Relative Contribution of Diagnostic Testing to the Diagnosis of Respiratory Syncytial Virus in Hospitalized Adults in the United States.

BACKGROUND: Respiratory syncytial virus (RSV) is a leading cause of acute respiratory illness (ARI) in older adults. Optimizing diagnosis could improve understanding of RSV burden. METHODS: We enrolled adults ≥50 years of age hospitalized with ARI and adults of any age hospitalized with congestive heart failure or chronic obstructive pulmonary disease exacerbations at two hospitals during two respiratory seasons (2018-2020). We collected nasopharyngeal (NP) and oropharyngeal (OP) swabs (n=1558), acute and convalescent sera (n=568), and expectorated sputum (n=153) from participants, and recorded standard-of-care (SOC) NP results (n=805). We measured RSV antibodies by two immunoassays and performed BioFire testing on respiratory specimens. RESULTS: Of 1,558 eligible participants, 92 (5.9%) tested positive for RSV by any diagnostic method. Combined NP/OP PCR yielded 58 positives, while separate NP and OP testing identified 11 additional positives (18.9% increase). Compared to Study NP/OP PCR alone, the addition of paired serology increased RSV detection by 42.9% (28 vs 40) among those with both specimen types, while the addition of SOC swab RT-PCR results increased RSV detection by 25.9% (47 vs 59). CONCLUSIONS: The addition of paired serology testing, SOC swab results, and separate testing of NP and OP swabs improved RSV diagnostic yield in hospitalized adults.

Detection of pathogenic bacteria and biomarkers in lung specimens from cystic fibrosis patients.

Diagnosing lung infections is often challenging because of the lack of a high-quality specimen from the diseased lung. Since persons with cystic fibrosis are subject to chronic lung infection, there is frequently a need for a lung specimen. In this small, proof of principle study, we determined that PneumoniaCheckTM, a non-invasive device that captures coughed droplets from the lung on a filter, might help meet this need. We obtained 10 PneumoniaCheckTMcoughed specimens and 2 sputum specimens from adult CF patients hospitalized with an exacerbation of their illness. We detected amylase (upper respiratory tract) with an enzymatic assay, surfactant A (lower respiratory tract) with an immunoassay, pathogenic bacteria by PCR, and markers of inflammation by a Luminex multiplex immunoassay. The amylase and surfactant A levels suggested that 9/10 coughed specimens were from lower respiratory tract with minimal upper respiratory contamination. The PCR assays detected pathogenic bacteria in 7 of 9 specimens and multiplex Luminex assay detected a variety of cytokines or chemokines. These data indicate that the PneumoniaCheckTMcoughed specimens can capture good quality lower respiratory tract specimens that have the potential to help in diagnosis, management and understanding of CF exacerbations and other lung disease.

Development and comparison of immunologic assays to detect primary RSV infections in infants.

Effective respiratory syncytial virus (RSV) vaccines have been developed and licensed for elderly adults and pregnant women but not yet for infants and young children. The RSV immune state of the young child, i.e., previously RSV infected or not, is important to the conduct and interpretation of epidemiology studies and vaccine clinical trials. To address the need for sensitive assays to detect immunologic evidence of past infection, we developed, characterized, and evaluated 7 assays including 4 IgG antibody enzyme immunoassays (EIAs), two neutralizing antibody assays, and an IFN-γ EliSpot (EliSpot) assay. The four IgG EIAs used a subgroup A plus subgroup B RSV-infected Hep-2 cell lysate antigen (Lysate), an expressed RSV F protein antigen (F), an expressed subgroup A G protein antigen (Ga), or an expressed subgroup B G protein (Gb) antigen. The two neutralizing antibody assays used either a subgroup A or a subgroup B RSV strain. The EliSpot assay used a sucrose cushion purified combination of subgroup A and subgroup B infected cell lysate. All seven assays had acceptable repeatability, signal against control antigen, lower limit of detection, and, for the antibody assays, effect of red cell lysis, lipemia and anticoagulation of sample on results. In 44 sera collected from children >6 months after an RSV positive illness, the lysate, F, Ga and Gb IgG EIAs, and the subgroup A and B neutralizing antibody assays, and the EliSpot assays were positive in 100%, 100%, 86%, 95%, 43%, and 57%, respectively. The Lysate and F EIAs were most sensitive for detecting RSV antibody in young children with a documented RSV infection. Unexpectedly, the EliSpot assay was positive in 9/15 (60%) of PBMC specimens from infants not exposed to an RSV season, possibly from maternal microchimerism. The Lysate and F EIAs provide good options to reliably detect RSV antibodies in young children for epidemiologic studies and vaccine trials.

