Doses Of Ag Research Articles

Molecular basis for potent B cell responses to antigen displayed on particles of viral size.

Multivalent viral epitopes induce rapid, robust and T cell-independent humoral immune responses, but the biochemical basis for such potency remains incompletely understood. We take advantage of a set of liposomes of viral size engineered to display affinity mutants of the model antigen (Ag) hen egg lysozyme. Particulate Ag induces potent 'all-or-none' B cell responses that are density dependent but affinity independent. Unlike soluble Ag, particulate Ag induces signal amplification downstream of the B cell receptor by selectively evading LYN-dependent inhibitory pathways and maximally activates NF-κB in a manner that mimics T cell help. Such signaling induces MYC expression and enables even low doses of particulate Ag to trigger robust B cell proliferation in vivo in the absence of adjuvant. We uncover a molecular basis for highly sensitive B cell responses to viral Ag display that is independent of encapsulated nucleic acids and is not merely accounted for by avidity and B cell receptor cross-linking.

Multistep IgE Mast Cell Desensitization Is a Dose- and Time-Dependent Process Partially Regulated by SHIP-1

Multistep mast cell desensitization blocks the release of mediators following IgE crosslinking with increasing doses of Ag. Although its invivo application has led to the safe reintroduction of drugs and foods in IgE-sensitized patients at risk for anaphylaxis, the mechanisms of the inhibitory process have remained elusive. We sought to investigate the kinetics, membrane, and cytoskeletal changes and to identify molecular targets. IgE-sensitized wild-type murine (WT) and FcεRIα humanized (h) bone marrow mast cells were activated and desensitized with DNP, nitrophenyl, dust mites, and peanut Ags. The movements of membrane receptors, FcεRI/IgE/Ag, actin, and tubulin and the phosphorylation of Syk, Lyn, P38-MAPK, and SHIP-1 were assessed. Silencing SHIP-1 protein was used to dissect the SHIP-1 role. Multistep IgE desensitization of WT and transgenic human bone marrow mast cells blocked the release of β-hexosaminidase in an Ag-specific fashion and prevented actin and tubulin movements. Desensitization was regulated by the initial Ag dose, number of doses, and time between doses. FcεRI, IgE, Ags, and surface receptors were not internalized during desensitization. Phosphorylation of Syk, Lyn, p38 MAPK, and SHIP-1 increased in a dose-response manner during activation; in contrast, only SHIP-1 phosphorylation increased in early desensitization. SHIP-1 phosphatase function had no impact on desensitization, but silencing SHIP-1 increased β-hexoxaminidase release, preventing desensitization. Multistep IgE mast cell desensitization is a dose- and time-regulated process that blocks β-hexosaminidase, impacting membrane and cytoskeletal movements. Signal transduction is uncoupled, favoring early phosphorylation of SHIP-1. Silencing SHIP-1 impairs desensitization without implicating its phosphatase function.

Astaxanthin, a Carotenoid without Vitamin A Activity, Augments Antibody Responses in Cultures Including T-helper Cell Clones and Suboptimal Doses of Antigen

Astaxanthin, a carotenoid without vitamin A activity, enhances T-dependent antigen (Ag)-specific humoral immune responses. We examined carotenoid actions on T-helper (Th) cell activity in a direct manner with reconstitution experiments; spleen Th cells were replaced with Ag-specific Type 1 and Type 2 (Th1 and Th2) Th cell clones. The Ag for the Th1 and Th2 clones were pigeon cytochrome C and rabbit γ-globulin, respectively. Astaxanthin and β-carotene augmented the number of IgM antibody (Ab)-secreting cells when unprimed B cells were incubated with Th clones and stimulated with suboptimal doses of Ag specific for each Th clone. The number of IgG Ab-secreting cells were greater with use of in vivo primed B cells than with unprimed B cells in both Th clones. Astaxanthin but not β-carotene augmented the number of IgG Ab-secreting cells when primed B cells and Th cell clones were stimulated with suboptimal doses of Ag specific for each Th clone. In the presence of optimal doses of Ag for each Th clone, neither carotenoid augmented the number of Ab-secreting cells. Astaxanthin and β-carotene may enhance the actions of both Th1 and Th2 cells for humoral immune responses with suboptimal Ag challenges; certain carotenoids may help maintain Ag-mediated immune responses at optimal levels.

