Age Related Alterations in Chemokine and Chemokine Receptor Gene Expression in Mouse Hearts

Data from our lab and others demonstrate that there is greater cardiac T cell infiltration with age and that these T cells play a role in pathogenic cardiac hypertrophy and fibrosis. However, how T cells are recruited to the heart with advanced age is unknown. PURPOSE: The purpose of this study was to assess age-related alterations in T cell recruiting chemokine and chemokine receptor gene expression in the heart. METHODS: This study was conducted using five young (4-6 months old) and five old (22-24 months old) mice. Following euthanasia, the heart tissue was snap frozen and homogenized. RNA was extracted using the phenol/chloroform method. Following cDNA synthesis, qPCR was performed for the chemokines Ccl2, Ccl5, Cxcl10; as well as the 18s gene which was used as an endogenous control. Additionally, we performed qPCR for chemokine receptors, specifically Ccr1, Ccr3, Ccr5, and Cxcr3. After qPCR, relative gene expression was determined using the delta delta CT method. Differences were assessed using an independent sample T test. All data are expressed as mean relative gene expression ± standard error. RESULTS: For the Cxcl10 cytokine, the relative gene expression was 1.43 ± 0.39 in young mice and 3.07 ± 0.52 in old mice (p=0.018). For the chemokine Ccl5, the relative gene expression was 1.03 ± 0.25 in young mice and 7.35 ± 4.37 in old mice (p=0.093). Additionally, the Ccl2 chemokine had a relative gene expression of 1.19 ± 0.47 in young mice and 1.44 ± 0.27 in old mice (p=0.328). Because we observed age related elevations in the Cxcl10 and Ccl5 chemokines, we assessed gene expression for the corresponding chemokine receptors. Ccl5 receptors, Ccr1 (1.73 ± 0.65, young vs. 2.89 ± 0.75, old p=0.140), Ccr3 (1.89 ± 0.73, young vs. 7.19 ± 4.74, old p=0.150), and Ccr5 (1.11± 0.13, young vs. 2.46 ± 0.51, old p=0.017) exhibited trends for elevation. Furthermore, the receptor Cxcr3 which corresponds to the Cxcl10 chemokine had a relative gene expression of 1.26 ± 0.28 in young mice and 8.73 ± 4.96 in old mice (p=0.086). CONCLUSION: In conclusion, these data suggest that gene expression for both the Ccl5 and Cxcl10 chemokine pathways is upregulated in hearts of the old mice. Further, Ccl5 and Cxcl10 may be responsible for the increased T cell recruitment to the aging heart. These chemokines represent a potential treatment target to preserve cardiovascular health in the elderly

Old Mice Exhibit Greater Skeletal Muscle Cxcl10 Gene Expression

Advanced age is associated with impairments in glucose tolerance increasing the risk for metabolic syndrome and type II diabetes. Preliminary data from our lab demonstrates greater T-cell infiltration in the skeletal muscle with age and that depletion of T-cells improves glucose tolerance in old mice. The mechanisms responsible for increased T-cell recruitment to the skeletal muscles with age is not known. PURPOSE: The purpose of this study was to determine whether gene expression of the T-cell recruiting chemokine’s Cxcl10, Ccl5, and Ccl2 are altered with advanced age in mouse skeletal muscle. METHODS: 4 young mice (~6months) and 5 old mice (~24 months) were euthanized and the gastrocnemius was snap frozen and kept -80°C until further use. Muscles were homogenized and the RNA was extracted using the phenol chloroform method. After the extraction of the RNA, the complimentary DNA (cDNA) was synthesized and used for qPCR to assess the gene expression of Ccl2, Cxcl10, Ccl5, and 18s. The 18s gene was used as the endogenous control. Relative gene expression was determined using the delta delta CT method. Group differences were assessed with an independent sample t-test. RESULTS: All data are expressed as mean ± standard error. Relative Ccl2 gene expression was 1.76 ± 0.82 in young mice and 1.96 ± 0.59 in old mice (p=0.42). Relative Cxcl10 gene expression was 0.88 ± 0.24 in young mice and 13.86 ± 1.25 in old mice (p=0.04). Relative Ccl5 gene expression was 3.88 ± 3.34 in young mice and 1.32± 0.28 in old mice (p=0.21). CONCLUSION: Overall, this study suggests that Cxcl10 but not Ccl2 and Ccl5 gene expression is greater in the gastrocnemius muscle of old mice. These data suggest that increased production of Cxcl10 may be responsible for skeletal muscle T cell infiltration with age. Our study suggests that the T-cell recruiting chemokine Cxcl10 may be a target to preserve metabolic health in older adults

