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Background:Impairment or deficiency of regulatory T cells (Treg) is associated with chronic inflammation and autoimmune diseases. Interleukin 2 (IL-2) is a cytokine indispensable for Treg expansion and immunosuppressive function. However, expansion of cytotoxic effector T (Teff) and NK cells and the associated vascular leakage side effect limit the use of IL-2 in autoimmune diseases [1].Objectives:Cugene developed a long-acting IL-2 variant with high Treg specificity and low toxicity to restore immune homeostasis and self-tolerance, and potentially cure autoimmune and inflammatory diseases.Methods:IL-2 variants were generated based on the quaternary structure of IL-2 and IL-2Rαβγ (alpha, beta, gamma) complex. Biological activity was determined by examining differential signaling activity in induction of STAT5 phosphorylation in defined lymphocyte populations of human PBMC using flow cytometry. Binding activity was evaluated by ELISA. Pharmacokinetics, pharmacodynamics, safety and tolerability were assessed in mice and cynomolgus monkeys. Treg suppressive function was determinedin vivo/ex vivo,and anti-inflammatory and anti-antibody production efficacy were determined in delayed-type hypersensitivity (DTH) and T-cell-dependent antibody response (TDAR) models.Results:Structure-based rational design and activity-guided fine-tuning generated an optimized IL-2 variant, CUG252. It demonstrated a strong and near wild-type IL-2 ability to stimulate STAT5 phosphorylation in IL-2Rαβγ dominant Treg cells but abolished activities in IL-2Rβγ dominant effector CD4, CD8 and NK cells. This was a result of biased binding activity to IL-2Rα while dramatically attenuated binding to IL-2Rβγ complex. In mice and monkeys, administration of CUG252 resulted in dose-dependent increases in Treg proliferation and expansion by more than 10- and 30-fold, respectively, with largely abolished activities in CD4+ T conventional, cytotoxic CD8+ Teff and NK cells. The ratio of Treg/Teff cells achieved was as high as 0.4 in mice and 1.2 in monkeys. Both CD4+ and CD8+ Tregs were expanded with preferential increases in memory over naïve subsets. A substantial increase in Treg-suppressive capacity over T effector cells was corroborated by enhanced expression of functional and inhibitory markers, including CD25, Foxp3, PD-1, CTLA-4, Tim3 and ICOS. In DTH and TDAR models, CUG252 strongly inhibited antigen-driven inflammation, B cell maturation, and antibody production. The sustained PK/PD profile supports monthly dosing or better in humans. CUG252 was well-tolerated and no changes in body weight, body temperature, clinical pathology or signs of vascular leakage were observed. Moreover, CUG252 demonstrated superior manufacturability.Conclusion:CUG252 demonstrates an emerging best-in-class profile among IL-2 variants. It displayed exquisite Treg-selectivity while retaining potency comparable to wild-type IL-2. It showed strong anti-inflammatory and anti-antibody production efficacy with significantly improved therapeutic index and manufacturability. Its favorable drug-like property and robust preclinical efficacy warrant further evaluation in patients with a variety of inflammation and autoimmune diseases.References:[1]Tahvildari M. et al. Low-Dose IL-2 Therapy in Transplantation, Autoimmunity, and Inflammatory Diseases. J Immunol. 2019; 203: 2749-2755Disclosure of Interests:Yao-te Hsieh Employee of: Cugene INC., CEDRIC HUBEAU Employee of: Cugene INC., Virginia MASSA Employee of: Cugene INC., WEN Li Employee of: Cugene INC., SANDRA FREI Employee of: Cugene INC., BEN CAPRARO Employee of: Cugene INC., ANDREA UMANA Employee of: Cugene INC., ANDREW AHERRERA Employee of: Cugene INC., YUESHENG LI Employee of: Cugene INC., JING XU Employee of: Cugene INC., LINGYUN RUI Employee of: Cugene INC.

Objective To evaluate/predict the offspring serum profile of antibodies against Porcine reproductive and respiratory syndrome virus (PRRSV) in an affected intensive herd, whereas sows are vaccinated, and determine the better sample time using pigs as sentinels. Methods Serum samples were collected from a total of 66 offspring pigs from a PRRSV endemic herd, whereas only sows were vaccinated. Six animals per grouped age were randomly selected with 0 (at perinatal time), 1, 2, 3, 6, 9, 12, 15, 18, 21 and 24 weeks of age. Individual and three pooled samples were tested by ELISA and PCR, respectively. Results The proportion of seropositive animals was 75.0% (18/24), 33.3% (6/18) and 95.8% (23/24) at farrowing (0–3 weeks), nursery (6–12 weeks) and growing/finishing (15–24 weeks) phases, respectively. It was 46 times more likely (P