Investigating the Effect of Three Antigens of Citrobacter freundii on Rabbit’s Immune Response

Citrobacter freundii (C. freundii) is responsible for a number of significant opportunistic infections. The present research was aimed to estimate the immune response of rabbits immunized with whole cell sonicated antigen (WCSA), lipopolysaccharide (LPS), and DNA antigens (Ag) extracted from C. freundii. Twenty-four Albino rabbits of both sexes, with 2-3 kg body weight, were divided randomly into four groups (6 rabbits for each). Two types of tests were performed including ELISA and skin test (delayed type hypersensitivity, DTH)). The 1st group was immunized with WCSA (1000 μg/mL). The 2nd group was immunized with LPS Ag at the same dose. The 3rd group was immunized with DNA extracted Ag (0.083 μg/mL). The 4th group (negative control) was injected with 1 mL PBS (pH 7.2) subcutaneously. After 14 days, rabbits were given booster doses of same Ag. The immunized animals showed significant increase of IgG and IL-6 concentration (P<0.05) following 28, 32, 46, 50 and 60 days of immunization in comparison with the negative control group. Concerning DTH, it showed an increase in the means of induration and erythema, with significant differences (P˂0.05) exerted by the concentrated antigens in all immunized groups after 24 h and 48 h compared with diluted Ag and negative control group. In conclusion, WCSA and LPS Ag, in comparison to DNA Ag, were observed to promote stronger humoral (IgG) and cellular (DTH and IL-6) immune responses. DNA Ag, on the other hand, elicited a weaker humoral and cellular immune response than other Ag.

Low Concentrations of Silver Nanoparticles and Silver Ions Perturb the Antioxidant Defense System and Nitrogen Metabolism in N2-Fixing Cyanobacteria.

Although toxic effects of silver nanoparticles (AgNPs) on aquatic organisms have been extensively reported, responses of nitrogen-fixing cyanobacteria to AgNPs/Ag+ under environmentally relevant concentrations are largely unknown. Here, cyanobacteria were exposed to different concentrations of AgNPs (0.01, 0.1, and 1 mg/L) or Ag+ (0.1, 1, and 10 μg/L) for 96 h. The impacts of AgNPs and Ag+ on photosynthesis and N2 fixation in cyanobacteria (Nostoc sphaeroides) were evaluated. In addition, gas chromatography-mass spectrometry (GC-MS)-based metabolomics was employed to give an instantaneous snapshot of the physiological status of the cells under AgNP/Ag+ exposure. Exposure to high doses of AgNPs (1 mg/L) or Ag+ (10 μg/L) caused growth inhibition, reactive oxygen species overproduction, malondialdehyde accumulation, and decreased N2 fixation. In contrast, low doses of AgNPs (0.01 and 0.1 mg/L) and Ag+ (0.1 and 1 μg/L) did not induce observable responses. However, metabolomics revealed that metabolic reprogramming occurred even at low concentrations of AgNP and Ag+ exposure. Levels of a number of antioxidant defense-related metabolites, especially phenolic acid and polyphenols (gallic acid, resveratrol, isochlorogenic acid, chlorogenic acid, cinnamic acid, 3-hydroxybenzoic acid, epicatechin, catechin, and ferulic acid), significantly decreased in response to AgNPs or Ag+. This indicates that AgNPs and Ag+ can disrupt the antioxidant defense system and disturb nitrogen metabolism even at low-dose exposure. Metabolomics was shown to be a powerful tool to detect "invisible" changes, not observable by typical phenotypic-based endpoints.

