JAK2 in human tumor cells was previously shown to increase susceptibility of these cells to NK cell lysis. In the present study, we examined the cellular mechanisms that mediate this effect in hematopoietic tumor cell lines and primary tumor cells. Incubation of tumor cells with supernatant from activated NK cells or interferon-gamma (IFNγ)-induced activation of pSTAT1 and increased expression of PD-L1 without altering expression of other activating or inhibitory NK cell ligands. These functional effects were blocked by chemical JAK inhibition or shRNAs targeting JAK1, JAK2 or STAT1. Inhibition of IFNγ signaling also prevented the upregulation of PD-L1 and blocking PD-L1 resulted in increased tumor lysis by NK cells. These results show that NK cell activation and secretion of IFNγ results in activation of JAK1, JAK2 and STAT1 in tumor cells, resulting in rapid up-regulation of PD-L1 expression. Increased expression of PD-L1 results in increased resistance to NK cell lysis. Blockade of JAK pathway activation prevents increased PD-L1 expression resulting in increased susceptibility of tumor cells to NK cell activity. These observations suggest that JAK pathway inhibitors as well as PD-1 and PD-L1 antibodies may work synergistically with other immune therapies by preventing IFN-induced inhibition of NK cell-mediated tumor cell lysis.

A single amino acid change in inhibitory killer cell Ig-like receptor results in constitutive receptor self-association and phosphorylation.

Signaling by immunoreceptors is often initiated by phosphorylation of cytosolic tyrosines, which then recruit effector molecules. In the case of MHC class I-specific inhibitory receptors, phosphorylation of cytosolic tyrosine residues within ITIMs results in recruitment of a protein tyrosine phosphatase that blocks activation signals. Recent work showed that signaling by an HLA-C-specific killer cell Ig-like receptor (KIR) is independent of signaling by activation receptors. It is not known how ITIM phosphorylation is initiated and regulated. In this article, we show that substitution of His-36 in the first Ig domain of KIR2DL1 with alanine (KIR2DL1-H36A) resulted in constitutive KIR2DL1 self-association and phosphorylation, as well as recruitment of tyrosine phosphatase SHP-1. Furthermore, substitution of His-36 with a similar bulky amino acid, phenylalanine, maintained the receptor in its unphosphorylated state, suggesting that steric hindrance by the His-36 side chain prevents constitutive KIR2DL1 self-association and ITIM phosphorylation. The equally strong phosphorylation of KIR2DL1 and KIR2DL1-H36A after inhibition of tyrosine phosphatase by pervanadate suggested that KIR2DL1-H36A is selectively protected from dephosphorylation. We propose that KIR phosphorylation is controlled by the accessibility of ITIM to tyrosine phosphatases and that KIR binding to HLA-C must override the hindrance that His-36 puts on KIR2DL1 self-association. Expression of KIR2DL1-H36A on NK cells led to stronger inhibition of lysis of HLA-C(+) target cells than did expression of wild-type KIR2DL1. These results revealed that ITIM phosphorylation is controlled by self-association of KIR and that His-36 serves as a gatekeeper to prevent unregulated signaling through KIR2DL1.

Teixobactin: a novel antibiotic in treatment of gram positive bacterial infections.

