Structure and reactivity of [RuII(terpy)(N^N)Cl]Cl complexes: consequences for biological applications

The crystal structures of [RuII(terpy)(bipy)Cl]Cl·2H2O and [RuII(terpy)(en)Cl]Cl·3H2O, where terpy = 2,2′:6′,2′′-terpyridine, bipy = 2,2′-bipyridine and en = ethylenediamine, were determined and compared to the structure of the complexes in solution obtained by multi-nuclear NMR spectroscopy in DMSOd-6 as a solvent. In aqueous solution, both chlorido complexes aquate fully to the corresponding aqua complexes, viz. [RuII(terpy)(bipy)(H2O)]2+ and [RuII(terpy)(en)(H2O)]2+, within ca. 2 h and ca. 2 min at 37 °C, respectively. The spontaneous aquation reactions can only be suppressed by chloride concentrations as high as 2 to 4 M, i.e. concentrations much higher than that found in human blood. The corresponding aqua complexes are characterized by pKa values of ca. 10 and 11, respectively, which suggest a more labile coordinated water molecule in the case of the [RuII(terpy)(en)(H2O)]2+ complex. Substitution reactions of the aqua complexes with chloride, cyanide and thiourea show that the [RuII(terpy)(en)(H2O)]2+ complex is 30-60 times more labile than the [RuII(terpy)(bipy)(H2O)]2+ complex at 25 °C. Water exchange reactions for both complexes were studied by 17O-NMR and DFT calculations (B3LYP(CPCM)/def2tzvp//B3LYP/def2svp and ωB97XD(CPCM)/def2tzvp//B3LYP/def2svp). Thermal and pressure activation parameters for the water exchange and ligand substitution reactions support the operation of an associative interchange (Ia) process. The difference in reactivity between these complexes can be accounted for in terms of π-back bonding effects of the terpy and bipy ligands and steric hindrance on the bipy complex. Consequences for eventual biological application of the chlorido complexes are discussed

TNF Drives Monocyte Dysfunction with Age and Results in Impaired Anti-pneumococcal Immunity

Monocyte phenotype and output changes with age, but why this occurs and how it impacts anti-bacterial immunity are not clear. We found that, in both humans and mice, circulating monocyte phenotype and function was altered with age due to increasing levels of TNF in the circulation that occur as part of the aging process. Ly6C+ monocytes from old (18–22 mo) mice and CD14+CD16+ intermediate/inflammatory monocytes from older adults also contributed to this “age-associated inflammation” as they produced more of the inflammatory cytokines IL6 and TNF in the steady state and when stimulated with bacterial products. Using an aged mouse model of pneumococcal colonization we found that chronic exposure to TNF with age altered the maturity of circulating monocytes, as measured by F4/80 expression, and this decrease in monocyte maturation was directly linked to susceptibility to infection. Ly6C+ monocytes from old mice had higher levels of CCR2 expression, which promoted premature egress from the bone marrow when challenged with Streptococcus pneumoniae. Although Ly6C+ monocyte recruitment and TNF levels in the blood and nasopharnyx were higher in old mice during S. pneumoniae colonization, bacterial clearance was impaired. Counterintuitively, elevated TNF and excessive monocyte recruitment in old mice contributed to impaired anti-pneumococcal immunity since bacterial clearance was improved upon pharmacological reduction of TNF or Ly6C+ monocytes, which were the major producers of TNF. Thus, with age TNF impairs inflammatory monocyte development, function and promotes premature egress, which contribute to systemic inflammation and is ultimately detrimental to anti-pneumococcal immunity

Book Review: Being and Owning: The Body, Bodily Material, and the Law by Jesse Wall

This is a book review of Being and Owning: The Body, Bodily Material, and the Law by Jesse Wall

Breakout Discussion: Pro Bono Business Law

MODERATOR Alicja Puchta, Osgoode Hall Law School PANELLISTS Susan Nickerson, Torys LLP John Walsh, OPTrust Andrea Boctor, Stikeman Elliott LLP Tamara Nachmani, Blakes, Cassels & Graydon LL

<i>Streptococcus pneumoniae</i> colonization disrupts the microbial community within the upper respiratory tract of aging mice

