Detection and Quantification of Calcitonin Gene-Related Peptide (CGRP) in Human Plasma Using a Modified Enzyme-Linked Immunosorbent Assay.

Calcitonin gene-related peptide (CGRP) is a vasoactive neuropeptide that plays a putative role in the pathophysiology of migraine headaches and may be a candidate for biomarker status. CGRP is released from neuronal fibers upon activation and induces sterile neurogenic inflammation and arterial vasodilation in the vasculature that receives trigeminal efferent innervation. The presence of CGRP in the peripheral vasculature has spurred investigations to detect and quantify this neuropeptide in human plasma using proteomic assays, such as the enzyme-linked immunosorbent assay (ELISA). However, its half-life of 6.9 min and the variability in technical details of assay protocols, which are often not fully described, have yielded inconsistent CGRP ELISA data in the literature. Here, a modified ELISA protocol for the purification and quantification of CGRP in human plasma is presented. The procedural steps involve sample collection and preparation, extraction using a polar sorbent as a means of purification, additional steps to block non-specific binding, and quantification via ELISA. Further, the protocol has been validated with spike and recovery and linearity of dilution experiments. This validated protocol can theoretically be used to quantify CGRP concentrations in the plasma of individuals not only with migraine, but also with other diseases in which CGRP may play a role.

Reduced quantity and function of pneumococcal antibodies are associated with exacerbations of COPD in SPIROMICS.

While hypogammaglobulinemia is associated with COPD exacerbations, it is unknown whether frequent exacerbators have specific defects in antibody production/function. We hypothesized that reduced quantity/function of serum pneumococcal antibodies correlate with exacerbation risk in the SPIROMICS cohort. We measured total pneumococcal IgG in n = 764 previously vaccinated participants with COPD. In a propensity-matched subset of n = 200 with vaccination within five years (n = 50 without exacerbations in the previous year; n = 75 with one, n = 75 with ≥2), we measured pneumococcal IgG for 23 individual serotypes, and pneumococcal antibody function for 4 serotypes. Higher total pneumococcal IgG, serotype-specific IgG (17/23 serotypes), and antibody function (3/4 serotypes) were independently associated with fewer prior exacerbations. Higher pneumococcal IgG (5/23 serotypes) predicted lower exacerbation risk in the following year. Pneumococcal antibodies are inversely associated with exacerbations, supporting the presence of immune defects in frequent exacerbators. With further study, pneumococcal antibodies may be useful biomarkers for immune dysfunction in COPD.

The role of proprotein convertase subtillisin/kexin type 9 in placental salvage and lipid metabolism in women with preeclampsia.

INTRODUCTION: Preeclampsia is associated with decreased maternal low-density lipoprotein cholesterol (LDL-c), which is essential for fetal growth. The underlying mechanisms for decreased LDL-c in preeclampsia remain unknown. Proprotein convertase subtillisin/kexin type 9 (PCSK9) regulates serum LDL-c via LDL receptor (LDL-R) degradation. We describe the possible role of PCSK9 in lipid metabolism in all compartments of the parturient (maternal blood, placental tissue, and fetal blood) in pregnancies with and without preeclampsia. METHODS: This is an observational study examining PCSK9 levels in maternal sera, umbilical cord blood, and PCSK9 protein content in placental tissue in three different locations (maternal placental interface, fetal placental interface, and umbilical cord) in women with and without preeclampsia at >23 weeks gestation. RESULTS: 68 parturients with preeclampsia and 55 without preeclampsia were enrolled. Maternal serum LDL-c (116.6 ± 48.9 mg/dL vs 146.1 ± 47.1 mg/dL, p = 0.0045) and PCSK9 (83 [61.8127.6] ng/mL vs 105.3 [83.5142.9] ng/mL, p = 0.011) were also reduced in the preeclamptics versus controls. There were no differences in PCSK9 protein content between preeclamptics and controls at comparative placental interfaces. However, PCSK9 protein content increased between the preeclampsia maternal placental interface (1.87 ± 0.62) and the preeclampsia umbilical cord (2.67 ± 1.08, p = 0.0243). DISCUSSION: PCSK9 levels are lower in maternal sera in preeclampsia when compared to controls. Placental PCSK9 protein content in preeclampsia increases from the maternal interface to the umbilical cord; however, this is not seen in controls. This suggests a potential compensatory mechanism for PCSK9 which allows for higher circulating fetal LDL-c levels in preeclampsia.

