Oklahoma City VA Medical Center

OBJECTIVE: Autophagy is a critical cellular system for removal of aggregated proteins and damaged organelles. Although dysregulated autophagy is implicated in the development of heart failure, the role of autophagy in the development of diabetic cardiomyopathy has not been studied. We investigated whether chronic activation of the AMP-activated protein kinase (AMPK) by metformin restores cardiac function and cardiomyocyte autophagy in OVE26 diabetic mice. RESEARCH DESIGN AND METHODS: OVE26 mice and cardiac-specific AMPK dominant negative transgenic (DN)-AMPK diabetic mice were treated with metformin or vehicle for 4 months, and cardiac autophagy, cardiac functions, and cardiomyocyte apoptosis were monitored. RESULTS: Compared with control mice, diabetic OVE26 mice exhibited a significant reduction of AMPK activity in parallel with reduced cardiomyocyte autophagy and cardiac dysfunction in vivo and in isolated hearts. Furthermore, diabetic OVE26 mouse hearts exhibited aggregation of chaotically distributed mitochondria between poorly organized myofibrils and increased polyubiquitinated protein and apoptosis. Inhibition of AMPK by overexpression of a cardiac-specific DN-AMPK gene reduced cardiomyocyte autophagy, exacerbated cardiac dysfunctions, and increased mortality in diabetic mice. Finally, chronic metformin therapy significantly enhanced autophagic activity and preserved cardiac functions in diabetic OVE26 mice but not in DN-AMPK diabetic mice. CONCLUSIONS: Decreased AMPK activity and subsequent reduction in cardiac autophagy are important events in the development of diabetic cardiomyopathy. Chronic AMPK activation by metformin prevents cardiomyopathy by upregulating autophagy activity in diabetic OVE26 mice. Thus, stimulation of AMPK may represent a novel approach to treat diabetic cardiomyopathy.

The activation and fusion of macrophages and of osteoclasts require the adaptor molecule DNAX-activating protein of 12 kD (DAP12), which contains immunoreceptor tyrosine-based activation motifs (ITAMs). TREM2 (triggering receptor expressed on myeloid cells-2) is the main DAP12-associated receptor in osteoclasts and, similar to DAP12 deficiency, loss of TREM2 in humans leads to Nasu-Hakola disease, which is characterized by bone cysts and dementia. Furthermore, in vitro experiments have shown that deficiency in DAP12 or TREM2 leads to impaired osteoclast development and the formation of mononuclear osteoclasts. Here, we demonstrate that the ligation of TREM2 activated phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase 1 (ERK1) and ERK2, and the guanine nucleotide exchange factor Vav3; induced the mobilization of intracellular calcium (Ca(2+)) and the reorganization of actin; and prevented apoptosis. The signaling adaptor molecule DAP10 played a key role in the TREM2- and DAP12-dependent recruitment of PI3K to the signaling complex. Src homology 2 (SH2) domain-containing inositol phosphatase-1 (SHIP1) inhibited TREM2- and DAP12-induced signaling by binding to DAP12 in an SH2 domain-dependent manner and preventing the recruitment of PI3K to DAP12. These results demonstrate a previously uncharacterized interaction of SHIP1 with DAP12 that functionally limits TREM2- and DAP12-dependent signaling and identify a mechanism through which SHIP1 regulates key ITAM-containing receptors by directly blocking the binding and activation of PI3K.

Recent research suggests that epigenetics, especially DNA methylation, plays a mechanistic role in aging. Epigenetic clocks, which measure changes in a few hundred specific CpG sites, can accurately predict chronological age in a variety of species, including humans. These clocks are currently the best biomarkers for predicting mortality in humans. Additionally, several studies have characterized the effects of aging across the methylome in a wide variety of tissues from humans and mice. A small fraction (~2%) of the CpG sites show age-related changes, either hypermethylation or hypomethylation with aging. Evaluation of non-CpG site methylation has only been examined in a few studies, with about ~0.5% of these sites showing a change with age. Therefore, while only a small fraction of cytosines in the genome show changes in DNA methylation with age, this represents 2 to 3 million cytosines in the genome. Importantly, the only study to compare the effect of aging on DNA methylation in male and female mice and humans found that >95% of the age-related changes in DNA methylation in the hippocampus were sexually divergent, i.e., the methylation did not differ between males and females at young age but age-related changes occurred in one sex but not the other. The age-related changes in DNA methylation tend to be enriched and under-represented in specific genomic contexts, with some commonalities between tissues and species that require further investigation. The strongest evidence that the age-related changes in DNA methylation play a role in aging comes from studies of anti-aging interventions (e.g., caloric restriction, dwarfism, and rapamycin treatment) in mice. These anti-aging interventions deaccelerate the epigenetic clocks and reverse/prevent 20 to 40% of the age-related changes in DNA methylation. It will be important in the future to demonstrate that at least some of the age-related changes in DNA methylation directly lead to alterations in the transcriptome of cells/tissues that could potentially contribute to aging.

