Although adoptive transfer of NK cells with IL-2 can induce complete remissions in 30-50% of patients with refractory AML, efficacy is limited by IL-2 mediated induction of Tregs and by lack of ...antigen specificity. Thus we generated a 161533 trispecific killer engager (TriKE) molecule containing an anti-CD16 scFv to engage NK cells, an anti-CD33 scFv to engage myeloid targets (including myelodysplastic syndrome MDS), and a modified IL-15 linker. We have previously shown that this molecule is superior to a 1633 bispecific killer engager (BiKE) in killing of AML targets and that it promotes enhanced survival and in vivo expansion of NK cells. We questioned the mechanism for the increased potency of the 161533 TriKE and if the TriKE could activate the dysfunctional and suppressed NK cells found in patients with MDS. Cryopreserved mononuclear cells (obtained from the NMDP Sample Repository) collected from 8 patients with advanced MDS were tested to investigate how the TriKE might enhance functionality in this setting. Previously we reported that MDS patients have significantly decreased frequencies of NK cells due to increased CD33+ myeloid derived suppressor cells. Our 161533 TriKE enhanced the function of NK cells derived from MDS patients against acute promyelocytic CD33+ leukemia HL-60 tumor targets (Figure 1A), when compared to 1633 BiKE, in a flow cytometry assay measuring NK cell degranulation (% CD107a: 41.8±3.8 vs. 30.3±3.2, p=0.004), and inflammatory cytokine production (% IFNg: 40.7±5.0 vs. 30.0±4.9, p=0.009; % TNFa: 36.9±5.5 vs. 28.4±4.8, p=0.009). State of the art microchip-based live cell imaging was then employed to evaluate NK cell function and contact-to-target dynamics (Figure 1B). Briefly, resting NK cells and HL-60 target cells were stained with distinct dyes and then co-incubated in the presence of BiKE or TriKE in microwells at 37°C and 5% CO2. Cells were then imaged for 12 hours using a Zeiss 880 microscope and analyzed by Matlab. In contrast to those incubated with BiKE, NK cells cultured in the presence of TriKE had augmented cytotoxicity (37%±6% vs. 59%±6%, p=0.02) and killed their targets remarkably faster (time to first target kill = 148±30 min vs. 75±26 min, p< 0.0001). In addition, NK cell serial killers were more common in the presence of TriKE compared to BiKE (number of killed targets ≥3: 18%±7% vs. 9%±1%, p=0.04). Having shown the robust killing dynamics of TriKE primed NK cells, we next designed an in vivo dose escalation study to evaluate HL-60 tumor control in our xenogeneic mouse model. NSG (NOD scid gamma) mice were conditioned with a sub-lethal dose of radiation (275 cGy) and engrafted with 750,000 HL-60luc cells, which allow for tracking of tumor growth using bioluminescent imaging (BLI). Mice were infused with 1 million fresh healthy donor human NK cells and treated daily with recombinant IL-15 (5 ug/injection), 20-200 ug of 161533 TriKE, or left untreated. Higher doses of TriKE provided better tumor control (6.3x109±2.9x109 200 ug 1615133 vs. 1.6 x1010±2.7x109 20 ug 161533 p/sec/cm2/sr) demonstrating dose responsiveness at days 14 and 21 This was not due to increased proliferation of effectors (NK cell numbers) induced by the IL-15 segment of the TriKE as the absolute PBNK cell counts in mice were not different across the TriKE concentrations. However, the day 14 and 21 absolute PBNK counts were higher in all of the TriKE groups when compared to the IL-15 treated mice, including in the 20 ug 161533 TriKE group which best matched the 5 ug IL-15 dose. These data indicated that the TriKE molecule mediates superior NK cell expansion or maintenance in vivo. Taken together the in vivo data suggests that 161533 TriKE not only mediates tumor control by inducing NK cell proliferation and survival in a methodology that exceeds signals provided by IL-15 alone, but also increases tumor killing through enhanced killing kinetics. In summary, we have shown that the 161533 TriKE can rescue dysfunctional NK cells suppressed in patients with MDS and can mediate potent in vivo tumor killing presumably through better NK cell maintenance and enhanced killing kinetics This is of translational importance and demonstrates that the cancer induced immune suppression is reversible by the activating signals induced by the TriKE molecule. The 161533 TriKE represents a promising modality to maximizing NK cell based immunotherapies against MDS and AML and will be in phase I clinical testing the first half of 2017.