Growth Differentiation Factor-15 Predicts Mortality and Heart Failure Exacerbation But Not Ventricular Arrhythmias in Patients With Cardiomyopathy.

Background Heart failure (HF) has been increasing in prevalence, and a need exists for biomarkers with improved predictive and prognostic ability. GDF-15 (growth differentiation factor-15) is a novel biomarker associated with HF mortality, but no serial studies of GDF-15 have been conducted. This study aimed to investigate the association between GDF-15 levels over time and the occurrence of ventricular arrhythmias, HF hospitalizations, and all-cause mortality. Methods and Results We used a retrospective case-control design to analyze 148 patients with ischemic and nonischemic cardiomyopathies and primary prevention implantable cardioverter-defibrillator (ICD) from the PROSe-ICD (Prospective Observational Study of the ICD in Sudden Cardiac Death Prevention) cohort. Patients had blood drawn every 6 months and after each appropriate ICD therapy and were followed for a median follow-up of 4.6 years, between 2005 to 2019. We compared serum GDF-15 levels within ±90 days of an event among those with a ventricular tachycardia/fibrillation event requiring ICD therapies and those hospitalized for decompensated HF. A comparator/control group comprised patients with GDF-15 levels available during 2-year follow-up periods without events. Median follow-up was 4.6 years in the 148 patients studied (mean age 58±12, 27% women). The HF cohort had greater median GDF-15 values within 90 days (1797 pg/mL) and 30 days (2039 pg/mL) compared with the control group (1062 pg/mL, both P<0.0001). No difference was found between the ventricular tachycardia/fibrillation subgroup within 90 days (1173 pg/mL, P=0.60) or 30 days (1173 pg/mL, P=0.78) and the control group. GDF-15 was also significantly predictive of mortality (hazard ratio, 3.17 [95% CI, 2.33-4.30]). Conclusions GDF-15 levels are associated with HF hospitalization and mortality but not ventricular arrhythmic events.

Effects of Ramelteon on the Prevention of Postoperative Delirium in Older Patients Undergoing Orthopedic Surgery: The RECOVER Randomized Controlled Trial.

OBJECTIVES: Postoperative delirium, associated with negative consequences including longer hospital stays and worse cognitive and physical outcomes, is frequently accompanied by sleep-wake disturbance. Our objective was to evaluate the efficacy and short-term safety of ramelteon, a melatonin receptor agonist, for the prevention of postoperative delirium in older patients undergoing orthopedic surgery. DESIGN: A quadruple-masked randomized placebo-controlled trial (Clinical Trials.gov NCT02324153) conducted from March 2017 to June 2019. SETTING: Tertiary academic medical center. PARTICIPANTS: Patients aged 65 years or older, undergoing elective primary or revision hip or knee replacement. INTERVENTION: Ramelteon (8 mg) or placebo MEASUREMENTS: Eighty participants were randomized to an oral gel cap of ramelteon or placebo for 3 consecutive nights starting the night before surgery. Trained research staff conducted delirium assessments for 3 consecutive days starting on postoperative day (POD) 0, after recovery from anesthesia, and on to POD2. A delirium diagnosis was based upon DSM-5 criteria determined by expert panel consensus. RESULTS: Of 80 participants, five withdrew consent (one placebo, four ramelteon) and four were excluded (four ramelteon) after randomization. Delirium incidence during the 2 days following surgery was 7% (5 of 71) with no difference between the ramelteon versus placebo: 9% (3 of 33) and 5% (2 of 38), respectively. The adjusted odds ratio for postoperative delirium as a function of assignment to the ramelteon treatment arm was 1.28 (95% confidence interval: 0.21-7.93; z-value 0.27; p-value = 0.79). Adverse events were similar between the two groups. CONCLUSION: In older patients undergoing elective primary or revision hip or knee replacement, ramelteon was not efficacious in preventing postoperative delirium.

