Limited impact of fingolimod treatment during the initial weeks of ART in SIV-infected rhesus macaques.

Antiretroviral therapy (ART) is not curative due to the persistence of a reservoir of HIV-infected cells, particularly in tissues such as lymph nodes, with the potential to cause viral rebound after treatment cessation. In this study, fingolimod (FTY720), a lysophospholipid sphingosine-1-phosphate receptor modulator is administered to SIV-infected rhesus macaques at initiation of ART to block the egress from lymphoid tissues of natural killer and T-cells, thereby promoting proximity between cytolytic cells and infected CD4+ T-cells. When compared with the ART-only controls, FTY720 treatment during the initial weeks of ART induces a profound lymphopenia and increases frequencies of CD8+ T-cells expressing perforin in lymph nodes, but not their killing capacity; FTY720 also increases frequencies of cytolytic NK cells in lymph nodes. This increase of cytolytic cells, however, does not limit measures of viral persistence during ART, including intact proviral genomes. After ART interruption, a subset of animals that initially receives FTY720 displays a modest delay in viral rebound, with reduced plasma viremia and frequencies of infected T follicular helper cells. Further research is needed to optimize the potential utility of FTY720 when coupled with strategies that boost the antiviral function of T-cells in lymphoid tissues.

Safety and Immunological Evaluation of Interleukin-21 Plus Anti-α4ß7 mAb Combination Therapy in Rhesus Macaques.

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections compromise gut immunological barriers, inducing high levels of inflammation and a severe depletion of intestinal CD4+ T cells. Expression of α4ß7 integrin promotes homing of activated T cells to intestinal sites where they become preferentially infected; blockade of α4ß7 with an anti-α4ß7 monoclonal antibody (mAb) prior to infection has been reported to reduce gut SIV viremia in rhesus macaques (RMs). Interleukin-21 (IL-21) administration in antiretroviral therapy-treated, SIV-infected RMs reduces gut inflammation and improves gut integrity. We therefore hypothesized that the combination of IL-21 and anti-α4ß7 mAb therapies could synergize to reduce inflammation and HIV persistence. We co-administered two intravenous doses of rhesus anti-α4ß7 mAb (50 mg/kg) combined with seven weekly subcutaneous infusions of IL-21-IgFc (100 µg/kg) in four healthy, SIV-uninfected RMs to evaluate the safety and immunological profiles of this intervention in blood and gut. Co-administration of IL-21 and anti-α4ß7 mAb showed no toxicity at the given dosages as assessed by multiple hematological and chemical parameters and did not alter the bioavailability of the therapeutics or result in the generation of antibodies against the anti-α4ß7 mAb or IL-21-IgFc. Upon treatment, the frequency of CD4 memory T cells expressing ß7 increased in blood and decreased in gut, consistent with an inhibition of activated CD4 T-cell homing to the gut. Furthermore, the frequency of T cells expressing proliferation and immune activation markers decreased in blood and, more profoundly, in gut. The combined IL-21 plus anti-α4ß7 mAb therapy is well-tolerated in SIV-uninfected RMs and reduces the gut homing of α4ß7+ CD4 T cells as well as the levels of gut immune activation.

Enhanced Rg3 negatively regulates Th1 cell responses.

BACKGROUND: Korean Red Ginseng (KRG; Panax ginseng Meyer) is a widely used medicinal herb known to exert various immune modulatory functions. KRG and one of its purified components, ginsenoside Rg3, are known to possess anti-inflammatory activities. How they impact helper T cell-mediated responses is not fully explored. In this study, we attempted to evaluate the effect of KRG extract (KRGE) and ginsenoside Rg3 on Th1 cell responses. METHODS: Using well-characterized T cell in vitro differentiation systems, we examined the effects of KRGE or enhanced Rg3 on the Th1-inducing cytokine production from dendritic cells (DC) and the naïve CD4+ T cells differentiation to Th1 cells. Furthermore, we examined the change of Th1 cell population in the intestine after treatment of enhanced Rg3. The influence of KRGE or enhanced Rg3 on Th1 cell differentiation was evaluated by fluorescence-activated cell sorting, enzyme-linked immunosorbent assay, and quantitative real-time polymerase chain reaction. RESULTS: KRGE significantly inhibited the production level of IL-12 from DCs and subsequent Th1 cell differentiation. Similarly, enhanced Rg3 significantly suppressed the expression of interferon gamma (IFNγ) and T-bet in T cells under Th1-skewing condition. Consistent with these effects in vitro, oral administration of enhanced Rg3 suppressed the frequency of Th1 cells in the Peyer's patch and lamina propria cells in vivo. CONCLUSION: Enhanced Rg3 negatively regulates the differentiation of Th1 cell in vitro and Th1 cell responses in the gut in vivo, providing fundamental basis for the use of this agent to treat Th1-related diseases.