Fungal Tolerance: An Alternative for the Selection of Fungi with Potential for the Biological Recovery of Precious Metals

The behavior of various filamentous fungi in the presence of metals such as Cu, Zn, Ni, Fe, Mn, and V has been widely reported. However, there is little information regarding metals such as Au, Ag and Pt that are not in the form of nanoparticles. The growth of eight filamentous fungi was evaluated at increasing doses of Au, Ag and Pt. The fungi were reactivated in Petri dishes with potato dextrose agar. Subsequently, individual mycelial disks from each strain were inoculated in PDA plates with the following doses of AuCl3, Ag2SO4 and PtCl4: 0, 50, 150 and 300 mg L−1, respectively. The plates were then incubated for 20 days—a period in which the diameter of the colony was measured every 24 h. Au showed the highest toxicity for the tested fungi. All silver doses decreased the growth of most of the fungi, while platinum did not cause any inhibitory effect on the growth of the eight tested fungi. With a simple test, it was possible to observe the effect of precious metals (PMs) on the growth of filamentous fungi and consider their possible biotechnological applications in the recovery of PMs from primary or secondary sources.

Study on the osteogenesis of rat mesenchymal stem cells and the long-term antibacterial activity of Staphylococcus epidermidis on the surface of silver-rich TiN/Ag modified titanium alloy.

The main causes of failure of orthopedic implants are infection and poor bone ingrowth. Surface modification of the implants to allow for long-term antibacterial and osteogenic functions is an effective solution to prevent failure of the implants. We developed silver-rich TiN/Ag nano-multilayers on the surface of titanium alloy with different doses of Ag+ . The antibacterial stability and osteogenesis of the silver-rich surface were determined by evaluating the adhesion and proliferation of Staphylococcus epidermidis, and the adhesion, proliferation, alkaline phosphatase activity, extracellular matrix mineralization, and the expression level of genes involved in osteogenic differentiation of rat bone mesenchymal stem cells (BMSCs). The results demonstrated that the antibacterial rates (Ra) of 5 × 1016 -Ag, 1 × 1017 -Ag, 5 × 1017 -Ag, and 1 × 1018 -Ag were respectively 46.21%, 85.66%, 94.99%, 98.48%, and 99.99%. After subcutaneous implantation in rats or immersion in phosphate buffered saline for up to 12 weeks, the silver-rich surface of the titanium alloy showed long-term stable inhibition of Staphylococcus epidermidis. Furthermore, in vitro and in vivo studies indicated that the Ag-implanted titanium did not show apparent cytotoxicity and that lower Ag+ implanted groups (5 × 1016 -Ag, 1 × 1017 -Ag) had better viability and biological safety when compared with higher Ag+ implanted groups. In addition, when compared with the Ti6Al4V-group, all Ag-implanted groups exhibited enhanced osteogenic indicators in rat BMSCs. Regarding osteogenic indicators, the surfaces of the 5 × 1017 -Ag group had better osteogenic effects than those of other groups. Therefore, the proper dose of Ag+ implanted TiN/Ag nano-multilayers may be one of the options for the hard tissue replacement materials with antibacterial activity and osteogenic functions.

Toxicity of Ag+ on microstructure, biochemical activities and genic material of Trifolium pratense L. seedlings with special reference to phytoremediation.

The objective of this research was to evaluate Ag+ toxicity in Trifolium pratense L. seedlings subjected to increasing doses of Ag+ by determining photosynthetic pigment and malondialdehyde (MDA) contents, microstructure and hereditary substance alterations, changes in activities of antioxidase-superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) as well as the content of total Ag absorbed in vivo with evaluation of root growth. Doses of approximately 80mgL-1 Ag+ severely affected photosynthetic efficiency in Trifolium pratense L. seedlings promoted by damages in photosynthetic apparatus evidenced by downward trend in photosynthetic pigment contents and obvious chlorosis. Alterations in enzymatic activity, lipid peroxidation, genic material damage and the presence of Ag+in vivo had impacted on photosynthetic machinery as well. A hormesis effect was observed at 60mgL-1 Ag+ for the photosynthetic pigments and antioxidase for Trifolium pratense L. seedlings. Tissue changes (i.e., roots, stems and leaves) observed in fluorescence microscope with obvious chlorosis, roots blackening and formation of agglomerated black particles, were related to the lesion promoted by excessive ROS in vivo. Asynchronous change of antioxidase activity corresponded to the alteration in the MDA content, indicating the synchronization in the elimination of ROS. The changes occurred in RAPD profiles of treated samples following Ag+ toxicity containing loss of normal bands, appearance of new bands and variation in band intensity compared to the normal plants with a dose-dependent effect. On average, the roots of Trifolium pratense L. immobilized 92.20% of the total Ag absorbed as a metal exclusion response. Root growth was significantly sensitive to Ag+ stress with obvious hormesis, which corresponded to the changes in Ag uptake, demonstrating the functional alterations in plants. To sum up, we suggest that modulating the genotype of Trifolium pratense L. seedlings to bear higher proportion of pollutants is conducive to contamination site treatment.