The National Institute on Aging Interventions Testing Program (ITP) evaluates agents hypothesized to increase healthy lifespan in genetically heterogeneous mice. Each compound is tested in parallel at three sites, and all results are published. We report the effects of lifelong treatment of mice with four agents not previously tested: Protandim, fish oil, ursodeoxycholic acid (UDCA) and metformin - the latter with and without rapamycin, and two drugs previously examined: 17-α-estradiol and nordihydroguaiaretic acid (NDGA), at doses greater and less than used previously. 17-α-estradiol at a threefold higher dose robustly extended both median and maximal lifespan, but still only in males. The male-specific extension of median lifespan by NDGA was replicated at the original dose, and using doses threefold lower and higher. The effects of NDGA were dose dependent and male specific but without an effect on maximal lifespan. Protandim, a mixture of botanical extracts that activate Nrf2, extended median lifespan in males only. Metformin alone, at a dose of 0.1% in the diet, did not significantly extend lifespan. Metformin (0.1%) combined with rapamycin (14 ppm) robustly extended lifespan, suggestive of an added benefit, based on historical comparison with earlier studies of rapamycin given alone. The α-glucosidase inhibitor, acarbose, at a concentration previously tested (1000 ppm), significantly increased median longevity in males and 90th percentile lifespan in both sexes, even when treatment was started at 16 months. Neither fish oil nor UDCA extended lifespan. These results underscore the reproducibility of ITP longevity studies and illustrate the importance of identifying optimal doses in lifespan studies.

In 2009, rapamycin was reported to increase the lifespan of mice when implemented later in life. This observation resulted in a sea-change in how researchers viewed aging. This was the first evidence that a pharmacological agent could have an impact on aging when administered later in life, i.e., an intervention that did not have to be implemented early in life before the negative impact of aging. Over the past decade, there has been an explosion in the number of reports studying the effect of rapamycin on various diseases, physiological functions, and biochemical processes in mice. In this review, we focus on those areas in which there is strong evidence for rapamycin's effect on aging and age-related diseases in mice, e.g., lifespan, cardiac disease/function, central nervous system, immune system, and cell senescence. We conclude that it is time that pre-clinical studies be focused on taking rapamycin to the clinic, e.g., as a potential treatment for Alzheimer's disease.

Inasmuch as the clinical features of the idiopathic inflammatory myopathies are not easily differentiated from those of other similar rheumatic and neurologic conditions, diagnosis is often difficult. Various classification criteria for polymyositis and dermatomyositis have been suggested by a number of investigators. The most commonly accepted and used criteria include symmetric proximal muscle weakness, serum elevations of muscle enzymes, the classic electromyographic and muscle biopsy findings of inflammatory myopathy, and the typical skin rash of dermatomyositis. Although these criteria are clinically useful, they can result in misdiagnoses and inappropriate therapies. They also result in heterogeneous patient groups being selected for clinical and laboratory studies. Furthermore, they do not include recent findings related to the myositis-specific autoantibodies and magnetic resonance imaging of muscle that have been found to be important adjuncts in assessing patients with muscle weakness or elevations of muscle enzymes. A modification to the Bohan and Peter criteria is proposed to include myositis-specific autoantibodies and magnetic resonance imaging. This proposal could initiate productive discussions and investigations of the sensitivity and specificity of new classification criteria for myositis and could ultimately enhance our treatment capabilities.

BACKGROUND: This study tested cortisol responses to a psychological stressor in controls (CT) versus patients who were diagnosed as alcohol dependent (AD) or alcohol and stimulant dependent (ADSD) by DSM-IV criteria and who were abstinent for 3 to 4 weeks from alcohol and illicit drugs. Alcohol increases cortisol secretion acutely and during withdrawal. However, there is little information about abnormalities of hypothalamic-pituitary-adrenocortical (HPA) reactivity in recovering alcoholics. METHODS: Accordingly, we tested HPA function in the laboratory between 7:00 and 9:30 AM on control versus stress days. Stress consisted of a 20-min public speaking challenge with preparation and delivery of two short speeches, ostensibly evaluated for quality of delivery, whereas control involved relaxing for the same period. Cortisol was measured in saliva collected at baseline, stress or control, and recovery period, and also at home at 9:00 PM on one of the two days. RESULTS: The three groups did not differ in diurnal patterns of cortisol secretion on the rest day and 9:00 PM sample, which indicated that AD and ADSD patients had intact diurnal HPA regulation at rest. During speech stress, the CT subjects showed the expected cortisol increase (p < 0.0001), whereas neither AD nor ADSD patients responded significantly. Cortisol values were not accounted for by covariates such as depression, posttraumatic stress disorder, glucose metabolism, or anthropometric or demographic characteristics. CONCLUSIONS: The apparent stress hyporesponsiveness of the AD and ADSD patients suggests a persistent disruption of HPA function, perhaps due to incomplete recovery from prior abuse, or to a preexisting alteration in neural systems that regulate HPA responses to stress.