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Cooley:Fate Therapeutics: Research Funding. Vallera:Oxis Biotech: Consultancy, Membership on an entity's Board of Directors or advisory committees. Miller:Fate Therapeutics: Consultancy, Research Funding; Oxis Biotech: Consultancy, Other: SAB.
Adiponectin, an adipocyte protein important in insulin sensitization and cardioprotection, has a strong genetic component. We hypothesized that variants in the adiponectin gene (adipocyte ...collagen-domain containing ACDC) contribute to adiponectin levels in a biracial adolescent cohort. We genotyped 11 ACDC single nucleotide polymorphisms (SNPs) in 631 non-Hispanic white and 553 African-American unrelated adolescents in grades 5-12 randomly selected from the Princeton School District Study. ACDC SNPs -11,391 (A allele), -10,068 (G allele), and +276 (T allele) were associated with higher adiponectin, adjusting for sex, puberty stage, BMI Z score, and waist Z score. Contiguous two-SNP haplotypes of promoter variants -11,391/-10,068 were significantly associated with adiponectin levels in whites and African Americans (P < 0.0001 and 0.03, respectively). Extended haplotypes from the promoter through the second intron (-11,391 to +349) strongly associated with adiponectin in whites (P = 6 x 10(-11)) and African Americans (P = 0.004), but haplotypes of first intron SNPs -4,521 to -657 did not (P > 0.2). Noncontiguous haplotypes or interactions between two-SNP (-11,391/-10,068) and three-SNP (+45, +276, and +349) haplotypes predicted adiponectin better than either region alone. Variants of ACDC are associated with adiponectin levels in whites and African Americans. Interactions between noncontiguous ACDC haplotypes strongly influence adiponectin levels, suggesting nonadditive and potentially cis relationships between these regions.
There is increasing evidence for variation in rates of nucleotide substitution among divergent taxonomic groups. Here, we summarize published rate data and show a strong relationship between ...substitution rate and body size. For instance, rates of nuclear and mtDNA evolution are slow in whales, intermediate in primates, and fast in rodents. A similar relationship exists for poikilothermic vertebrates. However, these taxa have slower mtDNA substitution rates overall than do homeotherms of similar size. A number of physiological and life history variables are highly correlated with body size. Of these, generation time and metabolic rate explain some patterns of rate heterogeneity equally well. In many cases, however, differences in metabolic rate explain important exceptions to the generation time model. Correlation between metabolic rate and nucleotide substitution may be mediated by (i) the mutagenic effects of oxygen radicals that are abundant by-products of aerobic respiration, and (ii) increased rates of DNA synthesis and nucleotide replacement in organisms with higher metabolic rates. Both of these factors increase mutation rate by decreasing the "nucleotide generation time," the average length of time before a nucleotide is copied either through replication or repair. Reconsideration of the generation time hypothesis to include physiological effects such as metabolic rate improves the theoretical underpinnings of molecular evolution.
Recent studies of stranded marine mammals indicate that exposure to underwater military sonar may induce pathophysiological responses consistent with decompression sickness (DCS). However, DCS has ...been difficult to diagnose in marine mammals. We investigated whether blood microparticles (MPs, measured as number/μl plasma), which increase in response to decompression stress in terrestrial mammals, are a suitable biomarker for DCS in marine mammals. We obtained blood samples from trained Steller sea lions (Eumetopias jubatus, 4 adult females) wearing time-depth recorders that dove to predetermined depths (either 5 or 50 meters). We hypothesized that MPs would be positively related to decompression stress (depth and duration underwater). We also tested the effect of feeding and exercise in isolation on MPs using the same blood sampling protocol. We found that feeding and exercise had no effect on blood MP levels, but that diving caused MPs to increase. However, blood MP levels did not correlate with diving depth, relative time underwater, and presumed decompression stress, possibly indicating acclimation following repeated exposure to depth.