Leveraging naturally occurring variation in financial stress to examine associations with inflammatory burden among older adults.

BACKGROUND: Financial strain is associated with earlier disability and mortality, but causal links are underexplored, partly because it is unethical to randomise people to financial stress. This study leverages naturally occurring random variation in days since monthly Social Security payment arrival among older adults to test associations with inflammatory biomarkers. METHODS: Biomarker data, including tumour necrosis factor (TNF)-α, interleukin (IL)-6 and C reactive protein (CRP), was collected from 2155 non-working healthy adults aged 70-79 years, participating in the Health, Aging and Body Composition Study. Days since payment arrival was independent of all demographic, socioeconomic or health characteristics measured in this study. Restricted cubic spline models estimated associations separately for each week of the month, stratified by financial strain status (interaction term p value for TNF-α model <0.05). RESULTS: Among financially strained older adults, more days since payment arrival was associated with higher TNF-α levels during the first week of the month (coefficient=0.102). Associations with IL-6 and CRP differed depending on the degree of financial strain (interaction term p values <0.05). Those with low, but not high, strain had lower levels of IL-6 (coefficient=-0.152) and CRP (coefficient=-0.179) during the first week. CONCLUSIONS: Days since monthly payments were associated with inflammatory cytokines among older adults who have difficulty making ends meet financially and associations depended on financial strain severity, suggesting that results are attributable to monthly variation in financial stress. Future research should examine whether more frequent Social Security disbursement would modify financial strain and inflammatory biomarkers.

Prevalence of Angiotensin II Type 1 Receptor Antibodies in Persons With Hypertension and Relation to Blood Pressure and Medication.

BACKGROUND: We aimed to determine the prevalence of antibodies against angiotensin II type 1 receptor (AT1RAb) in hypertensive adults and elucidate the relation of antihypertensive medication type to blood pressure (BP) among persons with and without AT1RAb. METHODS: Sera from participants in the Healthy Aging in Neighborhoods of Diversity across the Life Span (HANDLS) study with hypertension were tested for AT1RAb using a commercial Enzyme-linked immunosorbent assay (ELISA) (One Lambda; positive ≥17 units/ml). BP measurements, uncontrolled BP (systolic BP ≥140 and/or diastolic BP ≥90 mm Hg), and effect of BP medication type were compared for AT1RAb positive (+) vs. negative (-) participants using descriptive statistics and multivariable regression. RESULTS: One hundred and thirty-two (13.1%) participants were AT1RAb+. Compared with AT1RAb-, AT1RAb+ persons were more likely to be white (47% vs. 36.7%; P = 0.03) but had similar comorbid disease burden. In models adjusting for age, sex, and race, AT1RAb+ persons had higher diastolic BP (ß = 2.61 mm Hg; SE = 1.03; P = 0.01) compared with AT1RAb- participants. Rates of uncontrolled BP were similar between the groups. AT1RAb+ persons on an angiotensin receptor blocker (ARB; n = 21) had a mean of 10.5 mm Hg higher systolic BP (SE = 4.56; P = 0.02) compared with AT1RAb+ persons using other BP medications. The odds of uncontrolled BP among AT1RAb+ participants on an ARB was 2.05 times that of those on other medications. AT1RAb- persons prescribed an angiotensin-converting enzyme inhibitor (ACEi) had 1.8 mm Hg lower diastolic BP (SE = 0.81; P = 0.03) than AT1RAb- persons not prescribed an ACEi. CONCLUSIONS: AT1RAb was prevalent among hypertensive adults and was associated with higher BP among persons on an ARB.

Antibodies against Angiotensin II Type 1 and Endothelin A Receptors: Relevance and pathogenicity.

