Mitochondrial Transcription Factor A\u27s role in heart failure.

Background: An essential mitochondrial component, Mitochondrial Transcription Factor A (TFAM) is reduced within the failing heart. TFAM maintains regulatory functions of cardiomyocyte physiology and stability. Mitochondrial Transcription Factor A forms a mitochondrial nucleoid anti-oxidant structure and reduces pathophysiological abnormalities to structural and contractile proteins. Overall, this functions to physically stabilize mtDNA. Significant molecular changes such as a loss of TFAM drive pathophysiological concentrations of cytoplasmic Ca2+ and reactive oxygen species. Both of these factors induce proteolytic enzymes to degrade the physiological cardiomyocyte. In TFAMs absence, heart function deteriorates and hypertrophic expansion ensues: thereby changing the physiological myocardium to a pathological state. This alteration activates a remodeling cascade leading to cardiomyocyte decline. These maladaptive factors are major players in the pathological and time dependent formation of a failing myocardium. Heart failure is a leading cause of death in the United States and a multitude of molecular therapeutics individually target Ca2+ transporters or inhibit oxidative enzymes. However, the role of TFAM in reducing pathological cardiac remodeling vi maladaptive factors remains unclear. Furthermore, the purpose of this study is to assess TFAM’s role in inhibiting ROS and Ca2+ driven cardiomyocyte remodeling factors. Methods: In the first set of experiments, we studied the effect of TFAM overexpression vector and Crispr-cas9 knockdown on HL-1 cardiomyocytes. The cells were subjected to oxidative stress through hydrogen peroxide treatment and the analysis was compared to a standard control as well as control treatment groups; Lipofectamine and an empty vector. Molecular analysis of cardiomyocte remodeling factors Calpain1, MMP9 and NFAT4 and Ca2+ transporter SERCA2a was assessed via western blotting and immunocytochemistry. Statistical analysis was performed on graphpad prism software. In the second set of experiments, we studied the effects of aortic banding on TFAM transgenic mice. Trans-aortic constriction surgery with a 271/2g needle was performed on 8-10 week old mice. Prior to surgery, echocardiography ultrasound and blood pressure (coda tail cuff method) functional assays were performed and served as a baseline for post-surgical assessment. Eight weeks post-banding, ultrasound of aortic arch perfusion and turbulence were used to assess banding procedure. Cardiac hypertrophy, fibrosis and morphology were assessed via heart weight/body weight ratio, Mason Trichrome and Hematoxylin & Eosin Staining respectively. Molecular vii analysis of cardiac remodeling factors; Calpain1, MMP9, NFAT4 and Ca2+ transporter SERCA2a were assessed via western blotting and immunohistochemistry. Results: In vitro analysis of remodeling factors reveals that TFAM knockdown induces significant increases in maladaptive factors MMP9, Calpain1 and NFAT4. In HL-1 cardiomyocytes subjected to pathological concentrations of H2O2 (mimicking heart failure in vivo studies), TFAM over-expression mitigated hypertrophic stimulator and ROS inducer NFAT4 and decreased MMP9 expression. The TFAM transgenic animal model has reduced cardiac hypertrophy and morphology when compared to it’s WT surgical counterpart. Molecular analysis shows that TFAM over-expression reduces ROS propagated proteases (MMP9) and hypertrophic factors (NFAT4) in cardiac remodeling. Additionally, it was observed that TFAM over-expression does not increase SERCA2a protein expression and Calpain1 remains high in the TFAMTG TAC model. Conclusion: This study reveals that TFAM over-expression plays a vital role in pressure overload heart failure by inhibiting NFAT4 and MMP9. The Ca2+ induced Calpain1 pathway is not mitigated by TFAM over-expression in the TAC pressure overload model, but additional Ca2+ transporters may be affected by TFAM over-expression. TFAM loss or over-expression shifts the homeostatic balance held within the physiological myocardium

Resident Memory T Cells (TRM) Are Abundant in Human Lung: Diversity, Function, and Antigen Specificity