Abstract Background Optimal adherence with antihypertensive medications is crucial to prevent hypertension-related complications. This study evaluated whether the duration of initial antihypertensive ...prescription is associated with better medication adherence in a large sample of Chinese hypertensive patients. Methods and results From a validated clinical database which consists of all patients in the public healthcare sector in Hong Kong, all patients on their first-ever antihypertensive agent from 2001 to 2005 (N = 203,259) were included and followed-up for 12 months (and up to 5 years in separate analyses). The average age was 58.7 years (SD 17.3), and the overall rate of optimal adherence (as measured by having the Proportion of Days Covered ≥ 0.80) was 32.4%. The proportion of patients whose initial prescriptions lasted for ≤ 6 days; 7–14 days; 15–28 days and ≥ 29 days was 23.7%, 24.3%, 15.1% and 37.0%, respectively. The corresponding proportion of optimal adherence was 18.1%, 20.1%, 31.0% and 50.3%. The binary logistic regression analysis showed that after controlling for age, sex, socioeconomic status, service type, drug class, and district of residence, those whose initial prescription was 7–14 days (adjusted odds ratio AOR = 1.17, 95% C.I. 1.12–1.22); 15–28 days (AOR = 1.90, 95% C.I. 1.82–1.99) and ≥ 29 days (AOR = 4.13, 95% C.I. 3.96–4.31) were significantly more likely to be adherent than those who were prescribed for ≤ 6 days (all p < 0.001). These findings remained significant in separate analyses where the period of follow-up was extended to 5 years. Conclusions Shorter duration of first antihypertensive prescriptions was associated with poorer medication adherence, and this practice should be avoided if possible.
Bacterial antibiotic resistance is an emerging global crisis, and treatment of multidrug-resistant gram-negative infections, particularly those caused by the opportunistic human pathogen Pseudomonas ...aeruginosa, remains a major challenge. This problem is compounded by a lack of new antibiotics in the development pipeline: only two new classes have been developed since the 1960s, and both are indicated for multidrug-resistant gram-positive infections. A promising new approach to combat antibiotic resistance is by targeting bacterial virulence, rather than bacterial viability. The bacterial periplasmic protein DsbA represents a central point for antivirulence intervention because its oxidoreductase activity is essential for the folding and function of almost all exported virulence factors. Here we describe the three-dimensional structure of this DsbA target from P. aeruginosa, and we establish for the first time that a member of this enzyme family is capable of binding small molecules. We also describe biochemical assays that validate the redox activity of PaDsbA. Together, the structural and functional characterization of PaDsbA provides the basis for future studies aimed at designing a new class of antivirulence compounds to combat antibiotic-resistant P. aeruginosa infection.
Current dogma dictates that bacterial proteins with misoxidized disulfide bonds are shuffled into correctly oxidized states by DsbC. There are two proposed mechanisms for DsbC activity. The first ...involves a DsbC-only model of substrate disulfide rearrangement. The second invokes cycles of reduction and oxidation of substrate disulfide bonds by DsbC and DsbA respectively. Here, we addressed whether the second mechanism is important in vivo by identifying whether a periplasmic reductase could complement DsbC. We screened for naturally occurring periplasmic reductases in Bacteroides fragilis, a bacterium chosen because we predicted it encodes reductases and has a reducing periplasm. We found that the B. fragilis periplasmic protein TrxP has a thioredoxin fold with an extended N-terminal region; that it is a very active reductase but a poor isomerase; and that it fully complements dsbC. These results provide direct in vivo evidence that correctly folded protein is achievable via cycles of oxidation and reduction.
The androgen receptor (AR) gene exon 1 CAG repeat length has been proposed to be a determinant of between-individual variations in androgen action in target tissues, which might regulate phenotypic ...differences of human ageing. However, findings on its phenotypic effects are inconclusive.