Sir, Antimicrobial resistance have emerged as a serious concern worldwide. In spite of the best effort in developing novel antimicrobial agents employing advanced techniques like computer-aided drug design, proteomics, genomics, combinatorial synthesis and high- throughput screening, there is lack of newer antibiotics in the process of development [1]. Environmental microbiota has been an essential source of several antimicrobial agents. In a press release on 7th January, 2015, NovoBiotic Pharmaceuticals announced the discovery of teixobactin, a novel antibiotic from a previously uncultured and undescribed soil –bacteria belonging to β-proteobacteria provisionally named Eleftheria terrae, using isolation chip (iChip) method [1,2]. iChip uses an assembly of three (central, top and bottom plates) flat hydrophobic plastic polyoxymethylene plates containing multiple through-holes and polycarbonate membranes to compose an array of miniature diffusion chambers [3]. Each chamber is likely to capture a single cell when the assembly is dipped into a suitably diluted bacterial mixture. This novel method is being utilized to isolate and characterize microbes from unexplored environmental niche and the discovery of teixobactin could be considered as its first success [1]. Teixobactin is a cyclic depsipeptide containing an unusual amino acid enduracididine [1]. It is the first new class of antibiotic which acts on unique targets in cell wall synthesis pathway. It binds to a highly conserved non-peptide motiff of peptidoglycan precursor (lipid II) and teichoic acid precursor (lipid III), resulting in inhibition of cell wall synthesis and subsequent lysis. It has shown excellent activity against a wide range of gram positive bacteria, including multidrug resistant organisms such as Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin Intermediate S. aureus (VISA), Vancomycin – resistant enterococci (VRE), Clostridium difficile, Streptococcus pneumoniae and Mycobacterium tuberculosis [1]. However, it has inherent limitation against gram negative bacteria pertaining to their cell wall composition. Unlike other cell wall inhibitor antibiotics, it does not act on protein targets which may readily develop mutational resistance on continuous drug exposure. In fact, the absence of resistant mutants of Staphylococcus aureus and M. tuberculosis after prolonged exposure of teixobactin in sublethal concentration, have generated immense interest on its development and introduction for clinical use [1,2]. It has been effective in sepsis, pneumonia and soft tissue infection in animal model with a very low PD50 (50% protective dose) for MRSA in comparison to Vancomycin [1]. Since it has no deleterious action against mammalian cells due to lack of target sites and not been found to cause serious toxicity, haemolysis or DNA damage, it is likely to have wide therapeutic window. However, it has to prove its safety in human clinical trials. Although it has been expectantly described as ‘game-changing’ antibiotic because of its novel site of action precluding development of mutational resistance [4], it may not prevent resistance due to impermeable thickened cell wall which curtails its access to lipid II and lipid III, particularly in case of injudicious clinical use due to increasing drug promotion and marketing. In conclusion, the isolation of teixobactin from soil using iChip not only marks the development of a new antibiotic class with low risk of acquiring resistance, but also opens up infinite possibilities to discover useful molecules from unexplored strata of microbial kingdom.

Novel Small-Molecule Inhibitors Targeting Complement Factor D for Therapy of Paroxysmal Nocturnal Hemoglobinuria