Fibrinogen cleavage products and Toll-like receptor 4 promote the generation of programmed cell death 1 ligand 2-positive dendritic cells in allergic asthma.

BACKGROUND: Inhaled protease allergens preferentially trigger TH2-mediated inflammation in allergic asthma. The role of dendritic cells (DCs) on induction of TH2 cell responses in allergic asthma has been well documented; however, the mechanism by which protease allergens induce TH2-favorable DCs in the airway remains unclear. OBJECTIVE: We sought to determine a subset of DCs responsible for TH2 cell responses in allergic asthma and the mechanism by which protease allergens induce the DC subset in the airway. METHODS: Mice were challenged intranasally with protease allergens or fibrinogen cleavage products (FCPs) to induce allergic airway inflammation. DCs isolated from mediastinal lymph nodes were analyzed for surface phenotype and T-cell stimulatory function. Anti-Thy1.2 and Mas-TRECK mice were used to deplete innate lymphoid cells and mast cells, respectively. Adoptive cell transfer, bone marrow DC culture, anti-IL-13, and Toll-like receptor (TLR) 4-deficient mice were used for further mechanistic studies. RESULTS: Protease allergens induced a remarkable accumulation of TH2-favorable programmed cell death 1 ligand 2 (PD-L2)+ DCs in mediastinal lymph nodes, which was significantly abolished in mice depleted of mast cells and, to a lesser extent, innate lymphoid cells. Mechanistically, FCPs generated by protease allergens triggered IL-13 production from wild-type mast cells but not from TLR4-deficient mast cells, which resulted in an increase in the number of PD-L2+ DCs. Intranasal administration of FCPs induced an increase in numbers of PD-L2+ DCs in the airway, which was significantly abolished in TLR4- and mast cell-deficient mice. Injection of IL-13 restored the PD-L2+ DC population in mice lacking mast cells. CONCLUSION: Our findings unveil the "protease-FCP-TLR4-mast cell-IL-13" axis as a molecular mechanism for generation of TH2-favorable PD-L2+ DCs in allergic asthma and suggest that targeting the PD-L2+ DC pathway might be effective in suppressing allergic T-cell responses in the airway.

Clonal Expansion of Allergen-specific CD4+ T Cell in the Lung in the Absence of Lymph Nodes.

The expansion of allergen-specific CD4+ T cells is a critical step in inducing airway inflammation during allergic asthma. Such clonal expansion of T cells is initiated through the interaction between allergen-bearing dendritic cells and allergen-specific naïve T cells in the draining lymph nodes. Whether such T cell clonal expansion also occurs in the lung, the primary organ encountering inhaled allergens, remains unclear. Compared with wild-type mice, we found similar frequencies of CD4+ T cells in the lung of lymph node-deficient Rorgtgfp/gfp mice after repeated exposure to inhaled allergens. In addition, we observed an evident population of CD4+ T cells that underwent clonal expansion in the lung of allergen-challenged mice treated with an S1P antagonist FTY720 in an in vivo proliferation study with CFSE-labeled OT-II T cells. Moreover, the expansion of allergen-specific CD4+ T cells was significantly enhanced in the lungs of Rorgtgfp/gfp mice in comparison to that of wild-type mice. These results together demonstrate that the clonal expansion of allergen-specific CD4+ T cells occurs in the absence of the lymph nodes, indicating that the lung can act as a primary site of the clonal expansion of CD4+ T cells in response to inhaled allergens.

Colloidal polymers from dipolar assembly of cobalt-tipped CdSe@CdS nanorods.