Antibodies against two G-protein coupled receptors (GPCRs), angiotensin II type 1 receptor (AT1R) and endothelin A receptor (ETAR) are among a growing number of autoantibodies that are found to be associated with allograft dysfunction. AT1R antibodies (AT1Rabs) and ETAR antibodies (ETARabs) have been shown to activate their target receptors and affect signaling pathways. Multiple single center reports have shown an association between presence of these antibodies and acute or chronic rejection and graft loss in kidney, heart, liver, lung and composite tissue transplantations. However, the characteristics of patients that are most likely to develop adverse outcomes, the phenotypes associated with graft damage solely due to these antibodies, and the antibody titer required to cause dysfunction are areas that remain controversial. This review compiles existing knowledge on the effect of antibodies against GPCRs in other diseases in order to bridge the gap in knowledge within transplantation biology. Future areas for research are highlighted and include the need for functional assays and treatment protocols for transplant patients who present with AT1Rabs and ETARabs. Understanding how antibodies that activate GPCRs influence transplantation outcome will have direct clinical implications for preemptive evaluation of transplant candidates as well as the post-transplant care of organ recipients.

Angiotensin receptor autoantibodies as exposures that modify disease progression: Cross sectional, longitudinal and in vitro studies of prostate cancer.

Circulating angiotensin type I receptor (AT1R) agonistic autoantibodies (AT1RaAbs) that bind and chronically activate the receptor have been associated with a number of diseases suggesting that while the autoantibodies are not necessarily causative they may promote disease progression. The prostate has a local renin angiotensin system. The current study examines associations between AT1RaAbs and prostate cancer (PCA), disease-free survival (DFS), overall survival (OS) and AT1RaAb effects on PCA cell phenotype. In a cross-sectional set of serum obtained from 151 men diagnosed with PCA, nonmalignant prostate disease or no disease, higher serum AT1RaAb levels were associated with PCA and non-organ confined PCA. The odds ratio for PCA was 6.3 (95% confidence interval 2.2 to 18) for a positive 1:1600 titer and 18 (95% confidence interval 6.9 to 45) at AT1RaAb levels > 1.04 µg/ml, (p < 0.0001). In a longitudinal set of pre-diagnosis samples from 109 men, DFS hazard ratios of 2.2 (95% confidence interval 1.4 to 3.5) and 1.6 (95% confidence interval 1.0 to 2.5) for most proximal to diagnosis and most distal to diagnosis samples, respectively, were found for high versus low AT1RaAb groups. Hazard ratios for OS in most proximal and distal samples were 2.4 (95% confidence interval 1.6 to 3.6) and 1.8 (95% confidence interval 1.1 to 2.8), respectively. Accelerated failure modeling of survival indicated that a 1 µg/ml increase in AT1RaAb levels was associated with a reduction of DFS and OS by 20% at the most proximal time point and by 15% at the most distal time points. Adjusting for age, did not affect the association with DFS in proximal samples but changed distal time point DFS and OS to a 10% decrease for every 1 µg/ml increase in AT1RaAb. Additional adjustments for body mass index, systolic blood pressure and prostate-specific antigen did not appreciably alter these associations. AT1RaAb treatment of PC3, DU145, and LNCaP cells significantly increased the maximal growth rate approximately 2-fold and invasiveness approximately 3-fold. Conclusions: These observations provide evidence supporting AT1RaAbs as exposures that may modify prostate cancer progression and indicate they may be predictive markers for risk stratification.

Natriuretic Peptides in Post-mortem Brain Tissue and Cerebrospinal Fluid of Non-demented Humans and Alzheimer's Disease Patients.

Animal studies suggest the involvement of natriuretic peptides (NP) in several brain functions that are known to be disturbed during Alzheimer's disease (AD). However, it remains unclear whether such findings extend to humans. In this study, we aimed to: (1) map the gene expression and localization of NP and their receptors (NPR) in human post-mortem brain tissue; (2) compare the relative amounts of NP and NPR between the brain tissue of AD patients and non-demented controls, and (3) compare the relative amounts of NP between the cerebrospinal fluid (CSF) of AD patients and non-demented controls. Using the publicly available Allen Human Brain Atlas dataset, we mapped the gene expression of NP and NPR in healthy humans. Using immunohistochemistry, we visualized the localization of NP and NPR in the frontal cortex of AD patients (n = 10, mean age 85.8 ± 6.2 years) and non-demented controls (mean age = 80.2 ± 9.1 years). Using Western blotting and ELISA, we quantified the relative amounts of NP and NPR in the brain tissue and CSF of these AD patients and non-demented controls. Our results showed that NP and NPR genes were ubiquitously expressed throughout the brain in healthy humans. NP and NPR were present in various cellular structures including in neurons, astrocyte-like structures, and cerebral vessels in both AD patients and non-demented controls. Furthermore, we found higher amounts of NPR type-A in the brain of AD patients (p = 0.045) and lower amounts of NP type-B in the CSF of AD patients (p = 0.029). In conclusion, this study shows the abundance of NP and NPR in the brain of humans suggesting involvement of NP in various brain functions. In addition, our findings suggest alterations of NP levels in the brain of AD patients. The role of NP in the development and progression of AD remains to be elucidated.