INTRODUCTIONThe complement system is a pivotal player in multiple hematological conditions that include paroxysmal nocturnal hemoglobinuria (PNH). The current standard of care for PNH is intravenous infusion of eculizumab, a humanized monoclonal antibody that targets the terminal complement protein C5 and thereby efficiently impairs intravascular hemolysis. However, a significant fraction of PNH patients in clinical practice respond incompletely to eculizumab due to extravascular hemolysis by immune cells as eculizumab does not prevent erythrocyte opsonization. Additionally, a non-responsive sub-population has been identified with a rare genetic polymorphism in C5 that renders the variant incapable of binding eculizumab. Therefore, an unmet medical need remains for regimens that improve efficacy and that can be administered orally. To achieve this goal, we initiated a discovery program for small molecule inhibitors of complement factor D, a serine protease that is the rate limiting enzyme of the alternative complement pathway. We have obtained high resolution (1.5 Å) crystal structure of inhibitor-bound factor D. Through structure-guided optimization, we have discovered novel inhibitors of factor D that possess high potency and specificity as well as pharmacokinetic properties suitable for oral administration. Herein, we present a biochemical characterization of lead compounds and studies of their activities in various complement-mediated processes. METHODSThe binding affinity to factor D was determined by surface plasmon resonance (Biacore) analysis. The inhibitory effect on factor D protease was evaluated biochemically using both a nonspecific synthetic substrate and a natural substrate consisting of C3b and the complement factor B. The alternative pathway (AP) hemolytic assay was conducted with unsensitized rabbit erythrocytes, 8% human or non-human primate serum, and 10 mM MgEGTA. The classical pathway (CP) hemolytic assay was conducted with antibody-sensitized sheep erythrocytes and 0.5% human serum. To assess inhibition of CP activation through the AP-dependent amplification loop, inhibition of terminal complement complex (TCC) formation by 50% human serum was quantitated following activation by the CP-specific activator HAIGG. Inhibition of complement opsonization and lysis of PNH-like erythrocytes exposed to acidified serum (Ham test) was assessed ex vivo. Finally, selected lead compounds were administered to non-human primates to evaluate the efficacy of factor D inhibitors for blockade of alternative pathway-mediated events. RESULTSLead compounds obtained through optimization bound to factor D with Kd values below 1 nM. Tight binding to factor D of the compounds resulted in potent inhibition of factor D proteolytic activity assessed using a synthetic nonspecific substrate as well as a natural substrate. When evaluated in an AP hemolytic assay with human serum, the most potent compounds inhibited cell lysis with EC50 values below 10 nM. Similar inhibitory activity was observed when compounds were assessed in AP hemolytic assays with non-human primate serum, but not with rodent serum. The compounds showed no activity in a CP hemolytic assay using highly diluted human serum and antibody-coated erythrocytes, indicating specificity for factor D. When evaluated in the presence of 50% human serum and with TCC formation as an endpoint, however, the compounds blocked approximately 75% of TCC formation upon CP activation, a consequence of blockage of the AP-dependent amplification loop. Furthermore, the compounds prevented both opsonisation and lysis of PNH-like cells in the Ham test. Finally, when selected compounds were administered to non-human primates, potent inhibition of TCC formation following ex vivo AP activation was observed with EC50 values below 50 nM. CONCLUSIONSHighly potent and specific factor D inhibitors that block both the terminal pathway and opsonization were discovered using structure-guided design. These orally deliverable inhibitors hold promise as a novel therapeutic approach for treatment of complement-mediated hematological conditions including PNH. DisclosuresMorgan:Swedish Orphan Biovitrum: Consultancy, Research Funding; Glaxo Smith Kline: Consultancy; Achillion Pharmaceuticals: Consultancy, Research Funding. Thanassi:Achillion Pharmaceuticals: Employment. Podos:Achillion Pharmaceuticals : Employment. Phadke:Achillion Pharmaceuticals: Employment. Gadhachanda:Achillion Pharmaceuticals: Employment. Pais:Achillion Pharmaceuticals: Employment. Hashimoto:Achillion Pharmaceuticals: Employment. Wang:Achillion Pharmaceuticals: Employment. Chen:Achillion Pharmaceuticals: Employment. Wang:Achillion Pharmaceuticals: Employment. Agarwal:Achillion Pharmaceuticals: Employment. Deshpande:Achillion Pharmaceuticals: Employment. Huang:Achillion Pharmaceuticals: Employment. Wiles:Achillion Pharmaceuticals: Employment. Huang:Achillion Pharmaceuticals: Employment.

Preclinical Evaluation of Orally Bioavailable Small-Molecule Inhibitors of Complement Factor D As a Potential Treatment for Paroxysmal Nocturnal Hemoglobinuria

