Hemagglutination inhibition (HAI) titers against 2009 pandemic H1N1 viruses.

<p>Serum samples collected from US healthy volunteers vaccinated with 2009–2010 inactivated unadjuvanted trivalent influenza vaccine (TIV) were tested for cross-reactivity against 2009 pandemic H1N1 viruses by HAI assay using 0.5% turkey erythrocytes. The pandemic influenza specific seroprotection rates (the proportion of subjects having an HAI titer ≥40) before and 21 days after TIV administration and the seroconversion rates (the proportion of subjects having a ≥4-fold rise in HAI titers) were plotted according to the age distribution of vaccinees for A/California/07/2009 (H1N1) (A and F), A/Iraq/8529/2009 (H1N1) (B and G), A/Ontario/RV3226/2009 (H1N1) (C and H), A/South Carolina/18/2009 (H1N1) (D and I) and A/England/195/2009 (H1N1) (E and J), respectively. * indicates the corresponding seroconversion rate in panel F, G, H, I, and J.</p

Hemagglutination inhibition (HAI) titers against 2009–2010 seasonal vaccine strains.

<p>Serum samples collected from US healthy volunteers vaccinated with 2009–2010 inactivated unadjuvanted trivalent influenza vaccine (TIV) were tested by HAI assay using 0.5% turkey erythrocytes. The geometric mean titers (GMTs), the seroprotection rates (the proportion of subjects having an HAI titer ≥40) before and 21 days after TIV administration, and the seroconversion rates (the proportion of subjects having a ≥4-fold increase in HAI titers) were plotted according to the age distribution of vaccinees for A/Brisbane/59/2007 (H1N1) (A and D), A/Uruguay/716/2007 (H3N2) (B and E), and B/Brisbane/60/2008 (C and F), respectively. The 95% CI for individual HAI GMTs are shown as error bars. The dotted lines indicate HAI titer of 40.</p

Correlation of antibody titers specific for seasonal vaccine strains and pandemic viruses after seasonal vaccination.

<p>Serum samples were collected on 21 days after administration of 2009–2010 inactivated unadjuvanted trivalent influenza vaccine (TIV) in US healthy adults and elderly. An HAI assay was performed using 0.5% turkey erythrocytes. A correlation analysis between seasonal strain specific HAI titers and pandemic strain specific HAI titers was performed using nonparametric Spearman's ρ test by JMP Version 7. The scatterplots of HAI titers are shown in the presence of 95% bivariate normal density ellipses (indicating the distribution of 95% of individual data points plotted) and corresponding <i>p</i> values. A, A/California/07/2009 (H1N1) vs A/Uruguay/716/2007 (H3N2); B, A/South Carolina/18/2009 (H1N1) vs A/Uruguay/716/2007 (H3N2).</p

Comparative Glycomics Analysis of Influenza Hemagglutinin (H5N1) Produced in Vaccine Relevant Cell Platforms

Hemagglutinin (HA) is the major antigen in influenza vaccines, and glycosylation is known to influence its antigenicity. Embryonated hen eggs are traditionally used for influenza vaccine production, but vaccines produced in mammalian and insect cells were recently licensed. This raises the concern that vaccines produced with different cell systems might not be equivalent due to differences in their glycosylation patterns. Thus, we developed an analytical method to monitor vaccine glycosylation through a combination of nanoLC/MS^E and quantitative MALDI-TOF MS permethylation profiling. We then used this method to examine glycosylation of HAs from two different influenza H5N1 strains produced in five different platforms, including hen eggs, three different insect cell lines (High Five, <i>expres</i>SF+ and glycoengineered <i>expres</i>SF+), and a human cell line (HEK293). Our results demonstrated that (1) sequon utilization is not necessarily equivalent in different cell types, (2) there are quantitative and qualitative differences in the overall <i>N</i>-glycosylation patterns and structures produced by different cell types, (3) ∼20% of the <i>N</i>-glycans on the HAs produced by High Five cells are core α1,3-fucosylated structures, which may be allergenic in humans, and (4) our method can be used to monitor differences in glycosylation during the cellular glycoengineering stages of vaccine development

β_1/2 or M_2/3 Receptors Are Required for Different Gastrointestinal Motility Responses Induced by Acupuncture at Heterotopic or Homotopic Acupoints

