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Background: While chimeric antigen receptor T cells (CAR T-cells) induce dramatic remissions of refractory or recurrent B cell malignancies, the durability of these remissions is frequently limited by subsequent reduction in circulating CAR T-cells and/or by diminution of their effector function. We hypothesized that we could overcome this therapeutic limitation and increase the functional activity and longevity of CAR T-cells by selectively deriving them from virus-specific effector memory T cells. We have developed biologics we termed synTacs (artificial immunological synapse for T-cell activation), which selectively activate and expand antigen-specific CD8+ T cells in vitro and in vivo by recapitulating signals delivered at the immunological synapse. The synTacs consist of dimeric Fc domain scaffolds linking CD28- or 4-1BB-specific ligands to HLA-A2 MHC molecules covalently tethered to virus-derived peptides. Treatment of PBMCs from CMV-exposed donors with synTacs presenting a CMV-derived peptide (pp65-NLVPMVATV) induce vigorous and selective ex vivo and in vivo expansion of highly functional CMV-specific CD8+ T cells, with potent antiviral activity. We used these synTacs to selectively generate CAR T-cells from CMV-specific effector memory CD8+ T cells, which could be further expanded by restimulation with the CMV-specific synTacs. Methods: We treated PBMCs from CMV-exposed donors in media supplemented with either IL-2 or IL-7/12/15 with a synTac containing the CMV-derived pp65 peptide presented by HLA-A2 MHC molecules linked to ligands capable of stimulating CD28- or 4-1BB-dependent costimulatory pathways. PBMCs activated either with anti-CD3/CD28 or the CMV-specific synTacs were transduced with lentivirus expressing an anti-CD19 CAR and a GFP reporter gene. CMV-specific CD8+ T cells were quantified by tetramer staining and CAR T-cells were detected by GFP expression determined by flow cytometric analysis. The functional activity of the CD19 CAR T-cells was determined by a B cell-specific cytotoxic assay. Results: After 7 days, treatment of PBMCs with CMV-specific synTacs rapidly induced robust activation and >50-fold expansion of CMV-specific CD8+ T cells expressing effector memory markers. Treatment of the PBMCs with CMV-specific synTacs selectively activated CMV-specific T cells and enabled them to be specifically transduced with a CD19-specific CAR lentivirus and converted into CD19 CAR T-cells. These CMV-specific CD19 CAR T-cells displayed potent dose-responsive cytotoxic activity targeting purified primary B cells. Furthermore, these CMV-specific CD19 CAR T-cells could be selectively expanded by in vitro treatment with CMV-specific synTacs. Conclusions: SynTacs are versatile immunotherapeutics capable of selective in vitro and in vivo activation and expansion of virus-specific CD8+ T cells with potent antiviral cytotoxic activity. After selective lentiviral transduction and conversion into CD19 CAR T-cells, their co-expression of the CMV-specific T cell receptor enabled them to be potently stimulated and activated by in vitro treatment with CMV synTacs. The modular design of synTacs facilitates efficient coupling of other costimulatory ligands - such as OX40 or GITRL - or cytokines, such as IL-2, IL-7, or IL-15, to enable the selective in vivo delivery of defined costimulatory signals or cytokines to the CAR T-cells expressing CMV-specific TCR. This strategy has the potential to boost the in vivo activity of tumor-specific CAR T-cells after infusion and enable more durable and potent treatment of refractory/recurrent B cell malignancies. Disclosures Almo: Cue Biopharma: Current equity holder in publicly-traded company, Patents & Royalties: Patent number: 62/013,715, Research Funding. Goldstein:Cue Biopharma: Research Funding.