Short-term CD8+ T Cell Depletion Results in Decreased Large Artery Stiffness Via Decreased Collagen Content in Old Mice

Cardiovascular disease is the leading cause of death in the United States. Advanced age is associated with large artery stiffness, partially due to increased collagen deposition within the aorta. Previously we have found that T cells accumulate in the aortas of old animals. A large proportion of these cells are CD8+ and produce inflammatory cytokines. We have previously demonstrated that CD8+ T cell depletion results in improved large artery stiffness. However, the mechanisms by which CD8+ depletion improves large artery stiffness are unclear. Therefore, we sought to determine whether CD8+ T cell depletion in old mice results in blunted aortic collagen deposition compared to old CD8+ intact mice. PURPOSE: To test the hypothesis that old CD8+ T cell depleted mice would exhibit blunted collagen deposition and collagen gene expression in the aorta compared to old CD8+ T cell intact mice. METHODS: Old (22-24mo) mice (n=5-12) were injected every 5 days with anti-CD8+ or isotype (control) antibodies for 28 days. On the 28th day, the mice were euthanized, a 5mm section of the aorta was saved for histology and the remaining aorta was cleaned and saved for qPCR analysis. Aortas were paraffin embedded, sectioned, and stained using picrosirius red to assess collagen content. Collagen content was quantified using ImageJ. Aorta collagen gene expression was assessed by qPCR. Group differences were assessed by independent samples t test. Data is presented as mean ± SEM (normalized to old Isotype; Units Arbitrary (AU)). RESULTS: Old CD8+ depleted mice exhibited blunted collagen deposition in both the medial (Control: 1.00±0.06 AU, Anti-CD8:0.78±0.07 AU; p=0.02) and adventitial (Control: 1.00±0.05 AU, Anti-CD8:0.81±0.05 AU; p=0.01) layers of the aorta compared to isotype controls. However, there were no differences in the expression of genes responsible for collagen creation, Col1a1 (Control: 1.00±0.28 AU, Anti-CD8: 1.68±0.53AU, p=0.15) and Col3a1(Control: 1.00±0.46 AU, Anti-CD8: 0.89±0.35 AU, p=0.42). CONCLUSION: Short-term manipulation of CD8+ T cells results in blunted aortic collagen deposition but has no effect on collagen gene expression compared to old controls. The specific mechanisms in which old CD8+ T cells influence collagen deposition are unknown

Microscale rarefied gas dynamics and surface interactions for EUVL and MEMS applications.

A combined experimental/modeling study was conducted to better understand the critical role of gas-surface interactions in rarefied gas flows. An experimental chamber and supporting diagnostics were designed and assembled to allow simultaneous measurements of gas heat flux and inter-plate gas density profiles in an axisymmetric, parallel-plate geometry. Measurements of gas density profiles and heat flux are made under identical conditions, eliminating an important limitation of earlier studies. The use of in situ, electron-beam fluorescence is demonstrated as a means to measure gas density profiles although additional work is required to improve the accuracy of this technique. Heat flux is inferred from temperature-drop measurements using precision thermistors. The system can be operated with a variety of gases (monatomic, diatomic, polyatomic, mixtures) and carefully controlled, well-characterized surfaces of different types (metals, ceramics) and conditions (smooth, rough). The measurements reported here are for 304 stainless steel plates with a standard machined surface coupled with argon, helium, and nitrogen. The resulting heat-flux and gas-density-profile data are analyzed using analytic and computational models to show that a simple Maxwell gas-surface interaction model is adequate to represent all of the observations. Based on this analysis, thermal accommodation coefficients for 304 stainless steel coupled with argon, nitrogen, and helium are determined to be 0.88, 0.80, and 0.38, respectively, with an estimated uncertainty of {+-}0.02