The synthesis of a modular colloidal polymer system based on the dipolar assembly of CdSe@CdS nanorods functionalized with a single cobalt nanoparticle "tip" (CoNP-tip) is reported. These heterostructured nanorods spontaneously self-assembled via magnetic dipolar associations of the cobalt domains. In these assemblies, CdSe@CdS nanorods were carried as densely grafted side chain groups along the dipolar NP chain to form bottlebrush-type colloidal polymers. Nanorod side chains strongly affected the conformation of individual colloidal polymer bottlebrush chains and the morphology of thin films. Dipolar CoNP-tipped nanorods were then used as "colloidal monomers" to form mesoscopic assemblies reminiscent of traditional copolymers possessing segmented and statistical compositions. Investigation of the phase behavior of colloidal polymer blends revealed the formation of mesoscopic phase separated morphologies from segmented colloidal copolymers. These studies demonstrated the ability to control colloidal polymer composition and morphology in a manner observed for classical polymer systems by synthetic control of heterostructured nanorod structure and harnessing interparticle dipolar associations.

Directing the deposition of ferromagnetic cobalt onto Pt-tipped CdSe@CdS nanorods: synthetic and mechanistic insights.

A methodology providing access to dumbbell-tipped, metal-semiconductor and metal oxide-semiconductor heterostructured nanorods has been developed. The synthesis and characterization of CdSe@CdS nanorods incorporating ferromagnetic cobalt nanoinclusions at both nanorod termini (i.e., dumbbell morphology) are presented. The key step in the synthesis of these heterostructured nanorods was the decoration of CdSe@CdS nanorods with platinum nanoparticle tips, which promoted the deposition of metallic CoNPs onto Pt-tipped CdSe@CdS nanorods. Cobalt nanoparticle tips were then selectively oxidized to afford CdSe@CdS nanorods with cobalt oxide domains at both termini. In the case of longer cobalt-tipped nanorods, heterostructured nanorods were observed to self-organize into complex dipolar assemblies, which formed as a consequence of magnetic associations of terminal CoNP tips. Colloidal polymerization of these cobalt-tipped nanorods afforded fused nanorod assemblies from the oxidation of cobalt nanoparticle tips at the ends of nanorods via the nanoscale Kirkendall effect. Wurtzite CdS nanorods survived both the deposition of metallic CoNP tips and conversion into cobalt oxide phases, as confirmed by both XRD and HRTEM analysis. A series of CdSe@CdS nanorods of four different lengths ranging from 40 to 174 nm and comparable diameters (6-7 nm) were prepared and modified with both cobalt and cobalt oxide tips. The total synthesis of these heterostructured nanorods required five steps from commercially available reagents. Key synthetic considerations are discussed, with particular emphasis on reporting isolated yields of all intermediates and products from scale up of intermediate precursors.

Cell cycle-dependent induction of homologous recombination by a tightly regulated I-SceI fusion protein.

Double-strand break repair is executed by two major repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). Whereas NHEJ contributes to the repair of ionizing radiation (IR)-induced double strand breaks (DSBs) throughout the cell cycle, HR acts predominantly during the S and G2 phases of the cell cycle. The rare-cutting restriction endonuclease, I-SceI, is in common use to study the repair of site-specific chromosomal DSBs in vertebrate cells. To facilitate analysis of I-SceI-induced DSB repair, we have developed a stably expressed I-SceI fusion protein that enables precise temporal control of I-SceI activation, and correspondingly tight control of the timing of onset of site-specific chromosome breakage. I-SceI-induced HR showed a strong, positive linear correlation with the percentage of cells in S phase, and was negatively correlated with the G1 fraction. Acute depletion of BRCA1, a key regulator of HR, disrupted the relationship between S phase fraction and I-SceI-induced HR, consistent with the hypothesis that BRCA1 regulates HR during S phase.

Magnetic self-assembly of gold nanoparticle chains using dipolar core-shell colloids.

The preparation of gold nanoparticle (AuNP) assemblies was conducted by the synthesis and dipolar assembly of ferromagnetic core-shell nanoparticles composed of AuNP cores and cobalt NP shells. Dissolution of metallic Co phases with mineral acids afforded self-assembled AuNP chains and bracelets.

Synthesis and colloidal polymerization of ferromagnetic Au-Co nanoparticles into Au-Co3O4 nanowires.