Antibodies bound to human immunodeficiency virus type 1 (HIV-1) envelope protein expressed by infected cells mobilize antibody-dependent cellular cytotoxicity (ADCC) to eliminate the HIV-1-infected cells and thereby suppress HIV-1 infection and delay disease progression. Studies treating HIV-1-infected individuals with latency reactivation agents to reduce their latent HIV-1 reservoirs indicated that their HIV-1-specific immune responses were insufficient to effectively eliminate the reactivated latent HIV-1-infected T cells. Mobilization of ADCC may facilitate elimination of reactivated latent HIV-1-infected cells to deplete the HIV-1 reservoir and contribute to a functional HIV-1 cure. The most effective antibodies for controlling and eradicating HIV-1 infection would likely have the dual capacities of potently neutralizing a broad range of HIV-1 isolates and effectively mobilizing HIV-1-specific ADCC to eliminate HIV-1-infected cells. For this purpose, we constructed LSEVh-LS-F, a broadly neutralizing, defucosylated hexavalent fusion protein specific for both the CD4 and coreceptor gp120-binding sites. LSEVh-LS-F potently inhibited in vivo HIV-1 and simian-human immunodeficiency virus (SHIV) infection in humanized mouse and macaque models, respectively, including in vivo neutralization of HIV-1 strains resistant to the broadly neutralizing antibodies VRC01 and 3BNC117. We developed a novel humanized mouse model to evaluate in vivo human NK cell-mediated elimination of HIV-1-infected cells by ADCC and utilized it to demonstrate that LSEVh-LS-F rapidly mobilized NK cells to eliminate >80% of HIV-1-infected cells in vivo 1 day after its administration. The capacity of LSEVh-LS-F to eliminate HIV-1-infected cells via ADCC combined with its broad neutralization activity supports its potential use as an immunotherapeutic agent to eliminate reactivated latent cells and deplete the HIV-1 reservoir. IMPORTANCE Mobilization of antibody-dependent cellular cytotoxicity (ADCC) to eliminate reactivated latent HIV-1-infected cells is a strategy which may contribute to depleting the HIV-1 reservoir and achieving a functional HIV-1 cure. To more effectively mobilize ADCC, we designed and constructed LSEVh-LS-F, a broadly neutralizing, defucosylated hexavalent fusion protein specific for both the CD4 and coreceptor gp120-binding sites. LSEVh-LS-F potently inhibited in vivo HIV-1 and SHIV infection in humanized mouse and macaque models, respectively, including in vivo neutralization of an HIV-1 strain resistant to the broadly neutralizing antibodies VRC01 and 3BNC117. Using a novel humanized mouse model, we demonstrated that LSEVh-LS-F rapidly mobilized NK cells to eliminate >80% of HIV-1-infected cells in vivo 1 day after its administration. The capacity of LSEVh-LS-F to eliminate HIV-1-infected cells via ADCC combined with its broad neutralization activity supports its potential use as an immunotherapeutic agent to eliminate reactivated latent cells and deplete the HIV-1 reservoir.

We aimed to evaluate the association of preoperative plasma fibrinogen levels with the clinicopathological parameters, disease-free survival, and overall survival in patients with renal cell carcinoma. We retrospectively studied 286 patients with renal cell carcinoma who underwent radical nephrectomy from 2000 to 2003 at one center. The plasma fibrinogen was routinely determined before operation in all patients. The correlation of preoperative plasma fibrinogen levels with clinicopathological findings was evaluated by t-test or analysis of variance (ANOVA) methods. As well, univariate and multivariate analyses were used to determine the association between the preoperative level of plasma fibrinogen and survival duration. An elevated level of plasma fibrinogen was positively related to the Fuhrman grade (P

Inflammatory mediators and viral products produced by human immunodeficiency virus (HIV)-infected microglia and astrocytes perturb the function and viability of adjacent uninfected neuronal and glial cells and contribute to the pathogenesis of HIV-associated neurocognitive disorders (HAND). In vivo exposure to lipopolysaccharide (LPS) activates parenchymal microglia and astrocytes and induces cytokine and chemokine production in the brain. HIV-infected individuals display increased circulating LPS levels due to microbial translocation across a compromised mucosa barrier. We hypothesized that HIV-infected microglia and astrocytes display increased sensitivity to the proinflammatory effects of LPS, and this combines with the increased levels of systemic LPS in HIV-infected individuals to contribute to the development of HAND. To examine this possibility, we determined the in vivo responsiveness of HIV-infected microglia and astrocytes to LPS using our mouse model, JR-CSF/human cyclin T1 (JR-CSF/hu-cycT1) mice, which are transgenic for both an integrated full-length infectious HIV type 1 (HIV-1) provirus derived from the primary R5-tropic clinical isolate HIV-1JR-CSFregulated by the endogenous HIV-1 long terminal repeat and the hu-cycT1 gene under the control of a CD4 promoter. In the current report, we demonstrated that in vivo-administered LPS more potently activated JR-CSF/hu-cycT1 mouse microglia and astrocytes and induced a significantly higher degree of monocyte chemoattractant protein production by JR-CSF/hu-cycT1 astrocytes compared to that of the in vivo LPS response of control littermate mouse microglia and astrocytes. These results indicate that HIV infection increases the sensitivity of microglia and astrocytes to inflammatory stimulation and support the use of these mice as a model to investigate various aspects of the in vivo mechanism of HIV-induced neuronal dysfunction.