BACKGROUNDComplement factor D, a serine protease, plays an essential role in the activation of the alternative complement pathway and provides important amplification of the classical and lectin complement pathways. Cleavage of factor B by factor D generates C3 convertase that leads to opsonization of targeted surfaces with complement activation fragments and to the formation of the terminal complement complex (TCC); both events lead to cell lysis. Complement dysregulation underlies multiple hematological disorders including paroxysmal nocturnal hemoglobinuria (PNH), which is characterized by complement-mediated lysis of clonal populations of erythrocytes that lack glycophosphatidylinositol-anchored complement regulators. The current treatment for PNH is intravenous infusion of the anti-C5 monoclonal antibody eculizumab. Although eculizumab lessens intravascular hemolysis, it does not prevent opsonization of erythrocytes and subsequent extravascular hemolysis by immune cells. In contrast, factor D inhibitors are expected to inhibit both terminal complement pathway activation as well as opsonization and should, therefore, be well-positioned to potentially serve this unmet medical need. Herein, we present the preclinical evaluation of our small-molecule inhibitors of factor D including potency, off-target activities, metabolism, and pharmacokinetic properties.METHODS & RESULTS: Initial inhibitors were discovered through laboratory and virtual screening efforts. A high-resolution (1.5 Å) X-ray structure of an early proprietary inhibitor co-crystallized with factor D aided our optimization campaign that culminated in compounds with IC50 values below 100 nM in biochemical protease assays using natural and non-specific substrates. The potent inhibitory effect of the compounds on factor D protease activity translated to inhibition of cell lysis in an alternative pathway hemolytic assay with EC50 values as low as single-digit nM. These small molecules are highly selective for factor D and displayed no significant inhibitory effect on a panel of human serine proteases. In accordance with the high selectivity, the inhibitors showed minimal cellular toxicity, no effect on multiple human receptor-ligand interactions, and no inhibition of hERG potassium channel current. Finally, oral and intravenous administration of selected lead compounds to preclinical animal species showed pharmacokinetic properties that suggest their suitability for oral dosing in humans. CONCLUSIONSWe have discovered highly active small-molecule inhibitors of factor D that demonstrate oral bioavailability and low off-target activities. These key attributes, and the potential to inhibit both intravascular and extravascular hemolysis through inhibition of both terminal complement pathway and opsonization, position these inhibitors as promising development candidates for the oral treatment of PNH. DisclosuresWiles:Achillion Pharmaceuticals: Employment. Podos:Achillion Pharmaceuticals : Employment. Thanassi:Achillion Pharmaceuticals: Employment. Phadke:Achillion Pharmaceuticals: Employment. Gadhachanda:Achillion Pharmaceuticals: Employment. Pais:Achillion Pharmaceuticals: Employment. Hashimoto:Achillion Pharmaceuticals: Employment. Wang:Achillion Pharmaceuticals: Employment. Chen:Achillion Pharmaceuticals: Employment. Wang:Achillion Pharmaceuticals: Employment. Agarwal:Achillion Pharmaceuticals: Employment. Rivera:Achillion Pharmaceuticals: Employment. Elliot:Achillion Pharmaceuticals: Employment. Marlor:Achillion Pharmaceuticals: Employment. Zhang:Achillion Pharmaceuticals: Employment. Deshpande:Achillion Pharmaceuticals: Employment. Huang:Achillion Pharmaceuticals: Employment. Huang:Achillion Pharmaceuticals: Employment.

In silico and in vitro studies on the protein-protein interactions between Brugia malayi immunomodulatory protein calreticulin and human C1q.

Filarial parasites modulate effective immune response of their host by releasing a variety of immunomodulatory molecules, which help in the long persistence of the parasite within the host. The present study was aimed to characterize an immunomodulatory protein of Brugia malayi and its interaction with the host immune component at the structural and functional level. Our findings showed that Brugia malayi Calreticulin (BmCRT) is responsible for the prevention of classical complement pathway activation via its interaction with the first component C1q of the human host. This was confirmed by inhibition of C1q dependent lysis of immunoglobulin-sensitized Red Blood Cells (S-RBCs). This is possibly the first report which predicts CRT-C1q interaction on the structural content of proteins to explain how BmCRT inhibits this pathway. The molecular docking of BmCRT-C1q complex indicated that C1qB chain (IgG/M and CRP binding sites on C1q) played a major role in the interaction with conserved and non-conserved regions of N and P domain of BmCRT. Out of 37 amino acids of BmCRT involved in the interaction, nine amino acids (Pro126, Glu132, His147, Arg151, His153, Met154, Lys156, Ala196 and Lys212) are absent in human CRT. Both ELISA and in silico analysis showed the significant role of Ca+2 in BmCRT-HuC1q complex formation and deactivation of C1r2–C1s2. Molecular dynamics studies of BmCRT-HuC1q complex showed a deviation from ∼0.4 nm to ∼1.0 nm. CD analyses indicated that BmCRT is composed of 49.6% α helix, 9.6% β sheet and 43.6% random coil. These findings provided valuable information on the architecture and chemistry of BmCRT-C1q interaction and supported the hypothesis that BmCRT binds with huC1q at their targets (IgG/M, CRP) binding sites. This interaction enables the parasite to interfere with the initial stage of host complement activation, which might be helpful in parasites establishment. These results might be utilized for help in blocking the C1q/CRT interaction and preventing parasite infection.