Subacute co-exposure to low doses of ruthenium(III) changes the distribution, excretion and biological effects of silver ions in rats

Environmental contextAlthough ruthenium is a technology-critical element used in several new industries, little is known about its environmental impact or possible human health risks. We report a toxicological evaluation of laboratory animals during co-exposure to ruthenium and silver. We identified interactions between the two elements that could lead to unwanted biological effects. AbstractRuthenium is one of the rarest metals on Earth that is classified as a technology-critical element (TCE). Silver, however, is well known for its antibacterial and immunostimulant properties. The increasing use of Ru and Ag in medicine and daily life makes simultaneous exposure possible, with unknown pharmacokinetic or toxicological consequences for the human organism. Thus, the present study investigated the influence of co-exposure to RuIII on the subacute toxicity of Ag ions in rats after repeated daily administration for 28 days of low doses by oral gavage. The subacute toxicity was investigated by the evaluation of several biochemical and hematological parameters, evaluation of specific oxidative stress biomarkers from liver and kidney, and histopathological investigation of liver and kidney tissue samples after 28 days of exposure in female rats. In addition, Ag and Ru concentrations were determined by inductively coupled plasma mass spectrometry (ICP-MS) in urine, liver and kidney parenchyma in male rats. The obtained results showed that co-exposure to low doses of RuIII and Ag did not produce any significant biochemical, hematological or histopathological alterations in the treated female rat groups, except for an increased red cell distribution width (RDW) value. A decrease of urinary excretion of Ag and of the Ag concentration in kidneys was observed in the male rat group co-exposed to RuIII and Ag. This is the first invivo study investigating the toxic effect of co-exposure to low doses of Ag and Ru ions, and the obtained results may justify further research on this subject, mainly on the investigation of possible competitive mechanisms.

Study on the In Vitro and In Vivo Antibacterial Activity and Biocompatibility of Novel TiN/Ag Multilayers Immobilized onto Biomedical Titanium.

To determine the short- and long-term antibacterial properties of a novel biomedical titanium alloy to ensure excellent biocompatibility of the TiN/Ag multilayers loaded with different doses of Ag+. First, nanosized TiN/Ag multilayers were accumulated onto titanium alloy (Ti-6Al-4V) substrates via multi-arc ion plating. Then, the multilayers were implanted with different doses of silver ions (1×1017 ions/cm², 1×1018 ions/cm², 5×1016 ions/cm², and 5×1017 ions/cm²). Both short- and long-term antibacterial properties against Streptococcus mutans and Staphylococcus aureus were assessed via unique methods. Additionally, the response and behaviors of MC3T3-E1 and L929 cells on the different surfaces were evaluated by a variety of methods through comparison to a normal matched substrate (Ti-6Al-4V). In Vitro and In Vivo analyses revealed that the multilayers containing different doses of Ag ions effectively prevented bacterial adhesion and eliminated the majority of adhered bacteria in the initial period. In addition, the antibacterial activity of each TiN/Ag group improved with time, with the antibacterial rate (Ra) ultimately reaching 99% (antibacterial activity: 1 × 1018 ions/cm² > 5 × 1017 ions/cm² > 1 × 1017 ions/cm² > 5 × 1016 ions/cm²). All of the samples loaded with Ag+ exhibited good compatibility, as well as higher cell proliferation and lower apoptosis than the pure Ti-6Al-4V substrates. Considering both bacteriostasis and biocompatibility, 1 × 1017 ions/cm² and 5 × 1017 ions/cm² are the recommended doses for orthopedic and dental implants. The results indicate that all of the samples loaded with Ag+ possess excellent biocompatibility and antibacterial activity against common bacteria that cause implantation infection. The samples loaded with Ag+ can be implanted into soft and hard growing tissues to greatly improve the survival rate of orthopedic and dental implants.