Recent studies have shown that tissue resident memory T cells (TRM) are critical to antiviral host defense in peripheral tissues. This new appreciation of TRM that reside in epithelial tissues and mediate host defense has been studied most extensively in skin: adult human skin contains large numbers of functional TRM that express skin specific markers. Indeed, more than twice as many T cells reside in skin as in peripheral blood. This T cell population has a diverse T cell receptor repertoire, and can produce a broad array of cytokines. More recently, we have begun to examine other epithelial tissues for the presence of resident T cells. In the present study, we asked whether analogous populations of resident T cells could be found in human lung. We were able to demonstrate abundant resident T cells in human lung-more than 10 billion T cells were present. Lung T cells were largely of the effector memory T cell (TEM) phenotype, though small numbers of central memory T cells (TCM) and T regulatory cells (Treg) could be identified. Lung T cells had a diverse T cell receptor repertoire and subsets produced IL-17, IL-4, IFNγ, as well as TNFα. A significant number of lung TRM CD4+Th cells produced more than one cytokine, identifying them as “multifunctional” Th1 type cells. Finally, lung TRM, but not TRM resident to skin or T cells from blood, proliferated in response to influenza virus. This work suggests that normal human lung contains large numbers of TRM cells, and these cells are poised to respond to recall antigens previously encountered through lung mucosa. This population of T cells may contribute to the pathogenesis of asthma and other T cell mediated lung diseases

Screening a Peptide Library by DSC and SAXD: Comparison with the Biological Function of the Parent Proteins

We have recently identified the membranotropic regions of the hepatitis C virus proteins E1, E2, core and p7 proteins by observing the effect of protein-derived peptide libraries on model membrane integrity. We have studied in this work the ability of selected sequences of these proteins to modulate the Lβ-Lα and Lα-HII phospholipid phase transitions as well as check the viability of using both DSC and SAXD to screen a protein-derived peptide library. We demonstrate that it is feasible to screen a library of peptides corresponding to one or several proteins by both SAXD and DSC. This methodological combination should allow the identification of essential regions of membrane-interacting proteins which might be implicated in the molecular mechanism of membrane fusion and/or budding

Circulating microRNAs in sera correlate with soluble biomarkers of immune activation but do not predict mortality in ART treated individuals with HIV-1 infection: A case control study

Introduction: The use of anti-retroviral therapy (ART) has dramatically reduced HIV-1 associated morbidity and mortality. However, HIV-1 infected individuals have increased rates of morbidity and mortality compared to the non-HIV-1 infected population and this appears to be related to end-organ diseases collectively referred to as Serious Non-AIDS Events (SNAEs). Circulating miRNAs are reported as promising biomarkers for a number of human disease conditions including those that constitute SNAEs. Our study sought to investigate the potential of selected miRNAs in predicting mortality in HIV-1 infected ART treated individuals. Materials and Methods: A set of miRNAs was chosen based on published associations with human disease conditions that constitute SNAEs. This case: control study compared 126 cases (individuals who died whilst on therapy), and 247 matched controls (individuals who remained alive). Cases and controls were ART treated participants of two pivotal HIV-1 trials. The relative abundance of each miRNA in serum was measured, by RTqPCR. Associations with mortality (all-cause, cardiovascular and malignancy) were assessed by logistic regression analysis. Correlations between miRNAs and CD4+ T cell count, hs-CRP, IL-6 and D-dimer were also assessed. Results: None of the selected miRNAs was associated with all-cause, cardiovascular or malignancy mortality. The levels of three miRNAs (miRs -21, -122 and -200a) correlated with IL-6 while miR-21 also correlated with D-dimer. Additionally, the abundance of miRs -31, -150 and -223, correlated with baseline CD4+ T cell count while the same three miRNAs plus miR- 145 correlated with nadir CD4+ T cell count. Discussion: No associations with mortality were found with any circulating miRNA studied. These results cast doubt onto the effectiveness of circulating miRNA as early predictors of mortality or the major underlying diseases that contribute to mortality in participants treated for HIV-1 infection

Development and Validation of a Risk Score for Chronic Kidney Disease in HIV Infection Using Prospective Cohort Data from the D:A:D Study