<div><p>Acupuncture at homotopic acupoints or heterotopic acupoints is known to either inhibit or facilitate gastrointestinal motility, depending on the acupoint location. However, little effort has been made to investigate the roles of specific receptors (such as adrenergic and muscarinic acetylcholine receptors) in mediating the effects of acupuncture at heterotopic and homotopic acupoints. Different adrenergic receptor subtypes or cholinergic receptor subtypes are predominantly expressed in various sections of the gut, resulting in variations between the effects of acupuncture at heterotopic or homotopic acupoints on gastrointestinal motility. Here, we investigated the role of β₁/β₂ receptors and M₂/M₃ receptors in gastrointestinal motility regulated by acupuncture at ST37, a heterotopic acupoint, and ST25, a homotopic acupoint, by simultaneously recording intraluminal pressures in the distal colon and stomach or jejunum and examining fecal phenol red excretion in β_1/2 receptor-knockout mice and M_2/3 receptor-knockout mice. We found that knockout of the M_2/3 receptor significantly inhibited ST37 acupuncture-induced enhancement of gastric motility, jejunal motility, and colonic motility. Additionally, knocking out of the β_1/2 receptor significantly diminished the ST25 acupuncture-induced inhibition of gastric motility and jejunal motility without significantly altering the enhancement of colonic motility induced by acupuncture at ST25. Acupuncture at ST37 significantly accelerated gastrointestinal transition in β_1/2 receptor-knockout mice and their wild-type littermates. However, this acceleration of gastrointestinal transition was markedly diminished in M_2/3 receptor-knockout mice relative to their wild-type littermates. Acupuncture at ST25 significantly increased gastrointestinal transition in β_1/2 receptor-knockout mice and significantly decreased gastrointestinal transition in M_2/3 receptor-knockout mice without altering gastrointestinal transition in wild-type littermates of either. Our study revealed that M_2/3 receptors are required for the gastrointestinal motility associated with whole gastrointestinal transition enhanced by acupuncture at heterotopic acupoints, whereas β_1/2 receptors are required for the same gastrointestinal motility processes inhibited by acupuncture at homotopic acupoints. Therefore, our findings reveal important biological mechanisms underlying acupuncture treatment of disorders involving gastrointestinal motility dysfunction.</p></div

Effect of acupuncture at ST37 or ST25 on jejunal motility in β_1/2-AR KO mice and M_2/3-R KO mice.

<p><b>(A)</b> Representative traces of jejunal motility regulated by acupuncture at ST37 in β_1/2-AR KO mice and M_2/3-R KO mice. <b>(B)</b> Representative traces of jejunal motility regulated by acupuncture at ST25 in β_1/2-AR KO mice and M_2/3-R KO mice. <b>(C)</b> β_1/2-AR deletion did not change intrajejunal pressure increased by acupuncture at ST37 relative to WT littermates (unpaired <i>t</i>-test, n = 10 in each group). M_2/3-R deletion significantly reduced the increase in intrajejunal pressure caused by acupuncture at ST37 relative to WT littermates (* P < 0.05, unpaired <i>t</i>-test, n = 10 in each group). The dashed line denotes basal intrajejunal pressure before acupuncture. <b>(D)</b> β_1/2-AR or M_2/3-R deletion did not change the jejunal motility frequency induced by acupuncture at ST37 relative to their WT littermates (unpaired <i>t</i>-test, n = 10 in each group). The dashed line denotes basal intrajejunal frequency before acupuncture. <b>(E)</b> β_1/2-AR deletion significantly increased intrajejunal pressure reduced by acupuncture at ST25 relative to WT littermates (** P < 0.01, unpaired <i>t-</i>test, n = 10 in each group); M_2/3-R deletion did not significantly affect the decrease in intrajejunal pressure caused by acupuncture at ST25 relative to WT littermates (unpaired <i>t</i>-test, n = 10 in each group). The dashed line denotes basal intrajejunal pressure before acupuncture. <b>(F)</b> β_1/2-AR or M_2/3-R deletion did not change jejunal motility frequency mediated by acupuncture at ST25 relative to WT littermates (unpaired <i>t</i>-test, n = 10 in each group). The dashed line denotes basal jejunal frequency before acupuncture; MA: manual acupuncture.</p

Dry-wet ratio in β_1/2-AR KO mice and M_2/3-R KO mice.

<p>Dry-wet ratio in both β_1/2-AR KO mice and M_2/3-R KO mice was significantly higher than in their WT littermates (* P < 0.05, ** P < 0.01, unpaired <i>t</i>-test, n = 18 in each group).</p

Effect of acupuncture at ST37 or ST25 on phenol red excretion in β_1/2-AR KO mice and M_2/3-R KO mice.