To assess whether the AR CAG repeat length is associated with longitudinal changes in endpoints that are influenced by testosterone (T) levels in middle-aged and elderly European men.
Multinational European observational prospective cohort study.
A total of 1887 men (mean ± s.d. age: 63 ± 11 years; median follow up: 4.3 years) from centres of eight European countries comprised the analysis sample after exclusion of those with diagnosed diseases of the hypothalamic-pituitary-testicular (HPT) axis.
Longitudinal associations between the AR CAG repeat and changes in androgen-sensitive endpoints (ASEs) and medical conditions were assessed using regression analysis adjusting for age and centre. The AR CAG repeat length was treated as both a continuous and a categorical (6-20; 21-23; 24-39 repeats) predictor. Additional analysis investigated whether results were independent of baseline T or oestradiol (E
) levels.
The AR CAG repeat, when used as a continuous or a categorical predictor, was not associated with longitudinal changes in ASEs or medical conditions after adjustments. These results were independent of T and E
levels.
Within a 4-year time frame, variations in the AR CAG repeat do not contribute to the rate of phenotypic ageing, over and above, which might be associated with the age-related decline in T levels.
While in most muscles contraction is triggered by calcium effluxes, insect flight muscles are also activated by mechanical stretch. We are interested in understanding the role that the troponin C ...protein, usually the calcium sensor, plays in stretch activation. In the flight muscles of Lethocerus, a giant water bug often used as a model system, there are two isoforms of TnC, F1 and F2, present in an approximately 10:1 ratio. F1 TnC is responsible for activating the muscle following a stretch, whereas F2 TnC produces a sustained contraction, the magnitude of which depends on the concentration of Ca2+ in the fiber. We have previously shown that F1 TnC binds only one Ca2+ ion in its C-terminal domain and that interaction with troponin H, the insect ortholog of troponin I, is insensitive to Ca2+. Here, we have studied the effect of Ca2+ and Mg2+ on the affinities of the interaction of F2 TnC with troponin H peptides. We show that the presence of two Ca2+ ions, one in each of the globular domains, increases the affinity for TnH by at least 1 order of magnitude. The N lobe has a lower affinity for Ca2+, but it is also sensitive to Mg2+. The C lobe is insensitive to Mg2+ as previously demonstrated by mutations of the individual EF-hands. The interaction with TnH seems also to have significant structural differences from that observed for the F1 TnC isoform. We discuss how our findings could account for stretch activation.
At the forest landscape/region level, based on annual Forest Inventory and Analysis plot data from 1999 to 2010, oak decline and mortality trends for major oak species (groups) were examined in the ...Ozark Highlands of Arkansas and Missouri. Oak decline has elevated cumulative mortality of red oak species to between 11 and 15 percent in terms of relative density and basal area of standing dead oak trees, respectively. These values are three to five times higher than for white oak group and non-oak species. Oak decline and associated escalating mortality have occurred primarily in red oak species while the white oak group has maintained a relatively stable mortality rate that is comparable to non-oak species. Cross-correlation analyses indicate that mortality in the red oak group was significantly correlated with the growing season Palmer drought severity index (PDSI) and usually lagged two to three years following single drought events. Moreover, based on the past 17 years PDSI data, it appears that the cumulative impacts of drought may last up to 10 years. The Ozark Highlands experienced a severe drought extending from 1998 to 2000 and another milder drought from 2005 to 2006. These drought events triggered the escalation of mortality starting around year 2000. Spatially, high red oak mortality sites (hot spots with proportional basal area mortality > 0.12) initially occurred in the central Ozarks and spread gradually over most of the Ozark Highlands as regional droughts continued. In contrast, sites with elevated white oak and non-oak mortality occurred sporadically, mainly in the southern portion (Arkansas) of the Ozark Highlands. During the most recent inventory period (2006–2010), over 60%, 7% and 5% of red oak, white oak and non-oak groups, respectively, had relative mortality rates of > 12%.
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