Systemic lupus erythematosus (SLE), a complex polygenic autoimmune disease, is associated with increased complement activation. Variants of genes encoding complement regulator factor H (CFH) and five CFH-related proteins (CFHR1-CFHR5) within the chromosome 1q32 locus linked to SLE, have been associated with multiple human diseases and may contribute to dysregulated complement activation predisposing to SLE. We assessed 60 SNPs covering the CFH-CFHRs region for association with SLE in 15,864 case-control subjects derived from four ethnic groups. Significant allelic associations with SLE were detected in European Americans (EA) and African Americans (AA), which could be attributed to an intronic CFH SNP (rs6677604, in intron 11, P(meta) = 6.6×10(-8), OR = 1.18) and an intergenic SNP between CFHR1 and CFHR4 (rs16840639, P(meta) = 2.9×10(-7), OR = 1.17) rather than to previously identified disease-associated CFH exonic SNPs, including I62V, Y402H, A474A, and D936E. In addition, allelic association of rs6677604 with SLE was subsequently confirmed in Asians (AS). Haplotype analysis revealed that the underlying causal variant, tagged by rs6677604 and rs16840639, was localized to a ~146 kb block extending from intron 9 of CFH to downstream of CFHR1. Within this block, the deletion of CFHR3 and CFHR1 (CFHR3-1Δ), a likely causal variant measured using multiplex ligation-dependent probe amplification, was tagged by rs6677604 in EA and AS and rs16840639 in AA, respectively. Deduced from genotypic associations of tag SNPs in EA, AA, and AS, homozygous deletion of CFHR3-1Δ (P(meta) = 3.2×10(-7), OR = 1.47) conferred a higher risk of SLE than heterozygous deletion (P(meta) = 3.5×10(-4), OR = 1.14). These results suggested that the CFHR3-1Δ deletion within the SLE-associated block, but not the previously described exonic SNPs of CFH, might contribute to the development of SLE in EA, AA, and AS, providing new insights into the role of complement regulators in the pathogenesis of SLE.