In this work we explored whether DNA methyltransferase 3a (Dnmt3a) targeted to the HBV X promoter (XP) causes epigenetic suppression of hepatitis B virus (HBV). The C-terminus of Dnmt3a (Dnmt3aC) was fused to a six-zinc-finger peptide specific to XP to form a fused DNA methyltransferase (XPDnmt3aC). The binding and methyl-modifying specificity of XPDnmt3aC were verified with an electrophoretic mobility shift assay and methylation-specific PCR, respectively. XP activity and HBV expression were clearly downregulated in HepG2 cells transfected with plasmid pXPDnmt3aC. The injection of XPDnmt3aC into HBV transgenic (TgHBV) mice also showed significant inhibition, leading to low serum HBV surface protein (HBsAg) levels and a reduced viral load. Thus, XPDnmt3aC specifically silenced HBV via site-selective DNA methylation delivered by zinc-finger peptides. This study establishes the foundation of an epigenetic way of controlling HBV-related diseases.

Summary In our previous study, we showed that treatment with an anti-interleukin (IL)-12/23p40 antibody inhibits acute cardiac allograft rejection via inhibiting production of interferon (IFN)-γ and IL-17a. However, the impact of this antagonistic anti-p40 antibody on chronic cardiac rejection was unclear. Hearts of B6.C-H2bm12/KhEg mice were transplanted into major histocompatibility complex (MHC) class II-mismatched C57Bl/6J mice (wild-type, γδTCR –/– and IL-17–/–), which is an established murine model of chronic allograft rejection without immunosuppression. The mice were treated with control immunoglobulin (Ig)G or 200 µg anti-p40 monoclonal antibody on post-operative days, respectively. Abdominal palpation and echocardiography were used to monitor graft survival. The mice administered with anti-p40 antibody showed a significant promotion in graft survival (median survival time >100 days), and histological analyses revealed that cardiac allograft rejection was attenuated. Quantitative real-time polymerase chain reaction (qRT–PCR) and immunofluorescence analyses demonstrated that anti-p40 antibody down-regulated the level of ingraft cytokine and chemokine expression (IL-6, IFN-γ, IL-17a, CCL2 and CCL20). Flow cytometry analyses showed that γδ T cells are an important ingraft source of IFN-γ and IL-17a and inhibit the production of inflammation cytokine by anti-p40 antibody. Compared with the wild-type group, the graft survival time in the γδ T cell receptor–/– and IL-17–/– mice was prolonged significantly. Therefore we propose that, in the chronic allograft rejection model, treatment with anti-p40 antibody prolongs graft survival possibly by reducing the amount of reactive inflammatory cells, especially γδ T cells.

As the emergence of the Net-Centric Warfare (NCW), the military information system has been evolved from platform-centric to be net-centric, which brings great challenges for System of Systems (SoS) engineering in the net centric environment. A major task of system engineering is to build system architecture. Although classical system engineering deals very well with architecting problems for a single system in which user requirements are well defined, it has no good solutions for SoS architecting problems. In this paper, existing architecture development methods are briefly reviewed, and a novel capability-based method (CBM) for architecture design is proposed, which not only enables SoS architecting with the new kind of capability-based development process, but also ensures consistency to form integrated architectures. Index Terms—System of systems (SoS) engineering, architecture design, NCW.

The Lower Devonian to Upper Permian Stikine Assemblage is well exposed in northwestern British Columbia, near the west-central margin of the Stikine terrane. The stratigraphic base of the assemblage is not exposed and the Upper Triassic Stuhini Group disconformably overlies the assemblage locally. The assemblage consists mainly of various types of volcanic rocks, and there is a large, polyphase, Devono-Mississippian sub-volcanic plutonic complex. The assemblage includes a distinctive, but laterally discontinuous mid-Carboniferous limestone named the Arctic Lake Formation. Facies are mainly locally re-worked, well-bedded skeletal wackestone, but whole fossil floatstone and intraformational olistostromal deposits are present. Stratigraphic thickness varies from 32 to 80 metres. Stratigraphic contacts with bounding Lower and Upper Carboniferous successions are conformable and intergradational, but paraconformities may also be present. The formation records weak development of disjointed carbonate ramps on volcanic highlands. Neritic faunal assemblages have warm water, open marine affinity. The formation spans the Serpukhovian-Bashkirian boundary and encompasses microfacies assigned to Global Foraminiferal zones 17, 18 and 20. Integrated foraminiferal-coralconodont biostratigraphy and uranium-lead geochronology support a circa 350 Ma age for the Tournaisian-Visean boundary, and a circa 320 Ma age for the Serpukhovian-Bashkirian (Mississippian-Pennsylvanian) boundary.

