Rapid Estimation of Binding Activity of Influenza Virus Hemagglutinin to Human and Avian Receptors

A critical step for avian influenza viruses to infect human hosts and cause epidemics or pandemics is acquisition of the ability of the viral hemagglutinin (HA) to bind to human receptors. However, current global influenza surveillance does not monitor HA binding specificity due to a lack of rapid and reliable assays. Here we report a computational method that uses an effective scoring function to quantify HA-receptor binding activities with high accuracy and speed. Application of this method reveals receptor specificity changes and its temporal relationship with antigenicity changes during the evolution of human H3N2 viruses. The method predicts that two amino acid differences at 222 and 225 between HAs of A/Fujian/411/02 and A/Panama/2007/99 viruses account for their differences in binding to both avian and human receptors; this prediction was verified experimentally. The new computational method could provide an urgently needed tool for rapid and large-scale analysis of HA receptor specificities for global influenza surveillance.National Key Project (2008ZX10004-013)National Institutes of Health (U.S.) (grant AI07443)Singapore-MIT Alliance for Research and TechnologyMassachusetts Institute of Technology. International Science and Technology Initiatives Global Seed FundNational Basic Research Program (973 Program) (2009CB918503)National Basic Research Program (973 Program) (2006CB911002

High strength composites using interlocking carbon nanotubes in a polyimide matrix

Polyethylene was crystallized on carbon nanotubes (CNTs) resulting in the formation of nanosheets on the surface. The material was then carbonized in sulfuric acid at 20 degrees C for 24 h, resulting in a carbon shish-kebab (CSK) structure. Incorporating the CSKs in polyimide (PI) matrix produced a uniform dispersion in which interactions between the nodules producing an interlocking effect that significantly improved the load transfer between the matrix and the CNTs. Consequently, the CSK/PI composite showed a 27% increase in tensile strength, compared to one using pristine CNTs, and 100% increase in the strain compared to the pure PI membrane

A flagellin-derived toll-like receptor 5 agonist stimulates cytotoxic lymphocyte-mediated tumor immunity.

Toll-like receptor (TLR) mediated recognition of pathogen associated molecular patterns allows the immune system to rapidly respond to a pathogenic insult. The "danger context" elicited by TLR agonists allows an initially non-immunogenic antigen to become immunogenic. This ability to alter environment is highly relevant in tumor immunity, since it is inherently difficult for the immune system to recognize host-derived tumors as immunogenic. However, immune cells may have encountered certain TLR ligands associated with tumor development, yet the endogenous stimulation is typically not sufficient to induce spontaneous tumor rejection. Of special interest are TLR5 agonists, because there are no endogenous ligands that bind TLR5. CBLB502 is a pharmacologically optimized TLR5 agonist derived from Salmonella enterica flagellin. We examined the effect of CBLB502 on tumor immunity using two syngeneic lymphoma models, both of which do not express TLR5, and thus do not directly respond to CBLB502. Upon challenge with the T-cell lymphoma RMAS, CBLB502 treatment after tumor inoculation protects C57BL/6 mice from death caused by tumor growth. This protective effect is both natural killer (NK) cell- and perforin-dependent. In addition, CBLB502 stimulates clearance of the B-cell lymphoma A20 in BALB/c mice in a CD8(+) T cell-dependent fashion. Analysis on the cellular level via ImageStream flow cytometry reveals that CD11b(+) and CD11c(+) cells, but neither NK nor T cells, directly respond to CBLB502 as determined by NFκB nuclear translocation. Our findings demonstrate that CBLB502 stimulates a robust antitumor response by directly activating TLR5-expressing accessory immune cells, which in turn activate cytotoxic lymphocytes

TNF Receptor 1 Mediates Dendritic Cell Maturation and CD8 T Cell Response through Two Distinct Mechanisms

TNF-α and its two receptors (TNFR1 and 2) are known to stimulate dendritic cell (DC) maturation and T cell response. However, the specific receptor and mechanisms involved in vivo are still controversial. In this study, we show that in response to an attenuated mouse hepatitis virus infection, DCs fail to mobilize and up-regulate CD40, CD80, CD86, and MHC class I in TNFR1−/− mice as compared with the wild-type and TNFR2−/− mice. Correspondingly, virus-specific CD8 T cell response was dramatically diminished in TNFR1−/− mice. Adoptive transfer of TNFR1-expressing DCs into TNFR1−/− mice rescues CD8 T cell response. Interestingly, adoptive transfer of TNFR1-expressing naive T cells also restores DC mobilization and maturation and endogenous CD8 T cell response. These results show that TNFR1, not TNFR2, mediates TNF-α stimulation of DC maturation and T cell response to mouse hepatitis virus in vivo. They also suggest two mechanisms by which TNFR1 mediates TNF-α–driven DC maturation, as follows: a direct effect through TNFR1 expressed on immature DCs and an indirect effect through TNFR1 expressed on naive T cells.China. Ministry of Science and Technology (Grant number 2009CB522505)National Institutes of Health (U.S.) (Grant number AI69208

CBLB502 enhances the ability of splenocytes to control tumor growth <i>in vitro</i>.