Thrombophilia is the imbalance between the procoagulant systems and the regulatory mechanisms leading to excessive fibrin production and resulting thrombus formation.1,2 These imbalances can be caused by genetic or acquired factors or by a combination of the two.1 However, the process leading to excessive fibrin formation is not as simple as a single factor being disproportionate to the other factors, but rather a multifactorial model, in which many factors interact, leading to the development of thrombosis.1 Therefore, to attempt to determine the correct cause, an assessment of established risk factors must be made.2 Currently, this is an incomplete process because we do not know all of the responsible factors. But we do know several of the major factors.2 One of the main factors analyzed in most assessments is protein S (PS).2 A deficiency of PS seems to be a major contributor to thrombophilia, yet its biochemistry, physiology, and pathobiology are complex.4,5 PS functions as an antithrombotic factor by an activated protein C (APC)-dependent mechanism and an APC-independent mechanism.2,4,6 Its ultimate antithrombotic role is to down-regulate thrombin formation. PS also seems to have a role in fibrinolysis, complement regulation, and other defense mechanisms.6 The importance of any of these mechanisms in vivo is unknown.6 PS is a nonenzymatic cofactor that binds in equilibrium to a large complement protein, C4b-binding protein.6 Only the unbound (or “free”) form of PS has APC cofactor activity on the phospholipid surface to inactivate factors Va and VIIIa.2,4 Thrombophilia is a defined as a “complex disease” in which multiple factors together have a major role in the disease process. Today we understand only the basics of causation of the disease process. There are many ways to break down the disease process so that we can get a handle on the concept of thrombophilia. From a clinical perspective, the best way is to examine the disease is by dividing it into increasing complexity from the genetic defect to the clinical manifestations. To understand thrombophilia, the following definitions can help clinically define the disease process1: (1) genotype: the genetic makeup of an organism with reference to a single trait; the assumed cause of PS deficiency; (2) protein phenotype: the protein component made by the gene in question; the amount of PS synthesized by 3 different cell types that may or may not reflect the gene status; (3) laboratory phenotype: the measurable amount of protein produced by the gene in question. PS detection levels are measurable by various methods that may or may not correlate with the protein phenotype or genotype. (4) clinical phenotype: the expression of clinical characteristics of the protein (or lack of protein) made by a gene (with or without environmental and acquired interactions). Thrombus formation is the clinical phenotype that is assumed to be due to the PS deficiency, but many other factors may contribute to the clinical phenotype. An organism has an impressive ability to adjust its physiology to compensate for internal (genetic) errors and to respond to environmental changes. Clinical phenotype expression is the ultimate failure of the system of complex processes with numerous components to balance the system. In theory, this complexity should allow the overall process to tolerate moderate perturbations without causing an abnormal clinical phenotype. However, in some cases, the deficiency like PS deficiency seems to overwhelm the system and express the clinical phenotype.1 The initial question one must ask about thrombophilia is about the importance of the clinical phenotype compared with the genotype. Because thrombophilia is a complex disease, the most important clinical aspect is venous thrombosis because it can be diagnosed and treated irrespective of the genotype or the results of a laboratory assessment. However, this is usually after the occurrence of the disease process (venous thrombosis). The genetic and acquired causes of the thrombophilia are the second important aspect. However, at this time, it is not practical to identify the genotype(s) using molecular biologic methods. So we must rely on surrogate markers for the genetic defects by analyzing the protein levels found in plasma.2,4 This creates many issues associated with the identification of a genetic deficiency by laboratory phenotype alone. One of the major questions from a laboratory perspective is: Does a decreased level of PS (lower than the reference limit) truly constitute a genetic deficiency? This particular question is nicely addressed by Duebgen et al in this issue of the Journal.7 By using the clinical phenotype of thrombosis as the starting point, the authors identified the patients with levels of PS lower than the reference interval and subsequently analyzed for abnormalities in the PS gene. A number of very interesting conclusions come to light from their data. First, there is not a good correlation of low PS levels (PS activity or free PS antigen) and apparent PS genetic abnormalities. Second, Duebgen et al7 observed that patients with deletions or stop codons had significantly lower levels of PS compared with the laboratory values for patients with no detectable mutations but abnormal levels. Finally, Duebgen et al7 observed that the PS activity levels might be a better indicator of a genetic deficiency than free PS antigen. The definition of PS deficiency has not been definitively established.4,7–9 We have always assumed that establishing a normal reference limit (average ± 2 SD) constituted the true genetic deficiency.4,8,9 But from the article by Duebgen et al,7 how do we define PS deficiency? Defining PS deficiency has always been a problem not only because of the difficulty of assay performance but also because of the role of preanalytic variables, patient-acquired factors, and multiple factors affecting the plasma levels and the effect of postvenous thrombosis. Duebgen et al7 analyzed the results of the laboratory phenotype values compared with PS genotypes. They presented data indicating that a true PS deficiency based solely on laboratory assays may not accurately diagnose PS genotype abnormalities. This is a major observation that could affect most clinical laboratories performing thrombophilia workups. The bottom-line question is: Are we accurately diagnosing PS deficiency? We thought we were, but this article suggests we may not.7 More exploration must be made in the comparison of laboratory values of decreased PS levels and PS genotypes.4,8,9 Other factors may cause falsely low results in patients without a true PS genetic deficiency.2,4 Unfortunately, determining the genetics of PS deficiencies is really not yet a clinical laboratory option.2,4,7,8,10 The attempt by Duebgen et al7 to correlate genetic mutation of PS with the presence of decreased PS plasma levels is exciting. When they found mutations where no protein is made (large deletions and nonsense mutations), the laboratory values were significantly lower than for the group of patients with levels lower than the reference limit but with no detectable mutation.7 Is the presence of slightly abnormal plasma levels of PS activity or free PS antigen in patients with no detectable mutation truly a deficient state or is the actual lower limit of the “normal” range much lower4,7,9,11? There are a number of potential causes of low PS that are not related to a genetic defects: (1) The true reference range is wider than the established ranges performed by the standard statistical method (95th percentile). (2) Acquired states in people with previous thrombosis have lower levels. (3) Calibrators of the PS assay system are not accurate or are too high. (4) The current assay systems do not measure the true function(s) of the PS molecule. Duebgen et al7 found a correlation of the type of deficiency and the plasma level of PS. It is interesting that they found 19% to 26% PS activity and similar free PS antigen levels in the heterozygous deletions and nonsense mutations when one would expect levels of 40% to 60%.7 The cause of these lower-than-expected levels is unclear. For the analysis of PS in plasma, there are 3 available types of tests: PS activity, free PS antigen (which should correlate with activity), and total PS antigen.4 The total PS antigen, which is a measure of all PS antigen material in plasma combining the free and bound forms of PS, is not very helpful for the clinical diagnosis of PS deficiency.2,4 Many laboratories do not perform this assay because it does not contribute significantly to the diagnostic process.4 The other 2 assays are used to determine the presence or absence of a deficiency. However, both of these assays are sensitive to preanalytic variables and reagent instability.4,7,11 Acquired problems and patient variability can cause significant reductions of plasma PS levels measured by both assays.4,11 These factors can reduce the plasma PS levels, but it is unclear if they actually increase the clinical phenotype probability of thrombus formation. Duebgen et al7 state their case for the use of PS activity assay first, before measuring free PS antigen.7 They found that the activity was usually lower than the free PS antigen in known cases of genetic mutation.7 For most clinical coagulation evaluations, the main goal is to test for function (activity) and, if decreased, to confirm by antigenic assay to determine the type of deficiency (quantitative or qualitative).2 PS may be an exception because the PS activity assay is influenced by more factors than free PS antigen, including less stable reagents, assay inconsistencies, and unknown patient variables.2,4,11 In our studies, we have found that a high number of falsely low PS activity results are reported if the PS activity is used as the first assay.11 Of 4 US Food and Drug Administration–approved PS activity assays, the percentage of falsely low PS activity values in healthy people ranged from 10% to 20%.11 The results were sporadic and unpredictable and were normal if repeated on a new sample. In our laboratory and other special coagulation laboratories in the United States, the initial PS analysis is free PS antigen assay (a surrogate marker of PS activity).4,12 When the free PS antigen is less than the reference range, the PS activity assay is performed to confirm the deficiency.4,12 This algorithm of PS analysis will eliminate most misdiagnoses of PS deficiency in healthy people because falsely low PS activity values will be avoided.4,12 In addition, this algorithm results in significant cost savings by using less “reflex” and repeat testing.4 A minor disadvantage of free PS antigen might be the rare type II defect not being detected; however, Duebgen et al7 were able to identify the known PS mutations with the free PS antigen.4,7,12 The type of mutation in PS makes it more difficult to identify a correct diagnosis based on the laboratory assay. Based on the data reported by Duebgen et al,7 it is unclear about making a diagnosis using the laboratory phenotype as the major evidence to determine a lifelong deficiency. Can we rely on laboratory phenotype testing for the trueness of the PS deficiency? I do not believe so. The laboratory diagnosis of PS deficiency as familial PS deficiency remains difficult to determine. In the study by Duebgen et al,7 many of the laboratory deficiencies of PS did not have an associated PS gene mutation. We cannot conclude that these low laboratory values are true PS deficiencies, but we also cannot say that they are not. PS is probably the most difficult to assay to perform in coagulation studies. Laboratory phenotype determination is difficult owing to wide variations from preanalytic variables, assay method issues, and unstable reagents in conjunction with acquired abnormalities and patient variability. At this point, the laboratory cannot truly determine PS genotype with absolute certainty. Obtaining accurate genotype results is difficult with plasma-based PS activity and free PS antigen assays because the assays are prone to many variables from the patient to preanalytic variables to assay reagents. To better correlate the clinical phenotype with the PS genotype, we must circumvent the cause of these major variables by not using plasma-based assays. The most direct way to determine a true PS genetic deficiency is by genetic methods. However, direct genetic analysis is not a realistic method in today’s clinical laboratory. Probably, we will have to go to genetics as soon as possible to confirm that the clinical phenotype that may or may not be manifested by the laboratory result is actually caused by a genetic defect. Can we call a clinical phenotype (thrombosis) as being due to PS genetic deficiency if only the laboratory values are decreased? Must we confirm a genetic defect? These are the main questions addressed Duebgen et al.7 It seems that it is not correct to assume that moderately decreased levels are a true PS genetic deficiency. We are not even sure that these low PS levels contributed to the cause of the thrombosis. Based on the study by Duebgen et al,7 it seems that a lower cutoff for the PS reference interval should be considered. Using 3 SD from the average would eliminate a majority of the assumed PS deficiencies that are confined to a genetic mutation. These data and data from the mutation data bank for PS deficiencies should be compared with plasma PS levels to determine if moderately low levels of PS are associated with no mutations. Intermediate between genetics and expression of disease lies the laboratory testing aspect, which is where the problem of true identification of a defect occurs. We are still without true guidance on the use of plasma PS activity and free PS antigen levels in the diagnosis of PS deficiency. This article raises more questions for clinical laboratories but certainly is an insightful investigation into the genetics and measurement arenas. The article tells us that we need to be looking at the diagnostic criteria for PS deficiency from a new perspective.