Soluble Tumor Necrosis Factor Alpha Receptor 1, Bone Resorption, and Bone Mineral Density in the Year Following Hip Fractures: The Baltimore Hip Studies.

Although inflammation is known to influence bone turnover and bone mineral density (BMD), less is known about role of soluble tumor necrosis factor alpha receptor 1 (sTNFα-R1) in changes in bone turnover and BMD in the year after hip fracture. We studied 245 persons (117 men and 128 women) from the Baltimore Hip Studies. Bone turnover markers of resorption (carboxy-terminal type I collagen cross-links [CTX-I]) and formation (amino-terminal propeptide type I collagen [P1NP]), BMD of the contralateral hip, and sTNFα-R1 were measured within 15 days of hospitalization and 2, 6, and 12 months later. Latent class growth modeling was used to determine sTNFα-R1 trajectories. Weighted generalized estimating equations were used to examine the association of sTNFα-R1 trajectories with serum levels of CTX-I and P1NP and BMD; standardized beta coefficients (߈) are reported. Higher baseline sTNFα-R1 was significantly associated with a greater rate of CTX-I change (߈ = 0.26, p = 0.004). Four distinct sTNFα-R1 trajectories were identified. The two groups with higher sTNFα-R1 levels during the year following fracture had faster increasing levels of CTX-I compared to the group with lowest sTNFα-R1 levels (men: group 3: ߈ = 0.76, p = 0.02; group 4: ߈ = 1.4, p < 0.001; women: group 3; ߈ = 0.67, p = 0.02; group 4: ߈ = 1.3, p = 0.004). Men in the highest sTNFα-R1 group had a greater decline in BMD compared to the lowest sTNFα-R1 group (2-month ߈ = -0.01, p = 0.01; 6-month: ߈ = -0.09, p = 0.001; 12-months: ߈ = -0.1, p < 0.001). An increasing rate of CTX-I was associated with a steeper decline in total hip BMD in those within higher sTNFα-R1 trajectory groups (p < 0.001). CTX-I was significantly increased with sTNFα-R1 in both sexes. CTX-I and the highest sTNFα-R1 trajectory were significantly associated with declines in total hip BMD in men. Interventions that reduce systemic inflammation should be explored to reduce bone resorption and prevent a decline in BMD after hip fracture. © 2018 American Society for Bone and Mineral Research.

Topical Reformulation of Valsartan for Treatment of Chronic Diabetic Wounds.

Chronic wounds are among the most devastating and difficult to treat consequences of diabetes. Dysregulation of the skin renin-angiotensin system is implicated in abnormal wound healing in diabetic and older adults. Given this, we sought to determine the effects of topical reformulations of the angiotensin type 1 receptor blockers losartan and valsartan and the angiotensin-converting enzyme inhibitor captopril on wound healing in diabetic and aged mice with further validation in older diabetic pigs. The application of 1% valsartan gel compared with other tested formulations and placebo facilitated and significantly accelerated closure time and increased tensile strength in mice, and was validated in the porcine model. One percent of valsartan gel-treated wounds also exhibited higher mitochondrial content, collagen deposition, phosphorylated mothers against decapentaplegic homologs 2 and 3 and common mothers against decapentaplegic homolog 4, alpha-smooth muscle actin, CD31, phospho-vascular endothelial growth factor receptor 2, and p42/44 mitogen-activated protein kinase. Knockout of the angiotensin subtype 2 receptors abolished the beneficial effects of angiotensin type 1 receptor blockers, suggesting a role for angiotensin subtype 2 receptors in chronic wound healing.

