CD24-Fc suppression of immune related adverse events in a therapeutic cancer vaccine model of murine neuroblastoma.

Introduction: The combination of Myc-suppressed whole tumor cells with checkpoint inhibitors targeting CTLA-4 and PD-L1 generates a potent therapeutic cancer vaccine in a mouse neuroblastoma model. As immunotherapies translate from pre-clinical to clinical trials, the potential immune-related adverse events (irAEs) associated with induction of potent immunity must be addressed. The CD24-Siglec 10/G interaction is an innate checkpoint that abrogates inflammatory responses to molecules released by damaged cells, but its role in cancer immunology is not well defined. We investigate irAEs of an effective whole cell neuroblastoma vaccine and subsequently the effect of CD24-Fc, a CD24 and Fc fusion protein, on both the vaccine efficacy and induced irAEs in a mouse neuroblastoma model. Methods: To test whether the whole tumor cell vaccination leads to autoimmune responses in other organ systems we harvested lung, heart, kidney and colon from naïve mice (n=3), unvaccinated tumor only mice (n=3), and vaccinated mice with CD24 Fc (n=12) or human IgG-Fc control (n=12) after tumor inoculation and vaccination therapy at day 30. The Immune cell infiltrates and immunogenic pathway signatures in different organ systems were investigated using NanoString Autoimmune Profiling arrays. Nanostring RNA transcript results were validated with immunohistochemistry staining. Results: The whole tumor cell vaccine combined with immune checkpoint therapy triggers occult organ specific immune cell infiltrates, primarily in cardiac tissue and to a lesser extent in the renal and lung tissue, but not in the colon. CD24-Fc administration with vaccination partially impedes anti-tumor immunity but delaying CD24-Fc administration after initial vaccination reverses this effect. CD24-Fc treatment also ameliorates the autoimmune response induced by effective tumor vaccination in the heart. Discussion: This study illustrates that the combination of Myc suppressed whole tumor cell vaccination with checkpoint inhibitors is an effective therapy, but occult immune infiltrates are induced in several organ systems in a mouse neuroblastoma model. The systemic administration of CD24-Fc suppresses autoimmune tissue responses, but appropriate timing of administration is critical for maintaining efficacy of the therapeutic vaccine.

Separation of lead from aqueous phase by cucumber peel in column bioreactor: A phenomenon of interaction between biological and chemical system and its ecological importance.

This research work represents a holistic approach of separation of aqueous lead through dynamic adsorption on cucumber peel in fixed bed column bioreactor and highlights the biological perspective of mode of interaction between adsorbent and adsorbate. Additionally attempt has been made to elucidate the importance of this process in preservation of aquatic ecosystem. The study illustrates influence of design parameters, periodic surveillance of generated effluents, statistical and mathematical model analyses of results and desorption. The findings exhibited a direct association between quality of treated effluent and speed of the feed solution, feed concentration and height of the fixed bed. Lead removal percentage was at its best (99.25%) at 8 cm bed height, 20 mL min-1 (0.9 L h-1) flow rate and 50 mg L-1 feed concentration whereas adsorption capacity reached its peak (300.26 mg g-1) when feed concentration was doubled up to 100 mg L-1. Time bound monitoring confirmed concentration of lead in treated effluents remained within satisfactory level. Adsorbed lead was recovered up to ∼95%. Experimentation with actual industrial effluents demonstrated that lead removal percentage remained in the range of 99.97-99.46% and 99.96-99.17% up to the entire phase of bioreactor operation. In summation proper combination of design parameters of column bioreactor played important role in generating superior quality effluent, multiple reuse of the bioreactor bed was dependent on proper eluant treatment and practicability of the study was ascertained by its ability to maintain concentration of lead in actual industrial effluents within permissible limit for prolonged duration.

Tumor Apolipoprotein E is a key checkpoint blocking anti-tumor immunity in mouse melanoma.