UNLABELLED: BACKGROUND Hepatitis C viral (HCV) infection is the leading cause of death due to liver disease in the United States. Currently, pegylated interferon and ribavirin produce sustained viral remission in only 50% of patients. Additional agents are needed to increase the cure rate. In vitro experiments show strong antiviral effects of fluvastatin against HCV. OBJECTIVES: To assess the safety and antiviral effects of fluvastatin in chronic HCV carriers. METHODS: 31 veterans with chronic HCV were prospectively given oral doses of fluvastatin, 20 to 320 mg/day, for 2-12 weeks with weekly monitoring of HCV RNA and liver tests. Reductions of viral load (P < 0.01) versus a control group were considered suppressive. RESULTS: With 80 mg a day or less, 11/22 (50%) patients responded by lowering HCV RNA. The first lowering occurred within 4 weeks (9/11, 82%). The greatest weekly change in HCV RNA level was a 1.75 log(10) reduction. When lowered in responders, the viral load remained relatively constant for 2-5 weeks (7/9, 78%), or on the next test rebounded immediately to a non-significant change from, baseline (n = 2). Continued lowering of virus was seen in 2/19 (22 %) patients when the study ended. We found no evidence of liver tests worsening. CONCLUSIONS: FLV used as monotherapy in vivo showed suppressive effects of HCV clinically that are modest, variable, and often short-lived. These findings support "proof-of-concept" for pilot trials combining fluvastatin with standard therapy. Statins and fluvastatin, in particular, appear to be safe for use in hepatitis C.