Genetic Dissection of T4 Lysis

t is the holin gene for coliphage T4, encoding a 218-amino-acid (aa) protein essential for the inner membrane hole formation that initiates lysis and terminates the phage infection cycle. T is predicted to be an integral membrane protein that adopts an N(in)-C(out) topology with a single transmembrane domain (TMD). This holin topology is different from those of the well-studied holins S105 (3 TMDs; N(out)-C(in)) of the coliphage lambda and S68 (2 TMDs; N(in)-C(in)) of the lambdoid phage 21. Here, we used random mutagenesis to construct a library of lysis-defective alleles of t to discern residues and domains important for holin function and for the inhibition of lysis by the T4 antiholin, RI. The results show that mutations in all 3 topological domains (N-terminal cytoplasmic, TMD, and C-terminal periplasmic) can abrogate holin function. Additionally, several lysis-defective alleles in the C-terminal domain are no longer competent in binding RI. Taken together, these results shed light on the roles of the previously uncharacterized N-terminal and C-terminal domains in lysis and its real-time regulation.

Novel evidence for the specific interaction between cholesterol and α-haemolysin of Escherichia coli

Several toxins that act on animal cells present different, but specific, interactions with cholesterol or sphingomyelin. In the present study we demonstrate that HlyA (α-haemolysin) of Escherichia coli interacts directly with cholesterol. We have recently reported that HlyA became associated with detergent-resistant membranes enriched in cholesterol and sphingomyelin; moreover, toxin oligomerization, and hence haemolytic activity, diminishes in cholesterol-depleted erythrocytes. Considering these results, we studied the insertion process, an essential step in the lytic mechanism, by the monolayer technique, finding that HlyA insertion is favoured in cholesterol- and sphingomyelin-containing membranes. On the basis of this result, we studied the direct interaction with either of the lipids by lipid dot blotting, lysis inhibition and SPR (surface plasmon resonance) assays. The results of the present study demonstrated that an interaction between cholesterol and HlyA exists that seems to favour a conformational state of the protein that allows its correct insertion into the membrane and its further oligomerization to form pores.

Isolation, chemical characterization and biological activity of alfalfa (Medicago media Pers.) root saponins

Saponins from alfalfa (<em>Medicago media</em> Pers.) roots were isolated and their acid hydrolysis revealed several aglycones that were obtained in crystalline form and characterized. Medicagenic acid, hederagenin and soyasapogenols A. B, C. D, E and F were found. Crude saponins were separated into cholesterol-precipitable and nonprecipitable fractions. The precipitable fraction consisted of medicagenic acid glycosides: glucose, arabinose, xylose and rhamnose were found as their sugar chain components. The nonprecipitable fraction was a mixture of hederagenine and soyasapogenol glycosides, and glucose arthinose. xylose. galactose and glucuronic acid were found in the sugar component,. 7 he medicagenic acid glycosides made up 6% of root dry matter and showed high biological activity. They cuased red blood cells lysis (haemolytic index 3000), completely inhibited <em>Trichoderma viride</em> growth at the concentration of 2.5 Mg. 100 cm<sup>-3</sup> of growth medium and retarded wheat seedling growth at concentrations as low as 100 ppm. The cholesterol-nonprecipitable fraction caused no blood cell lysis and fungus growth inhibition, although it did inhibit seedling growth, but to a much lesser extent than medicagenic acrd glycosides. The detrimental effect of alfalfa root saponins on winter wheat crop after alfalfa is discussed.

Phosphatase activity and specific methanogenic activity in an anaerobic reactor treating sludge from a brackish recirculation aquaculture system

Anaerobic treatment of high salinity sludge from marine/brackish recirculation aquaculture systems is potentially limited by inhibition of enzymatic activities and cell lysis resulting from high osmotic pressures. To further address these limitations the following investigations were conducted: effect of salinity on phosphatase activity (PA), soluble microbial products (SMP) production, and presence of extracellular polymeric substances (EPS); effect of iron (III) chloride (FeCl3) on PA and specific methanogenic activity (SMA); effect of addition of the compatible solute glycine betaine (GB) and potassium on PA, as well as on SMP and EPS production, all under saline conditions. The results show that salinity has different effects on PA of anaerobes under starvation and feeding conditions. FeCl3 increased the SMA of the sludge by 22.5% at 100 mg FeCl3/L compared with a control group (0 mg FeCl3/L). Furthermore, results of analysis of variance tests show that betaine increased the polysaccharide content of EPS and polypeptide content of SMP. However, addition of 1 mM potassium chloride did not show a significant effect on EPS and SMP composition. In conclusion, anaerobic digestion of salty sludges from a brackish aquaculture recirculation system may not be negatively affected by FeCl3 addition to concentrate waste streams, whereas GB boosts the production of SMP and EPS.