Immunotherapy is a key modality in the treatment of cancer, but many tumors remain immune resistant. The classic mouse model of B16-F10 melanoma is immune resistant even in the face of checkpoint inhibition. Apolipoprotein E (apoE), a known immune suppressant is strikingly elevated in many human tumors, but its role in cancer immunology is not defined. We investigated the role of apoE in the immune micro-environment using a mouse melanoma model. We demonstrate that ApoE is -highly expressed in wild-type B16-F10 melanoma and serum levels progressively increase as tumors grow. The conditioned media from wild type ApoE secreting melanoma cells suppress T-cell activation in vitro while this suppressive effect is absent in conditioned media from ApoE knock out tumor cells. Mechanistically, apoE induces IL-10 secreting dendritic cells and stimulates T-cell apoptosis and arrest partially via the lrp8 receptor. Ablating ApoE in mice inoculated with tumor cells enabled tumor cell rejection and was associated with induction of immune pathway activation and immune cell infiltration. Tumor secreted apoE appears to be a potent immune cell checkpoint and targeting apoE is associated with enhanced tumor immunity in the mouse melanoma model.

Designing a single-mode anomalous dispersion silicon core fiber for temporal multiplet formation.

A highly nonlinear single-mode anomalous dispersion silicon core fiber (SCF) is suitably designed and optimized to generate a high repetition rate pulse train in the temporal domain from a single input pulse at a sufficiently shorter optimum length in comparison to silica-based standard fibers used for the same purpose. The large amount of Kerr-induced nonlinearity of a SCF is effectively utilized here such that input Gaussian pulses or pulse trains transform into a highly repetitive temporal multiplet. The effects of free-carrier generation-induced change in absorption and dispersion are included while studying the nonlinear pulse propagation through the SCF. To declare the generated pulse as a superior-graded triplet, a Q parameter, as a function of relative pulse parameters of the individual pulses of a triplet, is defined for the first time, to the best of our knowledge. Different pulse parameters are thoroughly optimized as well as the effect of external gain is examined from the perspective of requirement of shorter fiber length and development of quality triplets. Finally, the work is further extended for the formation of quadruplet pulses by the same type of SCF. It is to be mentioned here that such a methodical study for the generation of a temporal multiplet using a semiconductor core fiber has not been reported earlier.

Time-transformation technique for generation of optical pulse multiplets in a single mode anomalous dispersion optical fiber.

We present the generation of optical pulse multiplets in the temporal domain from a single pulse using the time-transformation technique for the first time, to the best of our knowledge. The generation of a pair of compressed, well-spaced, and identical Gaussian pulses from a third-order super-Gaussian input pulse when propagated through an anomalous dispersion fiber is reported. Detailed analysis on the effect of variation of input energy, pulse parameters, and optical gain in view of doublet formation leading to the possibility of high effective repetition rate (ERR) at smaller propagating length is reported. The formation of triplets and quadruplets from different input pulse shapes is investigated.

MYC oncogene is associated with suppression of tumor immunity and targeting Myc induces tumor cell immunogenicity for therapeutic whole cell vaccination.

BACKGROUND: MYC oncogene is deregulated in 70% of all human cancers and is associated with multiple oncogenic functions including immunosuppression in the tumor microenvironment. The role of MYC in the immune microenvironment of neuroblastoma and melanoma is investigated and the effect of targeting Myc on immunogenicity of cancer cells is evaluated. METHODS: Immune cell infiltrates and immunogenic pathway signatures in the context of MYCN amplification were analyzed in human neuroblastoma tumors and in metastatic melanoma. Dose response and cell susceptibility to MYC inhibitors (I-BET726 and JQ1) were determined in mouse cell lines. The influence of downregulating Myc in tumor cells was characterized by immunogenic pathway signatures and functional assays. Myc-suppressed tumor cells were used as whole cell vaccines in preclinical neuroblastoma and melanoma models. RESULTS: Analysis of immune phenotype in human neuroblastoma and melanoma tumors revealed that MYCN or c-MYC amplified tumors respectively are associated with suppressed immune cell infiltrates and functional pathways. Targeting Myc in cancer cells with I-BET726 and JQ1 results in cell cycle arrest and induces cell immunogenicity. Combining vaccination of Myc-inhibited tumor cells with checkpoint inhibition induced robust antitumor immunity and resulted in therapeutic cancer vaccine therapy in mouse neuroblastoma tumors. Despite vigorous antitumor immunity in the mouse melanoma model, upregulation of immunosuppressive pathways enabled tumor escape. CONCLUSIONS: This study demonstrates that the Myc oncogene is an appropriate target for inducing tumor cell immunogenicity and suggests that Myc-suppressed whole tumor cells combined with checkpoint therapy could be used for formulating a personalized therapeutic tumor vaccine.