The implicitly restarted generalized minimum residual method (IRGMRES) combined with the fast Fourier transform (FFT) technique is developed for solving three-dimensional (3-D) weak-form volume electric field integral equation of electromagnetic scattering problems. On several electromagnetic scattering problems, the resulted IRGMRES-FFT method converges two-three times faster than the conventional biconjugate gradient (BCG)-FFT method. Comparison with other Krylov-subspace iterative fast Fourier transforms methods also demonstrates the efficiency of the IRGMRES-FFT method.

In this article, the microstrip circuit is analyzed in terms of the mixed-potential integral equation (MPIE) by means of the rooftop-function expansion and the blaze-function testing technique, and the integral equation is solved using the loose generalized minimal residual fast Fourier transform (LGMRES-FFT) method. Our numerical calculations show that LGMRES-FFT can converge faster than the conjugate gradient-fast Fourier transform (CG-FFT) method. Some typical microstrip discontinuities are analyzed and the good results obtained demonstrate the validity of the proposed algorithm. © 2005 Wiley Periodicals, Inc. Int J RF and Microwave CAE, 2005.

Climate simulations and forecast experiments of increasingly large ensemble size are being performed to assess the predictive skill of a dynamic model on seasonal and longer timescales. Especially in the cases of ensemble climate simulation or forecast forced by observed or predicted sea surface temperatures, the model is expected to maximize potential predictability due to boundary forcing and to minimize internal variability generated from dynamic instability. In the light of small predictive skill in extratropics from boundary forcing, one must evaluate skill of the ensemble mean quantity against intersample variability or spread of the individual ensemble member. On the other hand, certain dominant signals in climate variability, such as E1 Nino-Southern Oscillation, have been documented. Predictability for these major signals is the hope of seasonal and climate forecasting using a dynamic model. It may be unrealistic to anticipate a model being able to simulate or forecast the full spectra of climate variability. The question is how to evaluate a model's performance in capturing the dominant climate signals in ensemble experiments with increasingly large sample size. These issues have motivated us to develop a compact methodology for assessing climate experiments with large ensemble size. This method treats the ensemble mean as signal and intersample variability as spread or noise in a common framework. Hence not only dominant signals from boundary forcing can be isolated, but also sensitivity of these signals to the forcing can be assessed. Other potential applications of the method to climate simulation and forecasting are also discussed.

In this paper, a multi-frequency lossless watermarking algorithm that the original image need not been destroyed is proposed by using bandelet and optimal matrix norms. The wavelet transform is first performed to the original image, the bandelet transform is then applied to the high and middle frequency parts of the transformed image, and the texture and edge traced by using the geometric flow via feature of the image to yield the high frequency lossless watermarking. For low frequency part of wavelet coefficients, by selecting optimal matrix norm, a low frequency watermarking scheme is also achieved. A lossless watermarking approach composed of multi-frequency information is then proposed. The experimental tests show the proposed watermarking system is evaluated against standard watermarking attacks.

Hmgb1, an evolutionarily conserved chromosomal protein, was recently re-discovered to be an innate immune-mediator contributing to both innate and adaptive immune responses. Here, we show a pivotal role for Hmgb1 in acute allograft rejection in a murine cardiac transplantation model. Extracellular Hmgb1 was found to be a potent stimulator for adaptive immune responses. Hmgb1 can be either passively released from damaged cells after organ harvest and ischemia/reperfusion insults, or actively secreted by allograft infiltrated immune cells. After transplantation, allografts show a significant temporal up-regulation of Hmgb1 expression accompanied by inflammatory infiltration, a consequence of graft destruction. These data suggest the involvement of Hmgb1 in acute allograft rejection. In line with these observations, treatment of recipients with rA-box, a specific blockade for endogenous Hmgb1, significantly prolonged cardiac allograft survival as compared to those recipients treated with either rGST or control vehicle. The enhanced graft survival is associated with reduced allograft expression of TNFalpha, IFNgamma and Hmgb1 and impaired Th1 immune response.