<p><b>(A)</b> β_1/2-AR deletion did not significantly increase phenol red excretion in feces (unpaired <i>t</i>-test, n = 10 in each group); acupuncture at ST37 increased fecal phenol red excretion significantly, relative to non-acupuncture, in both β_1/2-AR KO mice and their WT littermates (* P < 0.05, ** P < 0.01; unpaired <i>t</i>-test, n = 10 in each group). <b>(B)</b> β_1/2-AR deletion did not increase phenol red excretion in feces significantly (unpaired <i>t</i>-test, n = 10 in each group); Acupuncture at ST25 increased fecal phenol red excretion significantly relative to non-acupuncture in β_1/2-AR KO mice (** P < 0.01; unpaired <i>t</i>-test, n = 10 in each group); <b>(C)</b> M_<b>2/3</b>-R deletion increased phenol red excretion in feces significantly (## P < 0.01, unpaired <i>t</i>-test, n = 10 in each group); Acupuncture at ST37 facilitated fecal phenol red excretion significantly relative to non-acupuncture in WT littermates (** P < 0.01; unpaired <i>t</i>-test, n = 10 in each group). <b>(D)</b> M_2/3-R deletion increased phenol red excretion in feces significantly (## P < 0.01, unpaired <i>t</i>-test, n = 10 in each group); Acupuncture at ST25 decreased fecal phenol red excretion significantly relative to non-acupuncture in M_2/3-R KO mice (*P < 0.05; unpaired <i>t</i>-test, n = 10 in each group). <b>(E)</b> The rate of increase in phenol red excretion induced by acupuncture at ST37 in β_1/2-AR KO mice was not significantly different from that in WT littermates (unpaired <i>t</i>-test, n = 10 in each group). Deletion of M_2/3-Rs significantly abolished the increase in phenol red excretion induced by acupuncture at ST37 compared with that in WT littermates (** P < 0.01, unpaired <i>t</i>-test, n = 10 in each group). <b>(F)</b> β_1/2-AR knockout significantly diminished the decrease in phenol red excretion induced by acupuncture at ST25 relative to WT littermates (** P < 0.01, unpaired <i>t</i>-test, n = 10 in each group); the change in phenol red excretion induced by acupuncture at ST25 in M_2/3-R KO mice was significantly different from that in WT littermates (** P < 0.01, unpaired <i>t</i>-test, n = 10 in each group).</p

Effect of acupuncture at ST37 or ST25 on distal colonic motility in β_1/2-AR KO mice and M_2/3-R KO mice.

<p><b>(A)</b> Representative traces of distal colonic motility regulated by acupuncture at ST37 in β_1/2-AR KO mice and M_2/3-R KO mice. <b>(B)</b> Representative traces of distal colonic motility regulated by acupuncture at ST25 in β_1/2-AR KO mice and M_2/3-R KO mice. <b>(C)</b> β_1/2-AR deletion did not affect the increase in intracolonic pressure due to acupuncture at ST37, relative to WT littermates (unpaired <i>t</i>-test, n = 10 in each group). M_2/3-R deletion significantly reduced the increase in intracolonic pressure due to acupuncture at ST37 relative to WT littermates (* P < 0.05, unpaired <i>t</i>-test, n = 10 in each group). The dashed line denotes basal intracolonic pressure before acupuncture. <b>(D)</b> β_1/2-AR or M_2/3-R deletion did not affect the change in distal colonic motility frequency induced by acupuncture at ST37 relative to WT littermates (unpaired <i>t</i>-test, n = 10 in each group). The dashed line denotes basal intrajejunal frequency before acupuncture. <b>(E)</b> β_1/2-AR deletion did not significantly change the increase in intracolonic pressure due to acupuncture at ST25 relative to WT littermates (unpaired <i>t</i>-test, n = 10 in each group); M_2/3-R deletion significantly reduced the increase in intracolonic pressure resulting from acupuncture at ST25 relative to WT littermates (** P < 0.01, unpaired <i>t</i>-test, n = 10 in each group). The dashed line denotes basal intracolonic pressure before acupuncture. <b>(F)</b> β_1/2-AR or M_2/3-R deletion did not change distal colonic motility frequency induced by acupuncture at ST25, relative to WT littermates (unpaired <i>t</i>-test, n = 10 in each group). The dashed line denotes basal jejunal frequency before acupuncture; MA: manual acupuncture.</p