Efficient generation of triangular optical pulses in an erbium-doped chalcogenide fiber amplifier by exploiting the time transformation technique.

The time transformation (TT) technique is used to study the nonlinear pulse reshaping towards triangular pulse (TP) generation by efficiently optimizing the pulse parameters in an erbium-doped chalcogenide fiber amplifier (EDCFA), for the first time, to the best of our knowledge. The effects of input chirp parameter, optical gain, and dipole relaxation time are also depicted in the context of TP generation. The results obtained from the TT approach when compared with that from the nonlinear Schrodinger equation show excellent agreement. The study also reveals that spectral pulse doubling can be achieved at a shorter length when a pre-chirped Gaussian pulse is propagated through the proposed EDCFA.

Sprayable and biodegradable, intrinsically adhesive wound dressing with antimicrobial properties.

Conventional wound dressings are difficult to apply to large total body surface area (TBSA) wounds, as they typically are prefabricated, require a layer of adhesive coating for fixation, and need frequent replacement for entrapped exudate. Large TBSA wounds as well as orthopedic trauma and low-resource surgery also have a high risk of infection. In this report, a sprayable and intrinsically adhesive wound dressing loaded with antimicrobial silver is investigated that provides personalized fabrication with minimal patient contact. The dressing is composed of adhesive and biodegradable poly(lactic-co-glycolic acid) and poly(ethylene glycol) (PLGA/PEG) blend fibers with or without silver salt (AgNO3). in vitro studies demonstrate that the PLGA/PEG/Ag dressing has antimicrobial properties and low cytotoxicity, with antimicrobial silver controllably released over 7-14 days. In a porcine partial-thickness wound model, the wounds treated with both antimicrobial and nonantimicrobial PLGA/PEG dressings heal at rates similar to those of the clinical, thin film polyurethane wound dressing, with similar scarring. However, PLGA/PEG adds a number of features beneficial for wound healing: greater exudate absorption, integration into the wound, a 25% reduction in dressing changes, and tissue regeneration with greater vascularization. There is also modest improvement in epidermis thickness compared to the control wound dressing.

Third-order optical nonlinearity of the CuCo0.5Ti0.5O2 nanostructure under 120 fs laser irradiation.

Recently, titanium-based nanostructures with high nonlinear optical properties have found use in ultrafast photonic system applications. Here, we report a study of the third-order nonlinear optical property of the ${{\rm CuCo}_{0.5}}{{\rm Ti}_{0.5}}{{\rm O}_2}$CuCo0.5Ti0.5O2 (CCoTO) nanostructure synthesized via a simple chemical route. The 40-70 nm CCoTO nanoparticles with centrosymmetric crystalline structure show strong absorption in the 325-850 nm wavelength range due to the presence of different crystalline phases and surface vacancies. A Z-scan technique is used to study the electronic third-order nonlinearity of the synthesized nanoparticles, where a low-repetition-rate 120 fs laser source is employed to minimize thermal agitation-related nonlinearity. The CCoTO nanoparticles possess high surface defects due to oxygen- and copper-related vacancies, which are able to enhance the exciton oscillator strength resulting from the high value of third-order optical nonlinearity. The estimated values of nonlinear refractive index (${n_2}$n2) and nonlinear absorption coefficient ($\beta $ß) of the CCoTO are $ - {1.24}\; \times \;{{10}^{ - 15}}$-1.24×10-15 and ${3.79} \times {{10}^{ - 11}}$3.79×10-11, respectively, under ${188}\,\,{{\rm GW/cm}^2}$188GW/cm2 incident intensity. The intensity-dependent nonlinear optical property of the synthesized nanoparticles is also studied under different incident laser irradiation (62.7, 93, and ${188}\,\,{{\rm GW/cm}^2}$188GW/cm2). In the two-photon absorption (TPA)-dominated third-order nonlinear optical process, the values of ${n_2}$n2 and $\beta $ß of CCoTO are increased with intensifying the incident laser irradiation. The obtained high value of third-order optical nonlinearity of the synthesized nanostructure can be exploited in optical power limiters, pulse power reshaping, and optical switching applications.