A comparison between a submerged membrane bioreactor (MBR) and a conventional activated sludge process (CAS) was carried out under similar operational conditions. MBR demonstrated a more stable and excellent effluent quality than CAS. Its effluent COD concentration was 55.5 mg/L on average, much lower than that of CAS (79.7 mg/L). Soluble microbial products accumulated in the MBR during the first 120 days in operation due to membrane interception of macromolecules, but these accumulated substances were degraded at last with microbial acclimation. No similar phenomenon was observed in the CAS system. Compositions of the CAS effluent, MBR supernatant and membrane permeate were found quite different. In the CAS effluent and MBR supernatant, both macromolecules with MW60,000 and small molecules with MW3,000 were dominant and macromolecules had a much larger occupation in the MBR supernatant. In the membrane permeate, however, small molecules with MW3,000 were the major component. The relatively small floc size in the MBR was proved favorable to improve oxygen transfer rate.

Growth hormone (GH) has long been known to be a primary determinant of body height and an important regulator of body metabolism, yet the cellular and molecular bases for these effects of GH are only beginning to be understood. In 1993, GH receptor (GHR) was first observed to bind to the tyrosine kinase JAK2. GH increased JAK2's affinity for GHR, potently activated JAK2, and stimulated the phosphorylation of tyrosines within JAK2 and the cytoplasmic domain of GHR. In the intervening six years, a variety of signaling molecules have been identified that are tyrosyl phosphorylated in response to GH, presumably by the activated JAK2. These signaling molecules include 1) the latent cytoplasmic transcription factors--designated signal transducers and activators of transcription (Stats)--that have been implicated in the regulation of a variety of GH-dependent genes; 2) Shc proteins that lead to activation of the Ras-MAP kinase pathway: and 3) insulin receptor substrate (IRS) proteins that bind and thereby activate phosphatidylinositol 3' kinase and presumably other proteins. Recently, we have identified two additional signaling molecules for GH that bind to JAK2 and are phosphorylated on tyrosines in response to GH: SH2-B and signal regulated protein (SIRP). Based upon amino acid sequence analysis, SH2-B is presumed to be a cytoplasmic adapter protein. It binds with high affinity via its SH2 domain to phosphorylated tyrosines within JAK2. GH-induced binding of SH2-B to JAK2 via this site potently activates JAK2, leading to enhanced tyrosyl phosphorylation of Stat proteins and other cellular proteins. Because of its other potential protein-protein interaction domains and its recruitment and phosphorylation by kinases that are not activated by SH2-B, SH2-B is thought likely to mediate other, more-specific actions of GH, as yet to be determined. SIRP is a transmembrane protein that is now known to bind to integrin-associated protein. It appears to bind directly to JAK2 by a process that does not require tyrosyl phosphorylation, although is itself highly phosphorylated on tyrosines in response to GH. The phosphorylated SIRP recruits one or more molecules of the tyrosine phosphatase SHP2 that, in turn, de-phosphorylates SIRP and most likely JAK2. Thus, SIRP is predicted to be a negative regulator of GH action. It seems likely that the diverse actions of GH will be found to require coordinated interaction of all of these signaling proteins with each other as well as with other signaling molecules that are activated by GH and the numerous other ligands that are present at cells during a response to GH.

This experiment was designed to determine whether chronic hypoxia affects endothelium-dependent relaxation and cGMP content of rat pulmonary artery (PA). Both Ach and ATP were found to induce endothelium-dependent relaxation of PA; and this relaxation was not prevented by indomethacin, but was completely abolished by methylene blue. Chronic hypoxia significantly depressed the endothelium-dependent relaxation: the relaxation responses of intra-PA (IPA) and extra-PA (EPA) to 10(-6) mol/L Ach in the hypoxic group were 61.3% and 59.2% of those in control, and the relaxation responses of IPA and EPA to 1.8 x 10(-5) mol/L ATP in the hypoxic group were 64.9% and 55.2% of those in the control, respectively. Chronic hypoxia significantly decreased the basic level and Ach-induced accumulation of cGMP in the PA. Our data suggest that chronic hypoxia might depress rat pulmonary artery endothelium-dependent relaxation through the inhibition of cytosolic soluble guanylate cyclase in vascular smooth muscle cells.

The article reports an original technique of arterialisation of the portal vein in the rat, after porto-cava shunt, with termino-latreal anastomosis between the proximal stump of the portal vein and the aorta; compared with other techniques, this method has the advantage of avoiding right nephrectomy.

Reference is made to a personal series of vascular accesses for haemodialysis on 273 patients aged 6 months to 70 yr. During the course of 12 years, 533 operations were performed: 109 cannulations of the saphena, 152 by-pass, 251 arteriovenous fistulae (including 22 on patients under 10 yr of age), and 21 "difficult accesses". An assessment of the advantages and disadvantages of the techniques employed showed that the Cimino-Brescia fistula, combined, if necessary, with transient cannulation, is the soundest access owing to its longer life, lower incidence of complications, and better utilisation of the vascular material available.