Myeloid cells as effector cells for monoclonal antibody therapy of cancer

Monoclonal antibodies (mAbs) have become an important addition to chemo- and/or radiotherapy for the treatment of cancer. They have multiple effector functions that can lead to eradication of tumor, including induction of apoptosis, growth inhibition, and initiation of complement-dependent lysis. Furthermore, mAbs can recruit immune effector cells. Traditionally, natural killer cells have been considered as the main effector cell population in mAb-mediated tumor killing. Myeloid cells have potent cytotoxic ability, as well. Monocytes and macrophages have been shown to induce antibody-dependent cytotoxicity and phagocytosis of tumor cells in the presence of IgG anti-tumor mAb. Furthermore, neutrophils are the most abundant population of circulating white blood cells, and as such may constitute a formidable source of effector cells. However, when targeting neutrophils for tumor therapy, antibodies of the IgA subclass may be more effective. This article focuses on enlisting myeloid effector cells for mAb-based immunotherapy of cancer. Additionally, methods to study mAb-dependent phagocytosis of tumor cells by macrophages are compared.

Epithelial-to-Mesenchymal Transition and Autophagy Induction in Breast Carcinoma Promote Escape from T-cell–Mediated Lysis

Epithelial-to-mesenchymal transition (EMT) mediates cancer cell invasion, metastasis, and drug resistance, but its impact on immune surveillance has not been explored. In this study, we investigated the functional consequences of this mode of epithelial cell plasticity on targeted cell lysis by cytotoxic T lymphocytes (CTL). Acquisition of the EMT phenotype in various derivatives of MCF-7 human breast cancer cells was associated with dramatic morphologic changes and actin cytoskeleton remodeling, with CD24(-)/CD44(+)/ALDH(+) stem cell populations present exhibiting a higher degree of EMT relative to parental cells. Strikingly, acquisition of this phenotype also associated with an inhibition of CTL-mediated tumor cell lysis. Resistant cells exhibited attenuation in the formation of an immunologic synapse with CTLs along with the induction of autophagy in the target cells. This response was critical for susceptibility to CTL-mediated lysis because siRNA-mediated silencing of beclin1 to inhibit autophagy in target cells restored their susceptibility to CTL-induced lysis. Our results argue that in addition to promoting invasion and metastasis EMT also profoundly alters the susceptibility of cancer cells to T-cell-mediated immune surveillance. Furthermore, they reveal EMT and autophagy as conceptual realms for immunotherapeutic strategies to block immune escape.

Antibody-mediates inhibition of human C1s and the classical complement pathway

Disregulation of complement activation plays a critical role in numerous inflammatory diseases and therefore, inhibition of the complement pathway is of great therapeutic interest. In the classical complement pathway, immune complexes formed by IgM, IgG1, IgG2 and IgG3 antibodies result in the activation of the C1s protease that in turn cleaves C4 and then C4-bound-C2 yielding the proteolytic fragments C4b and C2a which associate to form a C3 convertase enzyme. We report here the engineering of a potent human antibody inhibitor of C1s protease activity. Phage panning of a very large synthetic (FAB) antibody fragment library using a truncated version of C1s, comprising the second CCP domain and serine protease domain (CCP2-SP) and expressed in insect cells, resulted in the isolation of a FAB that inhibited the catalytic activity of C1s. An affinity matured variant of the FAB format antibody displaying subnanomolar KD for C1s was shown to exhibit >80% inhibition of C2 processing at a 5:1 antibody:C1s molar ratio. We show that this engineered antibody, D.35, displays potent inhibition of complement deposition and lysis of Ramos cells by the anti-CD20 therapeutic antibody rituximab relative to the approved, but less-specific, human plasma-derived C1-inhibitor (CINRYZE). C1s inhibitory antibodies should be useful for delineating the role of the classical pathway in disease models and may hold promise as therapeutic agents.