<p>(A) SCCVII (a tumor line known to express TLR5 mRNA), A20 parental, luciferase-expressing, and RMAS parental and luciferase-expressing tumor cells were analyzed for TLR5 mRNA expression via RT-PCR. GAPDH was used as an internal loading control. (B) 2000 luciferase-expressing RMAS or A20 cells were cultured alone in 1 ml of complete media or co-cultured with 6×10⁶ C57BL/6 or BALB/c splenocytes, respectively. At 0 hr, the cells were treated with 10 µl (10 µg/mL) CBLB502 or media diluent. Tumor burden was measured by bioluminescence imaging after 72 hrs. Two tailed t-tests were used to determine significance (*P<0.05, **P<0.01). Shown are representative data from three independent experiments with similar results.</p

CBLB502 stimulates NK cell- and perforin-dependent tumor immunity.

<p>(A) Kaplan-Meier survival curve of WT C57BL/6 mice injected intravenously (IV) RMAS cells. Mice were treated with subcutaneous (SC) injections of either CBLB502 or PBS as described in Materials and Methods (PBS n = 8, CBLB502 n = 12). (B) Kaplan-Meier survival curve of WT C57BL/6 mice that were non-depleted or given either anti-CD8α antibody or anti-asialo GM1 antibody three days prior to IV injection with RMAS cells. PBS (n = 8), non-depleted CBLB502 (n = 12), NK depleted PBS (n = 8), NK depleted CBLB502 (n = 8), CD8 depleted PBS (n = 8), CD8 depleted CBLB502 (n = 8). (C) Peripheral blood was collected via eye bleeding 2 weeks after depletion with anti-asialo GM1 or anti-CD8α and the percentage of NK1.1⁺CD3⁻ and CD3⁺CD8⁺ T cells, respectively, were analyzed (n = 8 mice/group). (D) Kaplan-Meier survival curve of WT CBLB502 (n = 27), WT PBS (n = 27), Prf1^−/− CBLB502 (n = 15), Prf1^−/− PBS (n = 14). All asterisk (*) represent statistical significance as determined by Log-rank (Mantel-Cox) test versus non-depleted PBS control group (*P<0.05). All data shown are representative of one of at least three individual experiments, or data combined from multiple experiments.</p

CD11b⁺ CD11c⁻ and CD11b⁺ CD11c⁺ accessory cells, but not NK or T lymphocytes, directly respond to CBLB502 treatment.

<p>Nuclear translocation of the NFκB p65 subunit was used to evaluate TLR5 expression and functionality. Splenocytes were treated with 10 ng/mL TNF-α, 100 ng/mL LPS, 100 ng/mL CBLB502 or PBS for 1 hr. (A) Splenocytes were stained for CD4, CD8, or NK1.1 along with CD3. After cell surface staining, cells were permeabilized and stained intracellularly for NFκB p65. Target populations were then gated as CD3⁺CD4⁺, CD3⁺CD8⁺ and CD3⁻NK1.1⁺ and similarity score between DAPI and the FITC-labeled NFκB was measured. (B) Splenocytes were stained for CD11b, CD11c and NFκB as described in panel A. Target populations were then gated as CD11b⁺CD11c⁻ or CD11b⁺CD11c⁺ to quantitate the similarity score. (C) Representative images at 40× magnification of NK1.1⁺CD3⁻ cells and CD11b⁺ CD11c⁻ cells treated with PBS, TNF-α, LPS, or CBLB502. Two tailed t-tests were used to determine significance versus PBS-treated control samples (*P<0.05). Data are representative from one of least three experiments.</p

CD80 and CD86 are up-regulated <i>in vivo</i> after CBLB502 treatment in a TLR5 dependent manner.

<p>WT or TLR5^−/− C57BL/6 mice were injected s.c. with 1 µg CBLB502, 1 µg flagellin, or equivalent volume of PBS. 24 h post-injection splenocytes were harvested and stained for flow cytometry with CD11b, CD11c, CD80 and CD86 antibodies. For LPS injections, 10 µg LPS was injected intraperitoneally and splenocytes were harvested 6 hours later and stained with the aforementioned antibodies immediately. (A) CD80 expression on CD11b⁺CD11c⁻ and CD11b⁺CD11c⁺ cells from WT and TLR5^−/− mice after indicated treatments. (B) CD86 expression on these same cell subsets. Two tailed t-tests were used to determine significance versus PBS-treated controls (**P<0.01, n = 3–6 mice per group). Data are representative from one of least three experiments.</p

Plasma levels of pro-inflammatory cytokines are increased by CBLB502 treatment.

<p>C57BL/6 mice were treated s.c. with 5 µg CBLB502 and blood was collected via cardiac puncture from two mice per time point. Values depict concentration in plasma in pg/mL of various cytokines.</p