Chitotriosidase, a marker of innate immunity, is elevated in patients with primary breast cancer.

BACKGROUND: Cancer progression has been associated with altered immune cell function and activation. Neopterin, which is secreted by interferon-γ stimulated macrophages, exhibits an association with multiple cancer types and metastatic disease. Chitotriosidase, which is secreted by chronically activated macrophages and granulocyte-macrophage colony-stimulating factor stimulated neutrophils has not been studied in the setting of cancer. OBJECTIVE: The goal of this discovery study was to screen chitotriosidase for diagnostic capacity in detecting cancer and compare its operating characteristics with those of neopterin. METHODS: Serum from subjects with breast (n= 66) or prostate (n= 70) cancer, and from 204 subjects free of malignant disease were studied. Chitotriosidase was measured by enzyme activity assay, while neopterin was measured by a competitive enzyme immunoassay. Statistical analyses included group comparisons by Mann Whitney U test, diagnostic capacity by receiver operating characteristics (ROC) curve analysis and biomarker associations with physiologic and clinical measures by Spearman correlation. RESULTS: Chitotriosidase activity was significantly higher in both cancer types compared with gender matched controls, though only in breast cancer was the diagnostic capacity significant (area under the ROC curve of 0.97 ± 0.01). In contrast, neopterin was significantly elevated in prostate cancer and exhibited discriminatory capacity (area under the ROC curve of 0.76 ± 0.05). Age, BMI, % body fat and metastasis were variables that correlated with neopterin, but not chitotriosidase levels. CONCLUSIONS: The operating characteristics of serum chitotriosidase were different from neopterin and further analysis of chitotriosidase as a biomarker for breast cancer is warranted.

Discovery and Validation of Agonistic Angiotensin Receptor Autoantibodies as Biomarkers of Adverse Outcomes.

BACKGROUND: Agonistic angiotensin II type 1 receptor autoantibodies (AT1RaAbs) have not been associated with functional measures or risk for adverse health outcomes. AT1RaAbs could be used to stratify patient risk and to identify patients who can benefit from angiotensin receptor blocker treatment. METHODS: Demographic and physiological covariates were measured in a discovery set of community-dwelling adults from Baltimore (N=255) and AT1RaAb associations with physical function tests and outcomes assessed. A group from Chicago (N=60) was used for validation of associations and to explore the impact of angiotensin receptor blocker treatment. RESULTS: The Baltimore group had 28 subjects with falls, 32 frail subjects, and 5 deaths. Higher AT1RaAbs correlated significantly with interleukin-6 (Spearman r=0.33, P<0.0001), systolic blood pressure (Spearman r=0.28, P<0.0001), body mass index (Spearman r=0.28, P<0.0001), weaker grip strength (Spearman r=-0.34, P<0.01), and slower walking speed (Spearman r=-0.30, P<0.05). Individuals with high AT1RaAbs were 3.9 (95% confidence interval, 1.38-11.0) times more likely to be at high risk after adjusting for age (P<0.05). Every 1 µg/mL increase in AT1RaAbs increased the odds of falling 30% after adjusting for age, sex, body mass index, and blood pressure. The Chicago group had 46 subjects with falls and 60 deaths. Serum AT1RaAb levels were significantly correlated with grip strength (Spearman r=-0.57, P<0.005), walking speed (Spearman r=-0.47, P<0.005), and falls (Spearman r=0.30, P<0.05). Every 1 µg/mL increase in AT1RaAbs, decreased time to death by 9% after adjusting for age, sex, body mass index, and blood pressure. Chronic treatment with angiotensin receptor blockers was associated with better control of systolic blood pressure and attenuation of decline in both grip strength and time to death. CONCLUSIONS: In older individuals, higher AT1RaAb levels were associated with inflammation, hypertension, and adverse outcomes. Angiotensin receptor blocker treatment may blunt the harm associated with high levels of AT1RaAb.