A mixture toxicity approach to predict the toxicity of Ag decorated ZnO nanomaterials.

Nanotechnology is a rising field and nanomaterials can now be found in a vast variety of products with different chemical compositions, sizes and shapes. New nanostructures combining different nanomaterials are being developed due to their enhancing characteristics when compared to nanomaterials alone. In the present study, the toxicity of a nanostructure composed by a ZnO nanomaterial with Ag nanomaterials on its surface (designated as ZnO/Ag nanostructure) was assessed using the model-organism Daphnia magna and its toxicity predicted based on the toxicity of the single components (Zn and Ag). For that ZnO and Ag nanomaterials as single components, along with its mixture prepared in the laboratory, were compared in terms of toxicity to ZnO/Ag nanostructures. Toxicity was assessed by immobilization and reproduction tests. A mixture toxicity approach was carried out using as starting point the conceptual model of Concentration Addition. The laboratory mixture of both nanomaterials showed that toxicity was dependent on the doses of ZnO and Ag used (immobilization) or presented a synergistic pattern (reproduction). The ZnO/Ag nanostructure toxicity prediction, based on the percentage of individual components, showed an increase in toxicity when compared to the expected (immobilization) and dependent on the concentration used (reproduction). This study demonstrates that the toxicity of the prepared mixture of ZnO and Ag and of the ZnO/Ag nanostructure cannot be predicted based on the toxicity of their components, highlighting the importance of taking into account the interaction between nanomaterials when assessing hazard and risk.

Extracts and Fractions from Edible Roots of Sechium edule (Jacq.) Sw. with Antihypertensive Activity.

Sechium edule is traditionally used in Mexico as a therapeutic resource against renal diseases and to control high blood pressure. The purpose of this work is to evaluate the antihypertensive effect of the hydroalcoholic extract obtained from the roots of this plant, including its fractions and subfractions, on different hypertension models induced with angiotensin II (AG II). The hydroalcoholic extract was tested on an in vitro study of isolated aorta rings denuded of endothelial cells, using AG II as the agonist; this assay proved the vasorelaxant effect of this extract. Vagotomized rats were administered different doses of AG II as well as the Hydroalcoholic extract, which reduced blood pressure in 30 mmHg approximately; subsequently this extract was separated into two fractions (acetone and methanol) which were evaluated in the acute hypertension mouse model induced with AG II, where the acetone fraction was identified as the most effective one and was subsequently subfractioned using an open chromatographic column packed with silica gel. The subfractions were also evaluated in the acute hypertension model. Finally, the extract, fraction, and active subfraction were analyzed by MS-PDA-HPLC, identifying cinnamic derivative compounds like cinnamic acid methyl ester.

The pharmacokinetics of acyl, des-acyl, and total ghrelin in healthy human subjects