Adsorption of Lead on Lentil Husk in Fixed Bed Column Bioreactor.

This study depicts successful employment of fixed bed column bioreactor for adsorption of lead in continuous mode using lentil husk as sorbent. Design parameters considerably controlled the reactor performance, amongst which height of the fixed bed and flow rate were crucial in generating cleaner effluent. Adsorption capacity was found to shoot up to the level of 205.87 mg g-1 at 10 cm bed height, 100 mg L-1 feed concentration and 20 mL min-1 flow rate. Kinetic study done at regular intervals of time revealed high percentage removal of lead (99-96%) throughout entire span of reactor operation. Experimental data were well interpreted by Thomas model and Yoon-Nelson model. The reactor bed was regenerated after each adsorption and loaded metal was recovered up to the extent of ∼96%. The column reactor was efficient enough to treat lead containing actual industrial effluents.

Prospective use of a normally dispersive step-index chalcogenide fiber in nonlinear pulse reshaping.

In this work, several normally dispersive highly nonlinear chalcogenide optical fibers (NDHNCFs) with a step-index profile have been designed and optimized in view of efficient parabolic pulse (PP) generation. A typical NDHNCF is selected such that the group velocity dispersion is highest among them and the corresponding nonlinearity is also very high. The input pulse parameters are optimized to find the lowest possible optimum length (Lopt) of the fiber where the linearly chirped PP is obtained. Further, it is found that for a shorter input pulse width, PP can be generated at a sufficiently smaller length of the NDHNCF with a slight compromise for its misfit parameter. A detailed analysis of the effect of pre-chirping helps to identify the suitable amount of initial chirp for different chalcogenide fibers with a choice of input pulse parameters. Although no improvement in PP generation is found for normal and initially chirped hyperbolic secant pulses, a highly efficient triangular pulse is achieved for a particular value of input pulse energy and the initial chirp parameter. Finally, the comparative study substantiates that our optimized NDHNCF is capable enough to generate quality PP at a length almost 70% shorter than a standard silica-based fiber.

PD-L1 checkpoint inhibition and anti-CTLA-4 whole tumor cell vaccination counter adaptive immune resistance: A mouse neuroblastoma model that mimics human disease.

BACKGROUND: Adaptive immune resistance induces an immunosuppressive tumor environment that enables immune evasion. This phenomenon results in tumor escape with progression and metastasis. Programmed cell death-ligand 1 (PD-L1) expressed on tumors is thought to inhibit tumor-infiltrating lymphocytes (TILs) through programmed cell death 1 (PD1), enabling adaptive immune resistance. This study investigates the role of PD-L1 in both mouse and human neuroblastoma immunity. The consequence of PD-L1 inhibition is characterized in the context of an established whole tumor cell vaccine. METHODS AND FINDINGS: A mouse model of neuroblastoma was investigated using an Id2 knockdown whole cell vaccine in combination with checkpoint inhibition. We show that immunogenic mouse neuroblastoma acquires adaptive immune resistance by up-regulating PD-L1 expression, whereas PD-L1 is of lesser consequence in nonimmunogenic neuroblastoma tumors. Combining PD-L1 checkpoint inhibition with whole tumor cell/anti-CTLA-4 vaccination enhanced tumor cell killing, cured mice with established tumors, and induced long-term immune memory (6 months). From an evaluation of patient neuroblastoma tumors, we found that the inflammatory environment of the mouse neuroblastoma mimicked human disease in which PD-L1 expression was associated directly with TILs and lower-risk tumors. High-risk patient tumors were lacking both TILs and PD-L1 expression. Although a correlation in immunity seems to exist between the mouse model and human findings, the mouse tumor model is induced and not spontaneously occurring, and furthermore, the number of both mouse and human correlates is limited. CONCLUSIONS: This study demonstrates the role PD-L1 plays in neuroblastoma's resistance to immunity and defines the nonredundant effect of combination checkpoint inhibition with vaccine therapy in a mouse model. High-risk, nonimmunogenic human tumors display both diminished PD-L1 expression and adaptive immune resistance. Paradoxically, high-risk tumors may be more responsive to effective vaccine therapy because of their apparent lack of adaptive immune resistance.

Protein kinase Cα and Src kinase support human prostate-distributed dihydrotestosterone-metabolizing UDP-glucuronosyltransferase 2B15 activity.