Osteoblast function and bone histomorphometry in a murine model of Rett syndrome.

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder due to mutations affecting the neural transcription factor MeCP2. Approximately 50% of affected females have decreased bone mass. We studied osteoblast function using a murine model of RTT. Female heterozygote (HET) and male Mecp2-null mice were compared to wild type (WT) mice. Micro-CT of tibia from 5 week-old Mecp2-null mice showed significant alterations in trabecular bone including reductions in bone volume fraction (-29%), number (-19%), thickness (-9%) and connectivity density (-32%), and increases in trabecular separation (+28%) compared to WT. We also found significant reductions in cortical bone thickness (-18%) and in polar moment of inertia (-45%). In contrast, cortical and trabecular bone from 8 week-old WT and HET female mice were not significantly different. However, mineral apposition rate, mineralizing surface and bone formation rate/bone surface were each decreased in HET and Mecp2-null mice compared to WT mice. Histomorphometric analysis of femurs showed decreased numbers of osteoblasts but similar numbers of osteoclasts compared to WT, altered osteoblast morphology and decreased tissue synthesis of alkaline phosphatase in Mecp2-null and HET mice. Osteoblasts cultured from Mecp2-null mice, which unlike WT osteoblasts did not express MeCP2, had increased growth rates, but reductions in mRNA expression of type I collagen, Runx2 and Osterix compared to WT osteoblasts. These results indicate that MeCP2 deficiency leads to altered bone growth. Osteoblast dysfunction was more marked in Mecp2-null male than in HET female mice, suggesting that expression of MeCP2 plays a critical role in bone development.

The short-term and long-term impact of a brief aging research training program for medical students.

Summer training in aging research for medical students is a strategy for improving the pipeline of medical students into research careers in aging and clinical care of older adults. Johns Hopkins University has been offering medical students a summer experience of mentored research, research training, and clinical shadowing since 1994. Long-term outcomes of this program have not been described. The authors surveyed all 191 participants who had been in the program from 1994-2010 (60% female and 27% underrepresented minorities) and received a 65.8% (N = 125) response rate. The authors also conducted Google and other online searches to supplement study findings. Thirty-seven percent of those who have completed training are now in academic medicine, and program participants have authored or coauthored 582 manuscripts. Among survey respondents, 95.1% reported that participation in the Medical Student Training in Aging Research program increased their sensitivity to the needs of older adults. This program may help to build commitment among medical students to choose careers in aging.

Associations of inflammatory markers with AIDS and non-AIDS clinical events after initiation of antiretroviral therapy: AIDS clinical trials group A5224s, a substudy of ACTG A5202.

BACKGROUND: The association of inflammatory biomarkers with clinical events after antiretroviral therapy initiation is unclear. METHODS: A5202 randomized 1857 treatment-naive subjects to abacavir/lamivudine or tenofovir-DF/emtricitabine with efavirenz or atazanavir/ritonavir. Substudy A5224s measured inflammatory biomarkers on subjects with available plasma from baseline and week 24 or 96. An exploratory analysis of the association of high-sensitivity C-reactive protein, interleukin-6 (IL-6), soluble receptors of tumor necrosis factor α (sTNF)-RI, sTNF-RII, TNF-α, soluble vascular cellular adhesion molecules (sVCAM-1), and soluble intercellular adhesion molecules (sICAM-1) with times to AIDS and to non-AIDS events used Cox proportional hazards models. RESULTS: Analysis included 244 subjects; 85% men and 48% white non-Hispanic with median age 39 years, HIV-1 RNA of 4.6 log10 copies per milliliter, and CD4 of 240 cells per microliter. Overall, 13 AIDS events (9 opportunistic infections, 3 AIDS-cancers, and 1 recurrent bacterial pneumonia) and 18 non-AIDS events (6 diabetes, 4 cancers, 3 cardiovascular, and 5 pneumonias) occurred. Higher baseline IL-6, sTNF-RI, sTNF-RII, and sICAM-1 were significantly associated with increased risk of AIDS-defining events. Adjustment for baseline HIV-1 RNA did not change results, whereas adjusting for baseline CD4 count left only sTNF-RI and sICAM-1 significantly associated with increased risk. Time-updated values of IL-6, sTNFR-I and II, and sICAM-1 were also associated with an increased risk. For non-AIDS events, only higher baseline high-sensitivity C-reactive protein was significantly associated with increased risk, whereas higher IL-6 was marginally associated with higher risk. Analyses of time-updated biomarker values showed tumor necrosis factor α to be significantly associated with increased risk, even after adjustment for antiretroviral therapy, and CD4 count or HIV-1 RNA. CONCLUSIONS: Higher levels of several inflammatory biomarkers were independently associated with increased risk of AIDS and non-AIDS events.