Ghrelin stimulates GH secretion and regulates energy and glucose metabolism. The two circulating isoforms, acyl (AG) and des-acyl (DAG) ghrelin, have distinct metabolic effects and are under active investigation for their therapeutic potentials. However, there is only limited data on the pharmacokinetics of AG and DAG. To evaluate key pharmacokinetic parameters of AG, DAG, and total ghrelin in healthy men and women. In study 1, AG (1, 3, and 5 μg/kg per h) was infused over 65 min in 12 healthy (8 F/4 M) subjects in randomized order. In study 2, AG (1 μg/kg per h), DAG (4 μg/kg per h), or both were infused over 210 min in ten healthy individuals (5 F/5 M). Plasma AG and DAG were measured using specific two-site ELISAs (study 1 and 2), and total ghrelin with a commercial RIA (study 1). Pharmacokinetic parameters were estimated by non-compartmental analysis. After the 1, 3, and 5 μg/kg per h doses of AG, there was a dose-dependent increase in the maximum concentration (C(max)) and area under the curve (AUC(0-last)) of AG and total ghrelin. Among the different AG doses, there was no difference in the elimination half-life, systemic clearance (CL), and volume of distribution. DAG had decreased CL relative to AG. The plasma DAG:AG ratio was ~2:1 during steady-state infusion of AG. Infusion of AG caused an increase in DAG, but DAG administration did not change plasma AG. Ghrelin administration did not affect plasma acylase activity. The pharmacokinetics of AG and total ghrelin appears to be linear and proportional in the dose range tested. AG and DAG have very distinct metabolic fates in the circulation. There is deacylation of AG in the plasma but no evidence of acylation.

Silver release from decomposed hyperaccumulating Amanita solitaria fruit-body biomass strongly affects soil microbial community

Interaction of Ag with communities of soil saprotrophic organisms was studied in two different soils using a metagenomic approach. Three levels of Ag were applied to the soil samples: 0, 0.008 and 0.505μg Ag/g soil. Silver was applied in mineral form as well as naturally bound in dry fruit-body biomass of the Ag-hyperaccumulating ectomycorrhizal fungus Amanita solitaria. Contrasting behavior of fungi and bacteria in reaction to Ag dosages was observed. The majority of bacterial ribotypes tended to prefer the soil with low doses of Ag, the ribotypes of fungi were more abundant in untreated soils and soils treated with the highest Ag concentration. Organically bound and mineral forms of Ag did not differ substantially in their effects on microbes in samples. The results indicate that decomposing Ag-rich fungal biomass can significantly alter the soil microbiota. This can contribute to formation of spot-like non-homogeneities in soil microbial distribution.

CD70-Driven Costimulation Induces Survival or Fas-Mediated Apoptosis of T Cells Depending on Antigenic Load

Apoptosis plays an essential role in the removal of activated CD8 T cells that are no longer required during or postinfection. The Bim-dependent intrinsic pathway of apoptosis removes effector CD8 T cells upon clearance of viral infection, which is driven by withdrawal of growth factors. Binding of Fas ligand to Fas mediates activation-induced T cell death in vitro and cooperates with Bim to eliminate CD8 T cells during chronic infection in vivo, but it is less clear how this pathway of apoptosis is initiated. In this study, we show that the costimulatory TNFR CD27 provides a dual trigger that can enhance survival of CD8 T cells, but also removal of activated CD8 T cells through Fas-driven apoptosis. Using in vitro stimulation assays of murine T cells with cognate peptide, we show that CD27 increases T cell survival after stimulation with low doses of Ag, whereas CD27 induces Fas-driven T cell apoptosis after stimulation with high doses of Ag. In vivo, the impact of constitutive CD70-driven stimulation on the accumulation of memory and effector CD8 T cells is limited by Fas-driven apoptosis. Furthermore, introduction of CD70 signaling during acute infection with influenza virus induces Fas-dependent elimination of influenza-specific CD8 T cells. These findings suggest that CD27 suppresses its costimulatory effects on T cell survival through activation of Fas-driven T cell apoptosis to maintain T cell homeostasis during infection.

Silver(I), Mercury(II), Cadmium(II), and Zinc(II) Target Exposed Enzymic Iron-Sulfur Clusters when They Toxify Escherichia coli