Inflammaging, characterized by an increase in low-grade chronic inflammation with age, is a hallmark of aging and is strongly associated with various age-related diseases, including chronic liver disease (CLD) and hepatocellular carcinoma (HCC). Because necroptosis is a cell death pathway that induces inflammation through the release of DAMPs, we tested the hypothesis that age-associated increase in necroptosis contributes to chronic inflammation in aging liver. Phosphorylation of MLKL and MLKL oligomers, markers of necroptosis, as well as phosphorylation of RIPK3 and RIPK1 were significantly upregulated in the livers of old mice relative to young mice and this increase occurred in the later half of life (i.e., after 18 months of age). Markers of M1 macrophages, expression of pro-inflammatory cytokines (TNFα, IL6 and IL1β), and markers of fibrosis were all significantly upregulated in the liver with age and the change in necroptosis paralleled the changes in inflammation and fibrosis. Hepatocytes and liver macrophages isolated from old mice showed elevated levels of necroptosis markers as well as increased expression of pro-inflammatory cytokines relative to young mice. Short-term treatment with the necroptosis inhibitor, necrostatin-1s (Nec-1s), reduced necroptosis, markers of M1 macrophages, fibrosis, and cell senescence as well as reducing the expression of pro-inflammatory cytokines in the livers of old mice. Thus, our data show for the first time that liver aging is associated with increased necroptosis and necroptosis contributes to chronic inflammation in the liver, which in turn appears to contribute to liver fibrosis and possibly CLD.

In contrast to other mouse models that are deficient in antioxidant enzymes, mice null for Cu/Zn-superoxide dismutase ( Sod1 −/ − mice) show a major decrease in lifespan and several accelerated aging phenotypes. The goal of this study was to determine if cell senescence might be a contributing factor in the accelerated aging phenotype observed in the Sod1 −/ − mice. We focused on kidney because it is a tissue that has been shown to a significant increase in senescent cells with age. The Sod1 −/ − mice are characterized by high levels of DNA oxidation in the kidney, which is attenuated by DR. The kidney of the Sod1 −/ − mice also have higher levels of double strand DNA breaks than wild type (WT) mice. Expression (mRNA and protein) of p16 and p21, two of the markers of cellular senescence, which increased with age, are increased significantly in the kidney of Sod1 −/ − mice as is β-gal staining cells. In addition, the senescence associated secretory phenotype was also increased significantly in the kidney of Sod1 −/ − mice compared to WT mice as measured by the expression of transcripts for IL-6 and IL-1β. Dietary restriction of the Sod1 −/ − mice attenuated the increase in DNA damage, cellular senescence, and expression of IL-6 and IL-1β. Interestingly, the Sod1 −/ − mice showed higher levels of circulating cytokines than WT mice, suggesting that the accelerated aging phenotype shown by the Sod1 −/ − mice could result from increased inflammation arising from an accelerated accumulation of senescent cells. Based on our data with Sod1 −/ − mice, we propose that various bouts of increased oxidative stress over the lifespan of an animal leads to the accumulation of senescent cells. The accumulation of senescent cells in turn leads to increased inflammation, which plays a major role in the loss of function and increased pathology that are hallmark features of aging. • Sod1 − / − mice have high levels of oxidative damage and DNA double strand breaks. • Sod1 −/ − mice show increased cellular senescence, e.g., p16, p21 and β-gal+ cells. • Sod1 −/ − mice showed an increase in the senescence associated secretory phenotype. • Dietary restriction attenuated cellular senescence and inflammation in Sod1 −/− mice.

Ovarian aging leads to diminished fertility, dysregulated endocrine signaling and increased chronic disease burden. These effects begin to emerge long before follicular exhaustion. Female humans experience a sharp decline in fertility around 35 years of age, which corresponds to declines in oocyte quality. Despite a growing body of work, the field lacks a comprehensive cellular map of the transcriptomic changes in the aging mouse ovary to identify early drivers of ovarian decline. To fill this gap we performed single-cell RNA sequencing on ovarian tissue from young (3-month-old) and reproductively aged (9-month-old) mice. Our analysis revealed a doubling of immune cells in the aged ovary, with lymphocyte proportions increasing the most, which was confirmed by flow cytometry. We also found an age-related downregulation of collagenase pathways in stromal fibroblasts, which corresponds to rises in ovarian fibrosis. Follicular cells displayed stress-response, immunogenic and fibrotic signaling pathway inductions with aging. This report provides critical insights into mechanisms responsible for ovarian aging phenotypes. The data can be explored interactively via a Shiny-based web application.