The preparation of cobalt oxide nanowires with gold nanoparticle (AuNP) inclusions (Au-Co(3)O(4) nanowires) via colloidal polymerization of dipolar core-shell NPs is reported. Polystyrene-coated ferromagnetic NPs composed of a dipolar metallic cobalt shell and a gold NP core (PS-AuCoNPs) were synthesized by thermolysis of octacarbonyldicobalt [Co(2)(CO)(8)] in the presence of AuNP seeds and polymeric ligands. The colloidal polymerization process of these dipolar PS-AuCoNPs comprises dipolar nanoparticle assembly and solution oxidation of preorganized NPs to form interconnected cobalt oxide nanowires via the nanoscale Kirkendall effect, with AuNP inclusions in every repeating unit of the one-dimensional mesostructure. Calcination of the polymer-coated nanowires afforded polycrystalline Au-Co(3)O(4) nanowires that were determined to be electroactive. Nanocomposite materials were characterized by transmission electron microscopy, field-emission scanning electron microscopy, X-ray diffraction, vibrating sample magnetometry, and cyclic voltammetry. We demonstrate that the optical and electrochemical properties of Au-Co(3)O(4) nanowires are significantly enhanced in comparison with hollow Co(3)O(4) nanowires prepared via colloidal polymerization.

Colloidal polymerization of polymer-coated ferromagnetic nanoparticles into cobalt oxide nanowires.

The preparation of polystyrene-coated cobalt oxide nanowires is reported via the colloidal polymerization of polymer-coated ferromagnetic cobalt nanoparticles (PS-CoNPs). Using a combination of dipolar nanoparticle assembly and a solution oxidation of preorganized metallic colloids, interconnected nanoparticles of cobalt oxide spanning micrometers in length were prepared. The colloidal polymerization of PS-CoNPs into cobalt oxide (CoO and Co(3)O(4)) nanowires was achieved by bubbling O(2) into PS-CoNP dispersions in 1,2-dichlorobenzene at 175 degrees C. Calcination of thin films of PS-coated cobalt oxide nanowires afforded Co(3)O(4) metal oxide materials. Transmission electron microscopy (TEM) revealed the formation of interconnected nanoparticles of cobalt oxide with hollow inclusions, arising from a combination of dipolar assembly of PS-CoNPs and the nanoscale Kirkendall effect in the oxidation reaction. Using a wide range of spectroscopic and electrochemical characterization techniques, we demonstrate that cobalt oxide nanowires prepared via this novel methodology were electroactive with potential applications as nanostructured electrodes for energy storage.

Lanthanide(III)-doped magnetite nanoparticles.

Nearly monodisperse lanthanide-doped magnetite nanoparticles were obtained by thermally decomposing a mixture of Fe(acac)(3) and Ln(acac)(3) (acac = acetylacetonate; Ln = Sm, Eu, Gd) in the presence of passivating surfactants. Magnetic studies revealed room-temperature ferromagnetic behaviors of these doped nanoparticles, distinctly different from those of the undoped parent magnetite or the doped nanoparticles prepared by a coprecipitation method.

Synthesis and self-assembly of polymer-coated ferromagnetic nanoparticles.

We describe the synthesis and characterization of polymer-coated ferromagnetic cobalt nanoparticles (CoNPs). The synthesis of end-functionalized polystyrene surfactants possessing amine, carboxylic acid, or phosphine oxide end-groups was accomplished using atom-transfer radical polymerization. This versatile synthetic method enabled the production of multigram quantities of these polymeric surfactants that stabilized ferromagnetic CoNPs when dispersed in organic media. An in-depth investigation into the synthesis of polystyrene-coated ferromagnetic CoNPs was also conducted using various combinations of these polymeric surfactants in the thermolysis of dicobaltoctacarbonyl (Co(2)(CO)(8)). Moreover, the application of a dual-stage thermolysis with Co(2)(CO)(8) allowed for the preparation of large samples (200-820 mg) per batch of well-defined and dispersable ferromagnetic nanoparticles. Characterization of these functionalized nanoparticle materials was then done using transmission electron microscopy, X-ray diffraction, vibrating sample magnetometry, and thermogravimetric analysis. Self-assembly of these dipolar nanoparticles was investigated in solutions cast onto supporting substrates, where local nematic-like ordering of nanoparticle chains was observed along with a tendency of adjacent chains to form "zippering" configurations, both phenomena having been predicted by recent simulations of dipolar fluids in conjunction with van der Waals interactions.