The toxicity of soft metals is of broad interest to microbiologists, both because such metals influence the community structures in natural environments and because several metals are used as antimicrobial agents. Their potency roughly parallels their thiophilicity, suggesting that their primary biological targets are likely to be enzymes that contain key sulfhydryl moieties. A recent study determined that copper poisons Escherichia coli in part by attacking the exposed [4Fe-4S] clusters of dehydratases. The present investigation sought to test whether other soft metals also target these enzymes. In vitro experiments revealed that low-micromolar concentrations of Ag(I) and Hg(II) directly inactivated purified fumarase A, a member of the dehydratase family. The enzyme was also poisoned by higher levels of Cd(II) and Zn(II), but it was unaffected by even millimolar concentrations of Mn(II), Co(II), Ni(II), and Pb(II). Electron paramagnetic resonance analysis and measurements of released iron confirmed that damage was associated with destruction of the [4Fe-4S] cluster, and indeed, the reconstruction of the cluster fully restored activity. Growth studies were then performed to test whether dehydratase damage might underlie toxicity in vivo. Barely toxic doses of Ag(I), Hg(II), Cd(II), and Zn(II) inactivated all tested members of the [4Fe-4S] dehydratase family. Again, activity was recovered when the clusters were rebuilt. The metals did not diminish the activities of other sampled enzymes, including NADH dehydrogenase I, an iron-sulfur protein whose clusters are shielded by polypeptide. Thus, the data indicate that dehydratases are damaged by the concentrations of metals that initiate bacteriostasis.

Gads Regulates the Expansion Phase of CD8+ T Cell-Mediated Immunity

The Gads adaptor protein is critical for TCR-mediated Ca(2+) mobilization. We investigated the effect of Gads deficiency on the proliferation of CD8(+) T cells following peptide stimulation and in the context of infection with an intracellular pathogen. We stimulated CD8(+) T cells from Gads(+/+) OT-I and Gads(-/-) OT-I mice with cognate Ag (SIINFEKL) or altered peptide ligand. In vitro experiments revealed that Gads was required for optimal proliferation of CD8(+) T cells. This defect was most evident at the early time points of proliferation and when low doses of Ag were used as stimuli. Cell cycle analysis demonstrated that Gads(-/-) CD8(+) T cells had impaired TCR-mediated exit from the G(0) phase of the cell cycle. Furthermore, Gads(-/-) CD8(+) T cells had delayed expression of c-myc and CD69 upon the stimulation with SIINFEKL. We then investigated how Gads deficiency would impact CD8(+) T cell-mediated immunity in the context of infection with an intracellular pathogen. At early time points, Gads(+/+) and Gads(-/-) CD8(+) T cells proliferated to a similar extent, despite the fact that expression of CD69 and CD25 was reduced in the absence of Gads. After 5 d postinfection, Gads was required to sustain the expansion phase of the immune response; the peak response of Gads(-/-) cells was significantly lower than for Gads(+/+) cells. However, Gads was not required for the differentiation of naive CD8(+) T cells into memory cells. We conclude that the primary function of Gads is to regulate the sensitivity of the TCR to Ag ligation.

Axitinib (AG-013736; AG) combined with chemotherapy in patients (pts) with advanced non-small cell lung cancer (NSCLC) and other solid tumors