Oligoclonal CD8+ T Cells Play a Critical Role in the Development of Hypertension

Recent studies have emphasized a role of adaptive immunity, and particularly T cells, in the genesis of hypertension. We sought to determine the T-cell subtypes that contribute to hypertension and renal inflammation in angiotensin II-induced hypertension. Using T-cell receptor spectratyping to examine T-cell receptor usage, we demonstrated that CD8(+) cells, but not CD4(+) cells, in the kidney exhibited altered T-cell receptor transcript lengths in Vβ3, 8.1, and 17 families in response to angiotensin II-induced hypertension. Clonality was not observed in other organs. The hypertension caused by angiotensin II in CD4(-/-) and MHCII(-/-) mice was similar to that observed in wild-type mice, whereas CD8(-/-) mice and OT1xRAG-1(-/-) mice, which have only 1 T-cell receptor, exhibited a blunted hypertensive response to angiotensin II. Adoptive transfer of pan T cells and CD8(+) T cells but not CD4(+)/CD25(-) cells conferred hypertension to RAG-1(-/-) mice. In contrast, transfer of CD4(+)/CD25(+) cells to wild-type mice receiving angiotensin II decreased blood pressure. Mice treated with angiotensin II exhibited increased numbers of kidney CD4(+) and CD8(+) T cells. In response to a sodium/volume challenge, wild-type and CD4(-/-) mice infused with angiotensin II retained water and sodium, whereas CD8(-/-) mice did not. CD8(-/-) mice were also protected against angiotensin-induced endothelial dysfunction and vascular remodeling in the kidney. These data suggest that in the development of hypertension, an oligoclonal population of CD8(+) cells accumulates in the kidney and likely contributes to hypertension by contributing to sodium and volume retention and vascular rarefaction

Distribution of Mycobacterium ulcerans in Buruli Ulcer Endemic and Non-Endemic Aquatic Sites in Ghana

Mycobacterium ulcerans, the causative agent of Buruli ulcer, is an emerging environmental bacterium in Australia and West Africa. The primary risk factor associated with Buruli ulcer is proximity to slow moving water. Environmental constraints for disease are shown by the absence of infection in arid regions of infected countries. A particularly mysterious aspect of Buruli ulcer is the fact that endemic and non-endemic villages may be only a few kilometers apart within the same watershed. Recent studies suggest that aquatic invertebrate species may serve as reservoirs for M. ulcerans, although transmission pathways remain unknown. Systematic studies of the distribution of M. ulcerans in the environment using standard ecological methods have not been reported. Here we present results from the first study based on random sampling of endemic and non-endemic sites. In this study PCR-based methods, along with biofilm collections, have been used to map the presence of M. ulcerans within 26 aquatic sites in Ghana. Results suggest that M. ulcerans is present in both endemic and non-endemic sites and that variable number tandem repeat (VNTR) profiling can be used to follow chains of transmission from the environment to humans. Our results suggesting that the distribution of M. ulcerans is far broader than the distribution of human disease is characteristic of environmental pathogens. These findings imply that focal demography, along with patterns of human water contact, may play a major role in transmission of Buruli ulcer

Modulation of Heat Shock Transcription Factor 1 as a Therapeutic Target for Small Molecule Intervention in Neurodegenerative Disease

A yeast-based small molecule screen identifies a novel activator of human HSF1 and protein chaperone expression and which appears to alleviate the toxicity of protein misfolding diseases