Proteomic profiles and kinetics of development of bacteriophage T4 and its rI and rIII mutants in slowly growing Escherichia coli

Bacteriophage T4 survival in its natural environment requires adjustment of phage development to the slow bacterial growth rate or the initiation of mechanisms of pseudolysogeny or lysis inhibition (LIN). While phage-encoded RI and probably RIII proteins seem to be crucial players in pseudolysogeny and LIN phenomena, the identity of proteins involved in the regulation of T4 development in slowly growing bacteria has remained unknown. In this work, using a chemostat system, we studied the development of wild-type T4 (T4wt) and its rI (T4rI) and rIII (T4rIII) mutants in slowly growing bacteria, where T4 did not initiate LIN or pseudolysogeny. We determined eclipse periods, phage propagation times, latent periods and burst sizes of T4wt, T4rI and T4rIII. We also compared intracellular proteomes of slowly growing Escherichia coli infected with either T4wt or the mutants. Using two-dimensional PAGE analyses we found 18 differentially expressed proteins from lysates of infected cells. Proteins whose amounts were different in cells harbouring T4wt and the mutants are involved in processes of replication, phage-host interactions or they constitute virion components. Our data indicate that functional RI and RIII proteins - apart from their already known roles in LIN and pseudolysogeny - are also necessary for the regulation of phage T4 development in slowly growing bacteria. This regulation may be more complicated than previously anticipated, with many factors influencing T4 development in its natural habitat.

The role of complement in experimental autoimmune myasthenia gravis

Complement plays an important role in the pathophysiology of experimental autoimmune myasthenia gravis (EAMG). The deposition of IgG at the neuromuscular junction, followed by the activation and observance of C3 at the site, and finally the insertion of the membrane attack complex results in the destruction of the plasma membrane at the neuromuscular junction. Animal models of complement-deficient components show the importance of the mediated lysis in EAMG. These events have regulators that allow for the limitation in the cascade and the ability of the cell to inhibit complement at many places along the pathway. The complement regulatory proteins have many roles in reducing the activation of the complement cascade and the inflammatory pathways. Mice deficient in complement regulatory proteins, decay accelerating factor, and CD59 demonstrate a significant increase in the destruction at the neuromuscular junction. Inhibition of complement-mediated lysis is an attractive therapeutic in MG.

Recommendations for the diagnosis of paroxysmal nocturnal hemoglobinuria: a German-Austrian Consensus1)

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, life-threatening hematological disease. An acquired mutation in the PIG-A gene in hematopoietic precursor cells leads to a deficiency of glycosylphosphatidylinositol (GPI), a common membrane anchor for certain cell surface proteins. The consequence is a deficiency of the decay-accelerating factor (recognized by antibodies of the cluster CD55) and of the membrane inhibitor of reactive lysis (MIRL, recognized by CD59 antibodies) on the surface of blood cells leading to dysregulation of the complement cascade resulting in characteristic intravascular hemolysis and thromboembolic complications. The analysis of GPI-anchored proteins on blood cells is the recognized diagnostic gold standard. Some aspects of the recently published international guidelines are still a matter of discussion. On the basis of these guidelines, we discussed practical recommendations, which led to the German-Austrian consensus paper presented here. It contains specific proposals for indication of the test, preanalytical aspects, how to perform immunophenotyping, and for result interpretation. We propose a two-step diagnostic approach. Step 1 is a screening and step 2 is a confirmation of the diagnosis. The latter should be restricted to expert laboratories’ experience with PNH. According to this algorithm for a stepwise diagnostic approach, we recommend which antibody panel should be used in which step. The most frequent sources of error and how to avoid them are also summarized. This consensus should support medical laboratories to make an early and correct diagnosis of what is important to therapy and prognosis of the patient. Aspects of economic efficiency and feasibility were also included in our considerations.