3559 Background: AG is an oral, potent and selective inhibitor of VEGFRs 1, 2, 3. Anti-VEGF therapies combined with chemotherapy improve survival vs chemotherapy alone in pts with advanced solid tumors, including NSCLC. This phase I trial assessed AG combined with chemotherapy. Methods: Pts with advanced solid tumors without prior treatment with platins or taxanes received paclitaxel/carboplatin (P: 200 mg/m2 / C: AUC 6 mg*min/mL) every 3 weeks + AG at lead-in doses of 1, 3 or 5 mg BID. AG was increased to 5 mg BID post lead-in. Another cohort of pts with any prior chemotherapy for metastatic disease received AG 5 mg BID + gemcitabine/cisplatin (G: 1250 mg/m2 on days 1 and 8 / Cis: 80 mg/m2 on day 1) in 3-week cycles. For all cohorts, cycle 1 consisted of a 3–5-day lead-in period followed by AG for 18 days. On day 19, AG was held and then continued without interruption on day 3, cycle 2. Following determination of the maximum tolerated doses (MTDs) of AG + P/C or G/Cis, pts with squamous NSCLC were enrolled into an expansion cohort (prior anti-VEGF therapy not permitted) and received AG 5 mg BID + P/C. Dose-limiting toxicities (DLTs), adverse events (AEs), objective response rate (ORR), and pharmacokinetics (PK) were evaluated. Results: A total of 47 pts were enrolled; 26 pts were evaluable in the P/C cohort (including 12 pts with squamous NSCLC) and 21 pts in the G/Cis cohort. The MTD for AG in combination with standard doses of P/C and G/Cis was 5 mg BID. DLTs included fatigue, proteinuria and rash (1 each). Treatment-related AEs included hypertension (42%), diarrhea (35%) and fatigue (35%) in the P/C cohort, and headache (29%), hypertension (29%) and fatigue (24%) in the G/Cis cohort. There was no grade ≥3 hemoptysis among 12 pts with squamous NSCLC. The ORR was 29% and 26% for P/C (n = 24) and G/Cis (n = 19) cohorts, respectively. AG, P, C, G and Cis PK parameters and profiles were similar when administered alone and in combination (eg P: mean (%CV) AUCinf 20,822 (21) and 20,596 (21) ng.h/mL, respectively). Conclusions: AG 5 mg BID can be combined with standard P/C and G/Cis regimens with no apparent overlapping toxicities and unaltered PK. Both combinations have antitumor activity. In pts with squamous NSCLC, AG + P/C was well tolerated with no evidence of grade ≥3 hemoptysis. [Table: see text]

CD8+CD205+ Splenic Dendritic Cells Are Specialized to Induce Foxp3+ Regulatory T Cells

Foxp3(+)CD25(+)CD4(+) regulatory T cells (Treg) mediate immunological self-tolerance and suppress immune responses. A subset of dendritic cells (DCs) in the intestine is specialized to induce Treg in a TGF-beta- and retinoic acid-dependent manner to allow for oral tolerance. In this study we compare two major DC subsets from mouse spleen. We find that CD8(+) DEC-205/CD205(+) DCs, but not the major fraction of CD8(-) DC inhibitory receptor-2 (DCIR2)(+) DCs, induce functional Foxp3(+) Treg from Foxp3(-) precursors in the presence of low doses of Ag but without added TGF-beta. CD8(+)CD205(+) DCs preferentially express TGF-beta, and the induction of Treg by these DCs in vitro is blocked by neutralizing Ab to TGF-beta. In contrast, CD8(-)DCIR2(+) DCs better induce Foxp3(+) Treg when exogenous TGF-beta is supplied. In vivo, CD8(+)CD205(+) DCs likewise preferentially induce Treg from adoptively transferred, Ag-specific DO11.10 RAG(-/-) Foxp3(-)CD4(+) T cells, whereas the CD8(-)DCIR2(+) DCs better stimulate natural Foxp3(+) Treg. These results indicate that a subset of DCs in spleen, a systemic lymphoid organ, is specialized to differentiate peripheral Foxp3(+) Treg, in part through the endogenous formation of TGF-beta. Targeting of Ag to these DCs might be useful for inducing Ag-specific Foxp3(+) Treg for treatment of autoimmune diseases, transplant rejection, and allergy.

Actions of β-Carotene on T-Helper Cell Functions for Antibody Production

Abstract Actions of β-carotene isomers including all-trans-, 9-cis, and 13-cis-β-carotene and a β-carotene isomer mixture extracted from alga were examined in regard to T-helper (Th) cell function for antibody (Ab) production. β-carotene isomers did not significantly enhance in vitro Ab production by murine spleen cells, which may be partly due to the paucity of antigen (Ag)-specific Th cells. We thus replaced Th cells in spleen cell suspensions with Ag-specific Th clones in order to increase the frequency of Ag-specific Th cells. Then β-carotene isomers' effects on in vitro Ab production were reassessed. In this system, the enhancing actions of β-carotene isomers on Ab production were more evident compared to controls: (1) when cells were stimulated with suboptimal doses of Ag, and (2) when a type-2 T-helper (Th2) cell clone was used. The effects of all-trans-and 9-cis-β-carotene did not differ significantly, but the effects of 13-cis-β-carotene on Th clones was less than those exhibited by other β-carot...