WISP1/CCN4: a potential target for inhibiting prostate cancer growth and spread to bone.

Prostate cancer (PC) is a leading cause of death in men however the factors that regulate its progression and eventual metastasis to bone remain unclear. Here we show that WISP1/CCN4 expression in prostate cancer tissues was up-regulated in early stages of the disease and, further, that it correlated with increased circulating levels of WISP1 in the sera of patients at early stages of the disease. WISP1 was also elevated in the mouse prostate cancer model TRAMP in the hypoplastic diseased tissue that develops prior to advanced carcinoma formation. When the ability of anti-WISP1 antibodies to reduce the spread of PC3-Luc cells to distant sites was tested it showed that twice weekly injections of anti-WISP1 antibodies reduced the number and overall size of distant tumors developed after intracardiac (IC) injection of PC3-Luc cells in mice. The ability of antibodies against WISP1 to inhibit growth of PC3-Luc cancer cells in mice was also evaluated and showed that twice weekly injections of anti-WISP1 antibodies reduced local tumor growth when examined in xenografts. To better understand the mechanism of action, the migration of PC3-Luc cells through membranes with or without a Matrigel™ barrier showed the cells were attracted to WISP1, and that this attraction was inhibited by treatment with anti-WISP1 antibodies. We also show the expression of WISP1 at the bone-tumor interface and in the stroma of early grade cancers suggested WISP1 expression is well placed to play roles in both fostering growth of the cancer and its spread to bone. In summary, the up-regulation of WISP1 in the early stages of cancer development coupled with its ability to inhibit spread and growth of prostate cancer cells makes it both a potential target and an accessible diagnostic marker for prostate cancer.

Serum chitotriosidase, a putative marker of chronically activated macrophages, increases with normal aging.

BACKGROUND: Chitotriosidase (ChT) is secreted by chronically activated macrophages in Gaucher's disease. We hypothesize that circulating levels of ChT are altered with normal aging, reflecting age-related chronic macrophage activation. Potential sources that might contribute to altered levels were assessed by measuring systemic levels of ChT are α-naphthyl acetate esterase, a macrophage lysosomal enzyme; granulocyte-macrophage colony-stimulating factor (GM-CSF), which stimulates neutrophilic granule release of ChT; interleukin-6 (IL-6); and neopterin, a macrophage activation marker. METHODS: Serum was obtained from 315 healthy participants whose age ranged from 18 to 92 years. Anthropometric measures included percent body fat and body mass index. ChT and α-naphthyl acetate esterase levels were measured by enzyme activity assays. GM-CSF, IL-6, and neopterin concentrations were measured by commercial enzyme-linked immunosorbent assays. Serum marker values were statistically analyzed using nonparametric tests. RESULTS: Six percent of the participants had undetectable ChT levels. A positive association with age was observed for ChT and IL-6, whereas a negative correlation with age was seen for α-naphthyl acetate esterase and GM-CSF. ChT values were not associated with α-naphthyl acetate esterase or GM-CSF levels. ChT was independently associated with IL-6 and neopterin levels, but statistical significance was attenuated when controlled for age. CONCLUSIONS: The data are consistent with increased serum ChT activity not arising from altered macrophage lysosomal enzyme trafficking or GM-CSF-stimulated release of neutrophil granule stores. The association of ChT with age remains significant after controlling for neopterin and IL-6 changes with age, suggesting that ChT levels reflect a macrophage state distinct from acute macrophage activation or inflammatory state.