HIV-1-infected monocyte/macrophages located in lymph nodes and tissues are highly productive sources of HIV-1 and may function as a persistent reservoir contributing to the rebound viremia observed after highly active antiretroviral therapy is stopped. Mechanisms activating latently infected, primary monocyte/macrophages to produce HIV-1 were investigated using monocytes isolated from a transgenic mouse line carrying a full-length proviral clone of a monocyte-tropic HIV-1 isolate, HIV-1(JR-CSF), regulated by the endogenous long terminal repeat (LTR) (JR-CSF mice). Granulocyte-macrophage colony-stimulating factor (GM-CSF) combined with lipopolysaccharide (LPS) induced infectious HIV-1 production by JR-CSF mouse monocytes over 10-fold and 100-fold higher than that stimulated by GM-CSF or LPS alone, respectively. We examined mechanisms of GM-CSF synergy with LPS and demonstrated that GM-CSF up-regulated the LPS receptor, TLR-4, and also synergized with LPS to activate mitogen-activated protein (MAP) kinase/ERK kinase and the Sp1 transcription factor. Inhibitors of either MAP kinase/ERK kinase or p38 kinase but not PI 3-kinase potently suppressed GM-CSF and LPS-induced HIV-1 production by JR-CSF mouse monocytes. Because Sp1 is activated by both the MAP kinase/ERK kinase and p38 kinase pathways, we postulate that synergistic activation of these pathways by GM-CSF and LPS induced sufficient levels of Sp1 to activate the HIV-1 LTR in a Tat-independent manner and induced HIV-1 production by JR-CSF mouse monocytes. Thus, our study delineated the pathway of HIV-1 LTR activation by GM-CSF and LPS and indicated that JR-CSF transgenic mice may provide a new in vitro and in vivo system for investigating the mechanism by which inflammatory and infectious stimuli activate HIV-1 production from latently infected monocytes.

Two simple and robust designs for an external high-temperature reference electrode are described. The first external reference electrode uses a movable polytetrafluoroethylene (PTFE) piston to maintain the pressure balance between the reference solution and test solution. The second external reference electrode uses the test solution as reference solution, and this is feasible if the test solution contains a minimum amount of chloride. For both electrodes it is necessary to determine the potential drop across the (cooled) salt bridge, which connects the reference electrode to the autoclave. It is shown that a simple correction algorithm can be used to calculate this potential drop and that the corrections are in agreement with literature values. It is also shown that this correction algorithm is independent of the solution composition for a wide range of neutral and alkaline solutions. The potential difference between two standard hydrogen electrodes (SHE) at different temperatures has been determined. This potential difference has been used to extend the correction algorithm to be able to calculate the potential against the SHE at 25°C.

To investigate the erosion–corrosion behavior of the Cu-alloys, i.e. CuCrZr and CuAl25-candidate materials for the manufacture of the diverter cooling channels for ITER, a test loop has been developed for testing erosion–corrosion behavior under high heat flux conditions. The heat loads on the test specimen surface amounts to 10 MW/m 2 . Under adverse testing conditions in oxidizing water (5–10 ppm H 2 O 2 ) at 100 °C, with a flow velocity of 10 m/s and intermittent heat loading, followed by cooling cycles under oxidizing conditions maintained, CuCrZr alloy has shown the formation of a thick surface oxide films (essentially cupric oxide, CuO). This surface layer seems loosely adhesive, and detachable by mechanical or thermal shocks, or by fluid flow velocity effects. Indicated with a rough morphology, surface attacks were somewhat observed in areas after the surface oxide layer spalled off. Present results suggest the necessity for, such as, strict hydrogen water chemistry (HWC), in order to limit the copper corrosion rate or at least the release of copper into the cooling water circuit.