Although males with systemic lupus erythematosus (SLE) represent 4-22% of all SLE patients, it may not be appropriate that these cases should be subordinated to females with SLE in terms of most health-related issues. Over the past few decades, some distinctive features of male lupus have been observed with regard to genetic and environmental aspects of sex differences, clinical features, and outcome. In addition, recent insights into sex disparities in this disease have brought forth a few plausible and novel pathogenetic hypotheses. This review discusses these findings and sex disparities in SLE that appear to be especially noteworthy and pertinent to our understanding of male SLE.

OBJECTIVE: More than 80% of autoimmune disease predominantly affects females, but the mechanism for this female bias is poorly understood. We suspected that an X chromosome dose effect accounts for this, and we undertook this study to test our hypothesis that trisomy X (47,XXX; occurring in ∼1 in 1,000 live female births) would be increased in patients with female-predominant diseases (systemic lupus erythematosus [SLE], primary Sjögren's syndrome [SS], primary biliary cirrhosis, and rheumatoid arthritis [RA]) compared to patients with diseases without female predominance (sarcoidosis) and compared to controls. METHODS: All subjects in this study were female. We identified subjects with 47,XXX using aggregate data from single-nucleotide polymorphism arrays, and, when possible, we confirmed the presence of 47,XXX using fluorescence in situ hybridization or quantitative polymerase chain reaction. RESULTS: We found 47,XXX in 7 of 2,826 SLE patients and in 3 of 1,033 SS patients, but in only 2 of 7,074 controls (odds ratio in the SLE and primary SS groups 8.78 [95% confidence interval 1.67-86.79], P = 0.003 and odds ratio 10.29 [95% confidence interval 1.18-123.47], P = 0.02, respectively). One in 404 women with SLE and 1 in 344 women with SS had 47,XXX. There was an excess of 47,XXX among SLE and SS patients. CONCLUSION: The estimated prevalence of SLE and SS in women with 47,XXX was ∼2.5 and ∼2.9 times higher, respectively, than that in women with 46,XX and ∼25 and ∼41 times higher, respectively, than that in men with 46,XY. No statistically significant increase of 47,XXX was observed in other female-biased diseases (primary biliary cirrhosis or RA), supporting the idea of multiple pathways to sex bias in autoimmunity.

OBJECTIVE: To assess the value of the routine electrocardiogram (EKG) in the management of older (> 65 years) patients with cardiac disease during return visits to the ambulatory clinic. DESIGN: Retrospective chart analysis. SUBJECTS: Seventy-one patients older than 65 years of age, with cardiac diagnoses, presenting for follow-up visits. METHODS: Chart notes from 254 encounters with 71 patients in a cardiology clinic were analyzed. Excluded were new patients as well as patients followed in special arrhythmia clinics for pacemakers or significant arrhythmias. All patients underwent an EKG during each clinic visit, irrespective of their clinical status, according to the clinic's protocol. The frequency and nature of therapeutic decisions made in the clinic, the contribution of the EKG to the decision-making process, the appropriateness of the EKG, and the physician response to the EKG were assessed. MAIN RESULTS: Therapeutic decisions, the most common of which (28%) was medication changes, were made in 78 (31%) patients. The routine EKG was considered inappropriate in 60%. Unexpected diagnostic information not obtained from history or examination was not present in any encounter. EKG findings were not addressed by physicians in 22%. CONCLUSIONS: Most older patients seen in the cardiology clinic for return visits with stable symptoms do not benefit from EKGs. Unexpected diagnostic information from the EKG leading to major therapeutic decisions is rare in older people with stable symptoms.

AIM: The aim of this paper was to synthesize the literature related to meaning and present a conceptual analysis of searching for meaning in unexpected, significant, negative events. BACKGROUND: Events or situations that are perceived as unexpected and negative (e.g. chronic and/or life-threatening illness) can disrupt life plans and lead to emotional distress. Nurses are in a key position to aid in the coping process by assisting individuals to integrate the meaning of an unexpected, negative event into the life experience. METHODS: In May 2004, a comprehensive literature search using the terms meaning, search for meaning, meaning-making, making meaning, life change events, adaptation-psychological, stress, and coping was carried out. Of 339 possible references, 86 were included in the analysis. FINDINGS: Global meaning refers to an individual's beliefs, values, and purpose/goals. Situational meaning refers to the meaning the individual attaches to a situation. When the meaning attached to the situation is not congruent with global meaning, meaning is considered shattered and a search for meaning ensues. In the search for meaning, people may attempt to change the meaning of the event (through reattributions or creating illusions) or change global meaning (through positive reappraisal, problem-solving coping, or revaluing ordinary events). CONCLUSION: Nurses are in a key position to assist people through negative events. The process of searching for meaning provides a solid foundation for the development of nursing interventions to assist individuals through difficult times. Research is needed to develop nursing interventions based on the process of the search for meaning, as well as measures reflective of the search for meaning.