Because human prostate-distributed UDP-glucuronosyltransferase (UGT) 2B15 metabolizes 5α-dihydrotestosterone (DHT) and 3α-androstane-5α,17ß-diol metabolite, we sought to determine whether 2B15 requires regulated phosphorylation similar to UGTs already analyzed. Reversible down-regulation of 2B15-transfected COS-1 cells following curcumin treatment and irreversible inhibition by calphostin C, bisindolylmaleimide, or röttlerin treatment versus activation by phorbol 12-myristate 13-acetate indicated that 2B15 undergoes PKC phosphorylation. Mutation of three predicted PKC and two tyrosine kinase sites in 2B15 caused 70-100 and 80-90% inactivation, respectively. Anti-UGT-1168 antibody trapped 2B15-His-containing co-immunoprecipitates of PKCα in 130-140- and >150-kDa complexes by gradient SDS-PAGE analysis. Complexes bound to WT 2B15-His remained intact during electrophoresis, whereas 2B15-His mutants at phosphorylation sites differentially dissociated. PKCα siRNA treatment inactivated >50% of COS-1 cell-expressed 2B15. In contrast, treatment of 2B15-transfected COS-1 cells with the Src-specific activator 1,25-dihydroxyvitamin D(3) enhanced activity; treatment with the Src-specific PP2 inhibitor or Src siRNA inhibited >50% of the activity. Solubilized 2B15-His-transfected Src-free fibroblasts subjected to in vitro [γ-(33)P]ATP-dependent phosphorylation by PKCα and/or Src, affinity purification, and SDS gel analysis revealed 2-fold more radiolabeling of 55-58-kDa 2B15-His by PKCα than by Src; labeling was additive for combined kinases. Collectively, the evidence indicates that 2B15 requires regulated phosphorylation by both PKCα and Src, which is consistent with the complexity of synthesis and metabolism of its major substrate, DHT. Whether basal cells import or synthesize testosterone for transport to luminal cells for reduction to DHT by 5α-steroid reductase 2, comparatively low-activity luminal cell 2B15 undergoes a complex pattern of regulated phosphorylation necessary to maintain homeostatic DHT levels to support occupation of the androgen receptor for prostate-specific functions.

Regulated phosphorylation of a major UDP-glucuronosyltransferase isozyme by tyrosine kinases dictates endogenous substrate selection for detoxification.

Whereas UDP-glucuronosyltransferase-2B7 is widely distributed in different tissues, it preferentially detoxifies genotoxic 4-OH-estradiol and 4-OH-estrone (4-OHE(1)) with barely detectable 17ß-estradiol (E(2)) conversion following expression in COS-1 cells. Consistent with the UDP-glucuronosyltransferase requirement for regulated phosphorylation, we discovered that 2B7 requires Src-dependent tyrosine phosphorylation. Y236F-2B7 and Y438F-2B7 mutants were null and 90% inactive, respectively, when expressed in COS-1. We demonstrated that 2B7 incorporated immunoprecipitable [(33)P]orthophosphate and that 2B7His, previously expressed in SYF-(Src,Yes,Fyn)(-/-) cells, was Src-supported or phosphorylated under in vitro conditions. Unexpectedly, 2B7 expressed in SYF(-/-) and SYF(+/-) cells metabolized 4-OHE(1) at 10- and 3-fold higher rates, respectively, than that expressed in COS-1, and similar analysis showed that E(2) metabolism was 16- and 9-fold higher than in COS-1. Because anti-Tyr(P)-438-2B7 detected Tyr(P)-438-2B7 in each cell line, results indicated that unidentified tyrosine kinase(s) (TKs) phosphorylated 2B7 in SYF(-/-). 2B7-transfected COS-1 treated with increasing concentrations of the Src-specific inhibitor PP2 down-regulated 4-OHE(1) glucuronidation reaching 60% maximum while simultaneously increasing E(2) metabolism linearly. This finding indicated that increasing PP2 inhibition of Src allows increasing E(2) metabolism caused by 2B7 phosphorylation by unidentified TK(s). Importantly, 2B7 expressed in SYF(-/-) is more competent at metabolizing E(2) in cellulo than 2B7 expressed in COS-1. To confirm Src-controlled 2B7 prevents toxicity, we showed that 2B7-transfected COS-1 efficiently protected against 4-OH-E(1)-mediated depurination. Finally, our results indicate that Src-dependent phosphorylation of 2B7 allows metabolism of 4-OHE(1), but not E(2), in COS-1, whereas non-Src-phosphorylated 2B7 metabolizes both chemicals. Importantly, we determined that 2B7 substrate selection is not fixed but varies depending upon the TK(s) that carry out its required phosphorylation.