100-P

Aim KIRs are NK cell surface proteins that play an important role modulating the cytotoxicity of these cells. The balance of activating and inhibitory signals interactions results in induction or inhibition of NK-mediated target cell lysis. KIR genes can be present or absent into individual repertory and thus exhibit different combinations of 14 functional genes (8 encoding inhibitory receptors; 6 genes encoding activator receptors) and 2 pseudogenes setting genotypes. KIR frequencies and genotypes data may contribute to a better-understanding of evolution and mechanisms involved in NK-mediated cytotoxicity. Methods KIR genotyping was carried out through KIR SSO Genotyping Test (One Lambda Inc.) and PCR-SSOP of 93 Caucasian unrelated kidney donors from HUEC Hospital (Parana - Brazil). Results and analysis were produced with HLA Fusion ® (One Lambda Inc.) software package. We compare KIR gene carrier frequency (rxc Contingency Tables) from our sample with frequency data from other Caucasian populations (Brazilian and European) described in the literature. Results Gene frequencies found in the present study were: KIR3DL3(1.00), KIR2DS2(0.53), KIR2DL2(0.45), KIR2DL3(0.90), KIR2DP1(1.00), KIR2DL1(0.99), KIR3DP1(1.00), KIR2DL4(1.00), KIR3DL1(0.94), KIR3DS1(0.41), KIR2DL5(0.55), KIR2DS3(0.37), KIR2DS5(0.22), KIR2DS1(0.41), KIR2DS4(0.82) and KIR3DL2(1.00). These frequencies are very similar to frequencies found in another Brazilian population from a different region, as well as for the French and British Caucasian populations. Conclusions Gene frequencies found in the present study were: KIR3DL3(1.00), KIR2DS2(0.53), KIR2DL2(0.45), KIR2DL3(0.90), KIR2DP1(1.00), KIR2DL1(0.99), KIR3DP1(1.00), KIR2DL4(1.00), KIR3DL1(0.94), KIR3DS1(0.41), KIR2DL5(0.55), KIR2DS3(0.37), KIR2DS5(0.22), KIR2DS1(0.41), KIR2DS4(0.82) and KIR3DL2(1.00). These frequencies are very similar to frequencies found in another Brazilian population from a different region, as well as for the French and British Caucasian populations.

Synthesis and evaluation of 2,4,6-trisubstituted pyrimidine derivatives as novel antileishmanial agents

A series of new 2,4,6-trisubstituted pyrimidine derivatives 8(a–j) were synthesized by reacting substituted chalcones containing imidazole 6(a–d) and benzimidazole 7(a–f) with guanidine hydrochloride in the presence of strong base. Substituted chalcones were synthesized by reacting 4-(1H-imidazol-1-yl)benzaldehyde or 4-(1H-benzo[d]imidazol-1-yl)benzaldehyde with different substituted acetophenones in the presence of 40 % NaOH in methanol. The synthesized compounds were confirmed by IR, 1HNMR, and mass spectral data and screened for antileishmanial activity. Antileishmanial activity was performed against Leishmania donovani parasite, and percentage lysis inhibition were calculated by meglumine antimoliate taking a positive control and chloroform (0.1 % CHCl3) treatment served as control. Among all the compounds, 8h and 8j exhibited 50–57 % inhibition against promastigotes, thus providing new structural lead for antileishmanials.

Brachyury, a Driver of the Epithelial–Mesenchymal Transition, Is Overexpressed in Human Lung Tumors: An Opportunity for Novel Interventions against Lung Cancer

The epithelial-mesenchymal transition (EMT) is emerging as a critical factor for the progression and metastasis of carcinomas, as well as drug resistance. The T-box transcription factor Brachyury has been recently characterized as a driver of EMT in human carcinoma cells. The purpose of this study was to characterize Brachyury as a potential target for lung cancer therapy. The expression of Brachyury was evaluated by PCR and by immunohistochemistry in human lung tumors and adult normal tissues. Brachyury gene copy number and promoter methylation status were analyzed in tumor tissues with various levels of Brachyury expression. Lung carcinoma cells' susceptibility to T-cell lysis and EGF receptor (EGFR) kinase inhibition were also evaluated relative to the levels of Brachyury. Our results showed Brachyury protein expression in 41% of primary lung carcinomas, including 48% of adenocarcinomas and 25% of squamous cell carcinomas. With the exception of normal testis and some thyroid tissues, the majority of normal tissues evaluated in this study were negative for the expression of Brachyury protein. Brachyury-specific T cells could lyse Brachyury-positive tumors and the level of Brachyury corresponded to resistance of tumor cells to EGFR kinase inhibition. We hypothesize that the elimination of Brachyury-positive tumor cells may be able to prevent and/or diminish tumor dissemination and the establishment of metastases. The ability of Brachyury-specific T-cell lines to lyse Brachyury-positive tumor cells, in vitro, supports the development of Brachyury-based immunotherapeutic approaches for the treatment of lung cancer.