Apolipoprotein E ɛ4 allele is a common susceptibility gene for late-onset Alzheimer's disease. Brain vascular and metabolic deficits can occur in cognitively normal apolipoprotein E ɛ4 carriers decades before the onset of Alzheimer's disease. The goal of this study was to determine whether early intervention using rapamycin could restore neurovascular and neurometabolic functions, and thus impede pathological progression of Alzheimer's disease-like symptoms in pre-symptomatic Apolipoprotein E ɛ4 transgenic mice. Using in vivo, multimodal neuroimaging, we found that apolipoprotein E ɛ4 mice treated with rapamycin had restored cerebral blood flow, blood-brain barrier integrity and glucose metabolism, compared to age- and gender-matched wild-type controls. The preserved vasculature and metabolism were associated with amelioration of incipient learning deficits. We also found that rapamycin restored the levels of the proinflammatory cyclophilin A in vasculature, which may contribute to the preservation of cerebrovascular function in the apolipoprotein E ɛ4 transgenics. Our results show that rapamycin improves functional outcomes in this mouse model and may have potential as an effective intervention to block progression of vascular, metabolic and early cognitive deficits in human Apolipoprotein E ɛ4 carriers. As rapamycin is FDA-approved and neuroimaging is readily used in humans, the results of the present study may provide the basis for future Alzheimer's disease intervention studies in human subjects.

Psychological distress among cohabitating female partners of combat veterans with posttraumatic stress disorder (PTSD) was examined in a cross-sectional study using a modified version of the Health Belief Model. A convenience sample of 89 cohabitating female partners of male veterans in outpatient PTSD treatment was interviewed by telephone using a structured interview. Partners endorsed high levels of psychological distress with elevations on clinical scales at or exceeding the 90th percentile. Severe levels of overall psychological distress, depression, and suicidal ideation were prevalent among partners. Multivariate analyses revealed that perceived threat, recent mental health treatment, and level of involvement with veterans predicted global partner psychological distress. Partner burden was predicted by partner self-efficacy, perceived threat, barriers to mental health treatment, and partner treatment engagement. These findings are compelling since they demonstrate that partners of veterans with combat-related PTSD experience significant levels of emotional distress that warrant clinical attention. Psychological distress and partner burden were each associated with a unique combination of predictors, suggesting that although these constructs are related, they have distinct correlates and potentially different implications within the family environment. Future research should examine these constructs separately using causal modeling analyses to identify modifiable targets for interventions to reduce psychological distress among partners of individuals with PTSD.

BACKGROUND: The development of effective drug delivery systems capable of transporting small interfering RNA (siRNA) has been elusive. We have previously reported that colorectal cancer tumor xenograft growth was arrested following treatment with liposomal preparation of siDCAMKL-1. In this report, we have utilized Nanoparticle (NP) technology to deliver DCAMKL-1 specific siRNA to knockdown potential key cancer regulators. In this study, mRNA/miRNA were analyzed using real-time RT-PCR and protein by western blot/immunohistochemistry. siDCAMKL-1 was encapsulated in Poly(lactide-co-glycolide)-based NPs (NP-siDCAMKL-1); Tumor xenografts were generated in nude mice, treated with NP-siDCAMKL-1 and DAPT (γ-secretase inhibitor) alone and in combination. To measure let-7a and miR-144 expression in vitro, HCT116 cells were transfected with plasmids encoding the firefly luciferase gene with let-7a and miR-144 miRNA binding sites in the 3'UTR. RESULTS: Administration of NP-siDCAMKL-1 into HCT116 xenografts resulted in tumor growth arrest, downregulation of proto-oncogene c-Myc and Notch-1 via let-7a and miR-144 miRNA-dependent mechanisms, respectively. A corresponding reduction in let-7a and miR-144 specific luciferase activity was observed in vitro. Moreover, an upregulation of EMT inhibitor miR-200a and downregulation of the EMT-associated transcription factors ZEB1, ZEB2, Snail and Slug were observed in vivo. Lastly, DAPT-mediated inhibition of Notch-1 resulted in HCT116 tumor growth arrest and down regulation of Notch-1 via a miR-144 dependent mechanism. CONCLUSIONS: These findings demonstrate that nanoparticle-based delivery of siRNAs directed at critical targets such as DCAMKL-1 may provide a novel approach to treat cancer through the regulation of endogenous miRNAs.