Nasopharyngeal colonization by the Gram-positive bacterium Streptococcus pneumoniae is a prerequisite for pneumonia and invasive pneumococcal diseases. Colonization is asymptomatic, involving dynamic and complex interplay between commensals, the host immune system, and environmental factors. The elderly are at an increased risk of developing pneumonia, which might be due to changes in the respiratory microbiota that would impact bacterial colonization and persistence within this niche. We hypothesized that the composition of the upper respiratory tract (URT) microbiota changes with age and subsequently can contribute to sustained colonization and inefficient clearance of S. pneumoniae. To test this, we used a mouse model of pneumococcal colonization to compare the composition of the URT microbiota in young, middle-aged, and old mice in the naive state and during the course of colonization using nasal pharyngeal washes. Sequencing of variable region 3 (V3) of the 16S rRNA gene was used to identify changes occurring with age and throughout the course of S. pneumoniae colonization. We discovered that age affects the composition of the URT microbiota and that colonization with S. pneumoniae is more disruptive of preexisting communities in older mice. We have further shown that host-pathogen interactions following S. pneumoniae colonization can impact the populations of resident microbes, including Staphylococcus and Haemophilus. Together, our findings indicate alterations to the URT microbiota could be detrimental to the elderly, resulting in increased colonization of S. pneumoniae and decreased efficiency in its clearance.</p

Reducing TNF-regulated recruitment of Ly6C^high monocytes during <i>S</i>. <i>pneumoniae</i> colonization in old mice reduced nasopharyngeal bacterial loads.

<p>(A-B) TNF in the (A) nasopharnyx and (B) serum of young and old mice during <i>S</i>. <i>pneumoniae</i> colonization as measured by qPCR and ELISA, respectively (± SEM; <i>n</i> = 3–5). (C) CFUs in nasal lavages of old WT and old TNF mice on day 4 after colonization with <i>S</i>. <i>pneumoniae</i> (± SEM; <i>n</i> = 6–8, one independent experiment of two shown). (D) Ly6C^high monocytes as a percent of circulating CD45+ cells in old WT and TNF KO mice on day 4 of <i>S</i>. <i>pneumoniae</i> colonization (± SEM; <i>n</i> = 3–4, one independent experiment of two shown). Statistical significance was determined by two-tailed Mann-Whitney-Wilcoxon test, one-way ANOVA or two-way ANOVA with Fisher's LSD post-test where appropriate. * indicates <i>p</i> < .05, ** indicates <i>p</i> < 0.005, *** indicates <i>p</i> < 0.0005 and **** indicates p < 0.00005.</p

Depletion of inflammatory monocytes improves outcome to S. pneumoniae infection in old mice.

<p>Mice (n = 7-10/group) were injected with PS-MP day -1, 0, +1, +3 and +5 during colonization <i>with S</i>. <i>pneumoniae</i>. A) The percentage of Ly6C^high monocytes was significantly reduced in old mice treated with PS-MP (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005368#ppat.1005368.s003" target="_blank">S3 Fig</a>). B) Survival was significantly improved in old mice treated with PS-MP (p = 0.005, Mantel-Cox log-rank test). C) Both young and old mice treated with PS-MP lost less weight than their control counterparts (*,p<0.05, one-way ANOVA with uncorrected Fisher's LSD). Levels of <i>S</i>. <i>pneumoniae</i> in the D) nasal wash, E) lungs and F) spleen were lower in old mice treated with PS-MP. Fewer young mice had bacteria in their lungs and spleens when they were treated with PS-MP. (*,p<0.05, **,p<0.005 one-way ANOVA with uncorrected Fisher's LSD). CFU count for mice that reached endpoint before day 7 are not included.</p

Anti-TNF therapy can reverse the age-associated increase in circulating Ly6C^high monocytes.

<p>(A-B) Young mice were give 200 ng/ml of TNF intraperitoneally every other day for 3 weeks. Numbers of circulating Ly6C^high and Ly6C^low monocytes (A) and serum IL6 (B) were quantitated. The data represent the mean (± SEM) of 5 mice. (C) Young and old WT mice were treated for 3 weeks with a neutralizing TNF antibody or IgG control and total numbers of circulating Ly6C^high monocytes were quantitated by flow cytometry. The data represent the mean (± SEM) of 4 mice. (D) The mean CCR2 expression on circulating Ly6C^high monocytes in young and old mice treated with either anti-TNF or IgG was quantitated and found to be reduced with anti-TNF treatment (<i>n</i> = 4). (E) Intracellular staining of IL6 and TNF on blood monocytes after a 4 hour stimulation with LPS from young and old WT mice treated with either anti-TNF or IgG demonstrates that the number of monocytes that stain positive for IL6 or TNF are decreased with anti-TNF therapy(± SEM; <i>n</i> = of 4). (F) Serum IL6 is reduced in old mice treated with anti-TNF but not the IgG control. (G) IL6 production in whole blood following stimulation with LPS or a vehicle control after 24 hours from young and old WT mice given either anti-TNF or IgG (± SEM; <i>n</i> = 4). Statistical significance was determined by two-tailed Mann-Whitney-Wilcoxon test, one-way or two-way ANOVA with Fisher's LSD post-test where appropriate. * indicates <i>p</i> < .05, ** indicates <i>p</i> < 0.005, *** indicates <i>p</i> < 0.0005 and **** indicates <i>p</i> < 0.00005. (A-G) are representative of 1 experiment with n = 4 mice.</p