In the present work, the effects of dissolved oxygen and hydrogen peroxide on corrosion potential have been studied on type 316L stainless steel in a hot lithium hydroxide solution. The results indicate that the corrosion potential will remain at around −850 mV vs SCE if the dissolved oxygen in the solution is controlled at a level of less than 10 ppb. However, 316L stainless steel will become completely passivated when about 650 ppb of oxygen exists in the solution. As a result, the corrosion potential of 316L steel will rise to around −400 mV vs SCE, and eventually, with longer immersion times, to even higher values, e.g. −300 mV vs SCE. However, the presence of less than 500 ppb H 2 O 2 has no significant effect on the corrosion potential. A potential jump (from −850 to −500 mV vs SCE) is observed only when a large amount of H 2 O 2 , for instance 1 ppm, is present in the solution. Related to radiolysis of water and with potential-dependent stress corrosion cracking, the effects of dissolved oxygen and hydrogen peroxide on the corrosion potential of 316L stainless steel in the hot lithium hydroxide solution have been discussed.

The stress corrosion cracking (SCC) behavior of cold-worked AISI type 316L (UNS S31603) stainless steel (SS) in a concentrated lithium salt solution at elevated temperature was investigate...

The corrosion fatigue behavior of AISI 316L (UNS S31603) stainless steel was tested in concentrated aqueous lithium hydroxide solutions at elevated temperature. Fatigue tests were conducted in a 10 g LiOH/100cc H2O solution at 95°C at controlled electrochemical potentials. Experimental conditions met requirements of the aqueous Li salt blanket option for the international and European nuclear fusion reactor programs of the International Thermonuclear Experimental Reactor (ITER) and Next European Torus (NET). Results indicated 316L stainless steel was susceptible to corrosion fatigue in a narrow potential range of approximately 100 mV (vs SCE). Tests at lower or higher potentials (e.g., −80 and 200 mV), however, did not show susceptibility to corrosion fatigue cracking. Results were compared with the stress corrosion cracking behavior of 316L in the same environment.

This paper describes the effect of extended sensitization heat treatment (650°C) and applied potential on the corrosion behavior and the stress corrosion cracking (SCC) of low-carbon AISI 316L (UNS S31603) stainless steel (0.02%C) in a concentrated lithium hydroxide solution at 95°C. The results are compared to the behavior of solution-annealed material under the same conditions. In general, there is a fairly clear effect of prolonged heat treatment times on the material, and its susceptibility to intergranular attack (IGA) and SCC, as determined in slow strain rate tests, increases with increasing times of sensitization heat treatment. When the heat treatment extends to 100 h or more, the potential range in which IGA is observed extends to lower potentials (−300 mV SCE, open-circuit conditions) compared to the solution-annealed material. On the other hand, SCC is observed on both materials in approximately the same potential ranges (i.e., above approximately −100 mV SCE). However, both intergran...

The stress corrosion cracking (SCC) of AISI 316L stainless steel has been investigated in a concentrated LiOH solution at 95°C as a function of the electrochemical corrosion potential (ECP...

Molybdenum based alloys, such as TZM, Z6 and Mo-41Re, have been considered as possible divertor cooling-channel materials for NET. Preliminary corrosion investigation of these alloys and pure molybdenum in water with variable pH values from 2.9 to 11.1 has been carried out at 95°C. In this paper we present first results obtained from anodic potentiodynamic polarization measurements of molybdenum and its alloys, and from bimetallic corrosion experiments in which molybdenum base materials TZM and Z6 have been coupled with 316L stainless steel. The results indicate that the electrochemical corrosion potential of the molybdenum based alloys increases with decreasing pH value, and the coupling of type 316L stainless steel to the molybdenum alloys leads, depending upon the pH value, either to cathodic protection or to accelerated corrosion of the molybdenum alloys.

A possible concept for the blanket for next generation fusion devices is the lithium salt blanket, where lithium salt is dissolved in an aqueous coolant in order to provide for tritium. Type AISI 316L stainless steel has been considered as a structural material for such a blanket for NET (Next European Torus), and a systematic study of the corrosion behaviour of 316L stainless steel has been carried out in a number of lithium salt solutions. The experiments include cyclic potentiodynamic polarization measurement, crevice corrosion corrosion fatigue and stress corrosion cracking (SCC) tests. This paper presents a part of novel corrosion results concerning the compatibility of 316L steel and a series of other materials relevant to a fusion blanket environment. No major uniform corrosion problem has been observed, but localized corrosion, particularly corrosion fatigue and SCC, of 316L stainless steel have been found so far in a lithium hydroxide solution under some specific potential conditions. The critical electrochemical potential zones for SCC have been identified in the present study.