First season MWA EoR power spectrum results at redshift 7

The Murchison Widefield Array (MWA) has collected hundreds of hours of Epoch of Reionization (EoR) data and now faces the challenge of overcoming foreground and systematic contamination to reduce the data to a cosmological measurement. We introduce several novel analysis techniques, such as cable reflection calibration, hyper-resolution gridding kernels, diffuse foreground model subtraction, and quality control methods. Each change to the analysis pipeline is tested against a two-dimensional power spectrum figure of merit to demonstrate improvement. We incorporate the new techniques into a deep integration of 32 hours of MWA data. This data set is used to place a systematic-limited upper limit on the cosmological power spectrum of ∆2 ≤ 2.7×104 mK2 at k =0.27 h Mpc-1 and z = 7.1, consistent with other published limits, and a modest improvement (factor of 1.4) over previous MWA results. From this deep analysis, we have identified a list of improvements to be made to our EoR data analysis strategies. These improvements will be implemented in the future and detailed in upcoming publications

Ecology and Transmission of Buruli Ulcer Disease: A Systematic Review

Buruli ulcer is a neglected emerging disease that has recently been reported in some countries as the second most frequent mycobacterial disease in humans after tuberculosis. Cases have been reported from at least 32 countries in Africa (mainly west), Australia, Southeast Asia, China, Central and South America, and the Western Pacific. Large lesions often result in scarring, contractual deformities, amputations, and disabilities, and in Africa, most cases of the disease occur in children between the ages of 4–15 years. This environmental mycobacterium, Mycobacterium ulcerans, is found in communities associated with rivers, swamps, wetlands, and human-linked changes in the aquatic environment, particularly those created as a result of environmental disturbance such as deforestation, dam construction, and agriculture. Buruli ulcer disease is often referred to as the “mysterious disease” because the mode of transmission remains unclear, although several hypotheses have been proposed. The above review reveals that various routes of transmission may occur, varying amongst epidemiological setting and geographic region, and that there may be some role for living agents as reservoirs and as vectors of M. ulcerans, in particular aquatic insects, adult mosquitoes or other biting arthropods. We discuss traditional and non-traditional methods for indicting the roles of living agents as biologically significant reservoirs and/or vectors of pathogens, and suggest an intellectual framework for establishing criteria for transmission. The application of these criteria to the transmission of M. ulcerans presents a significant challenge

Multi-messenger observations of a binary neutron star merger

On 2017 August 17 a binary neutron star coalescence candidate (later designated GW170817) with merger time 12:41:04 UTC was observed through gravitational waves by the Advanced LIGO and Advanced Virgo detectors. The Fermi Gamma-ray Burst Monitor independently detected a gamma-ray burst (GRB 170817A) with a time delay of ~1.7 s with respect to the merger time. From the gravitational-wave signal, the source was initially localized to a sky region of 31 deg2 at a luminosity distance of 40+8-8 Mpc and with component masses consistent with neutron stars. The component masses were later measured to be in the range 0.86 to 2.26 Mo. An extensive observing campaign was launched across the electromagnetic spectrum leading to the discovery of a bright optical transient (SSS17a, now with the IAU identification of AT 2017gfo) in NGC 4993 (at ~40 Mpc) less than 11 hours after the merger by the One- Meter, Two Hemisphere (1M2H) team using the 1 m Swope Telescope. The optical transient was independently detected by multiple teams within an hour. Subsequent observations targeted the object and its environment. Early ultraviolet observations revealed a blue transient that faded within 48 hours. Optical and infrared observations showed a redward evolution over ~10 days. Following early non-detections, X-ray and radio emission were discovered at the transient’s position ~9 and ~16 days, respectively, after the merger. Both the X-ray and radio emission likely arise from a physical process that is distinct from the one that generates the UV/optical/near-infrared emission. No ultra-high-energy gamma-rays and no neutrino candidates consistent with the source were found in follow-up searches. These observations support the hypothesis that GW170817 was produced by the merger of two neutron stars in NGC4993 followed by a short gamma-ray burst (GRB 170817A) and a kilonova/macronova powered by the radioactive decay of r-process nuclei synthesized in the ejecta