Ristola M. on työryhmien DAD Study Grp ; Royal Free Hosp Clin Cohort ; INSIGHT Study Grp ; SMART Study Grp ; ESPRIT Study Grp jäsen.Background Chronic kidney disease (CKD) is a major health issue for HIV-positive individuals, associated with increased morbidity and mortality. Development and implementation of a risk score model for CKD would allow comparison of the risks and benefits of adding potentially nephrotoxic antiretrovirals to a treatment regimen and would identify those at greatest risk of CKD. The aims of this study were to develop a simple, externally validated, and widely applicable long-term risk score model for CKD in HIV-positive individuals that can guide decision making in clinical practice. Methods and Findings A total of 17,954 HIV-positive individuals from the Data Collection on Adverse Events of Anti-HIV Drugs (D:A:D) study with >= 3 estimated glomerular filtration rate (eGFR) values after 1 January 2004 were included. Baseline was defined as the first eGFR > 60 ml/min/1.73 m2 after 1 January 2004; individuals with exposure to tenofovir, atazanavir, atazanavir/ritonavir, lopinavir/ritonavir, other boosted protease inhibitors before baseline were excluded. CKD was defined as confirmed (>3 mo apart) eGFR In the D:A:D study, 641 individuals developed CKD during 103,185 person-years of follow-up (PYFU; incidence 6.2/1,000 PYFU, 95% CI 5.7-6.7; median follow-up 6.1 y, range 0.3-9.1 y). Older age, intravenous drug use, hepatitis C coinfection, lower baseline eGFR, female gender, lower CD4 count nadir, hypertension, diabetes, and cardiovascular disease (CVD) predicted CKD. The adjusted incidence rate ratios of these nine categorical variables were scaled and summed to create the risk score. The median risk score at baseline was -2 (interquartile range -4 to 2). There was a 1: 393 chance of developing CKD in the next 5 y in the low risk group (risk score = 5, 505 events), respectively. Number needed to harm (NNTH) at 5 y when starting unboosted atazanavir or lopinavir/ritonavir among those with a low risk score was 1,702 (95% CI 1,166-3,367); NNTH was 202 (95% CI 159-278) and 21 (95% CI 19-23), respectively, for those with a medium and high risk score. NNTH was 739 (95% CI 506-1462), 88 (95% CI 69-121), and 9 (95% CI 8-10) for those with a low, medium, and high risk score, respectively, starting tenofovir, atazanavir/ritonavir, or another boosted protease inhibitor. The Royal Free Hospital Clinic Cohort included 2,548 individuals, of whom 94 individuals developed CKD (3.7%) during 18,376 PYFU (median follow-up 7.4 y, range 0.3-12.7 y). Of 2,013 individuals included from the SMART/ESPRIT control arms, 32 individuals developed CKD (1.6%) during 8,452 PYFU (median follow-up 4.1 y, range 0.6-8.1 y). External validation showed that the risk score predicted well in these cohorts. Limitations of this study included limited data on race and no information on proteinuria. Conclusions Both traditional and HIV-related risk factors were predictive of CKD. These factors were used to develop a risk score for CKD in HIV infection, externally validated, that has direct clinical relevance for patients and clinicians to weigh the benefits of certain antiretrovirals against the risk of CKD and to identify those at greatest risk of CKD.Peer reviewe

Mechanism of TFAM Mediated Cardiomyocyte Protection*

Although mitochondrial transcription factor A (TFAM) is a protective component of mitochondrial DNA and a regulator of calcium and reactive oxygen species (ROS) production, the mechanism remains unclear. In heart failure (HF), TFAM is significantly decreased and cardiomyocyte instability ensues. TFAM inhibits Nuclear Factor of Activated T cells (NFAT), which reduces ROS production; additionally, TFAM transcriptionally activates SERCA2a to decrease free calcium. Therefore, decreasing TFAM vastly increases protease expression and hypertrophic factors, leading to cardiomyocyte functional decline. To examine this hypothesis, treatments of 1.0 micrograms of a TFAM vector and 1.0 micrograms of a CRISPR-Cas9 TFAM plasmid were administered to HL-1 cardiomyocytes via lipofectamine transfection. Western blotting and confocal microscopy analysis show that CRISPR-Cas9 knockdown of TFAM significantly increased proteases Calpain1, MMP9 and regulators Serca2a, and NFAT4 protein expression. CRISPR knockdown of TFAM in HL-1 cardiomyocytes upregulates degratory factors, leading to cardiomyocyte instability. Hydrogen peroxide oxidative stress decreased TFAM expression and increased Calpain1, MMP9, and NFAT4 protein expression. TFAM overexpression normalizes pathological hypertrophic factor NFAT4 in the presence of oxidative stress.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Nf-kappa-b And Hiv - Reply

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62943/1/343219a0.pd