The major chemical-detoxifying system of UDP-glucuronosyltransferases requires regulated phosphorylation supported by protein kinase C.

Finding rapid, reversible down-regulation of human UDP-glucuronosyltransferases (UGTs) in LS180 cells following curcumin treatment led to the discovery that UGTs require phosphorylation. UGTs, distributed primarily in liver, kidney, and gastrointestinal tract, inactivate aromatic-like metabolites and a vast number of dietary and environmental chemicals, which reduces the risk of toxicities, mutagenesis, and carcinogenesis. Our aim here is to determine relevant kinases and mechanism(s) regulating phosphorylation of constitutive UGTs in LS180 cells and 10 different human UGT cDNA-transfected COS-1 systems. Time- and concentration-dependent inhibition of immunodetectable [(33)P]orthophosphate in UGTs and protein kinase Cepsilon (PKCepsilon), following treatment of LS180 cells with curcumin or the PKC inhibitor calphostin-C, suggested UGT phosphorylation is supported by active PKC(s). Immunofluorescent and co-immunoprecipitation studies with UGT-transfected cells showed co-localization of UGT1A7His and PKCepsilon and of UGT1A10His and PKCalpha or PKCdelta. Inhibition of UGT activity by PKCepsilon-specific antagonist peptide or by PKCepsilon-targeted destruction with PKCepsilon-specific small interference RNA and activation of curcumin-down-regulated UGTs with typical PKC agonists verified a central PKC role in glucuronidation. Moreover, in vitro phosphorylation of nascent UGT1A7His by PKCepsilon confirms it is a bona fide PKC substrate. Finally, catalase or herbimycin-A inhibition of constitutive or hydrogen peroxide-activated-UGTs demonstrated that reactive oxygen species-related oxidants act as second messengers in maintaining constitutive PKC-dependent signaling evidently sustaining UGT phosphorylation and activity. Because cells use signal transduction collectively to detect and respond appropriately to environmental changes, this report, combined with our earlier demonstration that specific phospho-groups in UGT1A7 determined substrate selections, suggests regulated phosphorylation allows adaptations regarding differential phosphate utilization by UGTs to function efficiently.

Targeted inhibition of glucuronidation markedly improves drug efficacy in mice - a model.

Finding UDP-glucuronosyltransferases (UGT) require protein kinase C-mediated phosphorylation is important information that allows manipulation of this critical system. UGTs glucuronidate numerous aromatic-like chemicals derived from metabolites, diet, environment and, inadvertently, therapeutics to reduce toxicities. As UGTs are inactivated by downregulating PKCs with reversibly-acting dietary curcumin, we determined the impact of gastro-intestinal glucuronidation on free-drug uptake and efficacy using immunosuppressant, mycophenolic acid (MPA), in mice. Expressed in COS-1 cells, mouse GI-distributed Ugt1a1 glucuronidates curcumin and MPA and undergoes irreversibly and reversibly dephosphorylation by PKC-specific inhibitor calphostin-C and general-kinase inhibitor curcumin, respectively, with parallel effects on activity. Moreover, oral curcumin-administration to mice reversibly inhibited glucuronidation in GI-tissues. Finally, successive oral administration of curcumin and MPA to antigen-treated mice increased serum free MPA and immunosuppression up to 9-fold. Results indicate targeted inhibition of GI glucuronidation in mice markedly improved free-chemical uptake and efficacy using MPA as a model.

Increased synthesis of dihydroxybenzoic acid in the presence of aluminum by Rhizobium MO1.

Rhizobizum sp. MO1, a mung bean (Vigna radiata) symbiont, produces a catecholate type of siderophore, 2,3-dihydroxy benzoic acid (DHBA), in iron depleted medium. Addition of aluminum to the medium decreased the growth but increased the production of the siderophore.