Polymer-coated ferromagnetic colloids from well-defined macromolecular surfactants and assembly into nanoparticle chains.

A novel synthetic route to polymer-coated ferromagnetic colloids of metallic cobalt has been developed. Well-defined end-functional polystyrenes were synthesized using controlled radical polymerization and used as surfactants in the thermolysis of dicobaltoctacarbonyl to afford uniform ferromagnetic nanoparticles. The presence of the polymer shell enabled prolonged colloidal stability of dispersions in a wide range of organic solvents and formed glassy encapsulating coatings around ferromagnetic cores in the solid state. These polymer-coated colloids assembled into robust, micron-sized nanoparticle chains when cast onto supporting surfaces due to dipolar associations of magnetic cores. Hierarchical assemblies were also prepared by blending polystyrene-coated cobalt colloids with larger silica beads.

Control of sister chromatid recombination by histone H2AX.

Histone H2AX has a role in suppressing genomic instability and cancer. However, the mechanisms by which it performs these functions are poorly understood. After DNA breakage, H2AX is phosphorylated on serine 139 in chromatin near the break. We show here that H2AX serine 139 enforces efficient homologous recombinational repair of a chromosomal double-strand break (DSB) by using the sister chromatid as a template. BRCA1, Rad51, and CHK2 contribute to recombinational repair, in part independently of H2AX. H2AX(-/-) cells show increased use of single-strand annealing, an error-prone deletional mechanism of DSB repair. Therefore, the chromatin response around a chromosomal DSB, in which H2AX serine 139 phosphorylation plays a central role, "shapes" the repair process in favor of potentially error-free interchromatid homologous recombination at the expense of error-prone repair. H2AX phosphorylation may help set up a favorable disposition between sister chromatids.

Bladder mucosa pH and Pco2 as a minimally invasive monitor of hemorrhagic shock and resuscitation.

BACKGROUND: Continuous monitoring of pH, Pco2, and Po2 using fiberoptic sensor technology has been proposed recently as a clinical monitor of the severity of shock and impaired tissue perfusion. Surrogates of gut tissue perfusion such as gastric tonometry, although cumbersome, have been used to indirectly quantify the degree of gut ischemia. The purpose of this study was to demonstrate the feasibility of monitoring bladder mucosa (BM) and to compare urinary bladder mucosa and proximal jejunum mucosa interstitial pH and Pco2 during hemorrhagic shock and resuscitation. METHODS: Eleven male miniature swine (25-35 kg) (control, n = 4; shock, n = 7) underwent jejunal tonometry and cystostomy. A multisensor probe was placed adjacent to the BM. Urine was diverted. Normocarbia was maintained. Animals were hemorrhaged and kept at a mean arterial pressure of 40 mm Hg. When a constant infusion was required to maintain the mean arterial pressure at 40 mm Hg (decompensation), animals were resuscitated with shed blood plus two times the shed volume in lactated Ringer's solution (20 minutes) and observed for 2 hours. RESULTS: During decompensation, BM pH values decreased significantly from 7.33 +/- 0.08 to 7.01 +/- 0.2 (p < 0.01) and recovered to 7.11 +/- 0.19 at 120 minutes after completion of resuscitation. During decompensation, BM Pco2 values increased significantly compared with baseline (from 49 +/- 6 mm Hg to 71 +/- 19 mm Hg, p < 0.05) and returned to baseline with resuscitation. Jejunum mucosa and BM interstitial Pco2 correlated throughout shock and resuscitation (r = 0.49). Bland-Altman analysis demonstrated significant differences between jejunum mucosa (intramucosal pH) and BM interstitial pH. CONCLUSION: Shock-induced changes in the Pco2 of the BM are comparable to tonometric changes in the gut. These data suggest that continuous fiberoptic multisensor probe monitoring of the BM could potentially provide a minimally invasive method for the assessment of impaired tissue perfusion of the splanchnic circulation during shock and resuscitation.