The tissue culture of Saussurea involucrata (TCSauI) KAR. et KIR. was studied to determine its antiinflammatory and analgesic activities in experimental animals. Similar to wild S. involucrata, TCSauI at doses of 75—300 mg/kg i.g. for 7 d markedly inhibited hindpaw edema induced by carrageenin in rats, ear edema induced by dimethylbenzene, and increased capillary permeability in the mouse abdominal cavity induced by acetic acid. Moreover, TCSauI had inhibitory activities against the writhing reaction induced by acetic acid and the hot plate reaction in mice. The present study provided evidence first that TCSauI has antiinflammatory and analgesic activities, suggesting the potential of the tissue culture technique to substitute for wild S. involucrata in the pharmaceutical industry.

Basic fibroblast growth factor (bFGF) is implicated in the pathogenesis of several types of vascular and connective diseases. A key step in the discovery of bFGF receptor antagonists to mitigate these actions is to define the functional epitopes required for receptor binding of the growth factor. Using structure-based site-directed mutagenesis, two critical areas on the bFGF surface for the high affinity receptor binding have already been identified [Springer, B.A., Pantoliano, M.W., Barberal, F.A., Gunyuzlu, P.L., Thompson, L.D., Herblin, W.F., Rosenfeld, S.A. and Book, G.W. (1994) J. Biol. Chem., 269, 26879-26884; Zhu, H.Y., Ramnarayan, K., Anchin, J., Miao, Y., Sereno, A., Millman, L., Zheng, J., Balaji, V.N. and Wolff, M.E. (1995) J. Biol. Chem., 270, 21869-21874; Zhu, H.Y., Anchin, J., Ramnarayan, K., Zheng, J., Kawai, T., Mong, S. and Wolff, M.E. (1997) Protein Engng, 10, 417-421]. According to these studies, one receptor binding site includes two polar residues Glu96 and Asn104 on bFGF whereas the other includes four hydrophobic residues Tyr24, Tyr103, Leu140 and Met142. Using a protein modelling technique, we report here the identification of a new hydrophobic patch on bFGF which includes residues Tyr73, Val88 and Phe93. The role of this area on receptor binding affinity was evaluated by mutating each of these residues individually and determining the mutated protein's (mutein's) receptor binding affinity. In addition, we examined the role of two other hydrophobic residues, Phe30 and Leu138, on bFGF for high-affinity receptor binding. These two residues are the neighbors of the hydrophobic residues Tyr24 and Tyr103, respectively. Replacement of Val88 and Phe93 with alanine reduced the receptor binding affinity about 10- and 80-fold, respectively, compared with wild-type bFGF. In contrast, substitution of Phe30 and Leu138 with alanine has no effect on the receptor binding affinities. We conclude that the newly identified hydrophobic residues, Val88 and Phe93, are crucial for the receptor binding. The present data, together with the previous identification of four hydrophobic residues (Tyr24, Tyr103, Leu140 and Met142), suggests that there are two hydrophobic receptor binding sites on the bFGF surface. Our findings can be employed in the discovery and design of potent bFGF antagonists using computational methods.

Purine and pyrimidine are essential for cellular proliferation1, 2, 3. But, purine and pyrimidine are synthesized from glutamine in de novo synthesis pathway and cultured cancer cells do not require them in the medium. This is the reason that utilization of nucleosides by human cancer cells and their changes with 5 fluorouracil (5FU), the fluorinated analogue of uracil which is used in chemotherapy for various tumors2, 3, has not been reported. In this study, as a source of purine and pyrimidine, a solution containing a mixture of nucleosides and a nucleotide (OG-VI), developed recently as a nutritional supplement4, was used in the medium. It consists of purines, guanosine 5’-mono-phosphate (5’-GMP) and inosine, and pyrimidines, cytidine, uridine, and thymidine (TdR). It is known to increases hepatic regeneration and improve protein metabolism after partial hepatectomy in rats5, 6, and increase DNA synthesis in primary cultured hepatocytes7.