An RSV Live-Attenuated Vaccine Candidate Lacking G Protein Mucin Domains Is Attenuated, Immunogenic, and Effective in Preventing RSV in BALB/c Mice.

BACKGROUND: Respiratory syncytial virus (RSV) is a leading viral respiratory pathogen in infants. The objective of this study was to generate RSV live-attenuated vaccine (LAV) candidates by removing the G-protein mucin domains to attenuate viral replication while retaining immunogenicity through deshielding of surface epitopes. METHODS: Two LAV candidates were generated from recombinant RSV A2-line19F by deletion of the G-protein mucin domains (A2-line19F-G155) or deletion of the G-protein mucin and transmembrane domains (A2-line19F-G155S). Vaccine attenuation was measured in BALB/c mouse lungs by fluorescent focus unit (FFU) assays and real-time polymerase chain reaction (RT-PCR). Immunogenicity was determined by measuring serum binding and neutralizing antibodies in mice following prime/boost on days 28 and 59. Efficacy was determined by measuring RSV lung viral loads on day 4 postchallenge. RESULTS: Both LAVs were undetectable in mouse lungs by FFU assay and elicited similar neutralizing antibody titers compared to A2-line19F on days 28 and 59. Following RSV challenge, vaccinated mice showed no detectable RSV in the lungs by FFU assay and a significant reduction in RSV RNA in the lungs by RT-PCR of 560-fold for A2-line19F-G155 and 604-fold for A2-line19F-G155S compared to RSV-challenged, unvaccinated mice. CONCLUSIONS: Removal of the G-protein mucin domains produced RSV LAV candidates that were highly attenuated with retained immunogenicity.

Functional antibody-dependent cell mediated cytotoxicity (ADCC) responses to vaccine and circulating influenza strains following vaccination.

Novel cell-based assays were developed to assess antibody-dependence cellular cytotoxicity (ADCC) antibodies against both vaccine and a representative circulation strain HA and NA proteins for the 2014-15 influenza season. The four assays using target cells stably expressing one of the four proteins worked well. In pre- and post-vaccine sera from 70 participants in a pre-season vaccine trial, we found ADCC antibodies and a rise in ADCC antibody titer against target cells expressing the 4 proteins but a much higher titer for the vaccine than the circulating HA in both pre-and post-vaccine sera. These differences in HA ADCC antibodies were not reflected in differences in HA binding antibodies. Our observations suggested that relatively minor changes on the subtype HA can result in large differences in ADCC activity.

The development and kinetics of functional antibody-dependent cell-mediated cytotoxicity (ADCC) to SARS-CoV-2 spike protein.

Since the COVID-19 pandemic, functional non-neutralizing antibody responses to SARS-CoV-2, including antibody-dependent cell-mediated cytotoxicity (ADCC), are poorly understood. We developed an ADCC assay utilizing a stably transfected, dual-reporter target cell line with inducible expression of a SARS-CoV-2 spike protein on the cell surface. Using this assay, we analyzed 61 convalescent serum samples from adults with PCR-confirmed COVID-19 and 15 samples from healthy uninfected controls. We found that 56 of 61 convalescent serum samples induced ADCC killing of SARS-CoV-2 S target cells, whereas none of the 15 healthy controls had detectable ADCC. We then found a modest decline in ADCC titer over a median 3-month follow-up in 21 patients who had serial samples available for analysis. We confirmed that the antibody-dependent target cell lysis was mediated primarily via the NK FcγRIIIa receptor (CD16). This ADCC assay had high sensitivity and specificity for detecting serologic immune responses to SARS-CoV-2.

Predicting impacts of future climate change and hydropower development towards habitats of native and non-native fishes.

Climate change and hydropower development are two primary stressors affecting riverine ecosystems and both stressors facilitate invasions by non-native species. However, little study has focused on how habitats of native and non-native fishes may be affected by independent or combined impacts of such stressors. Here we used the Jinsha River as an example to predict habitat change and distributional shift of native and non-native fishes with species distribution models. The Jinsha River Basin has nearly 40 cascade dams constructed or planned and located in the Tibetan Plateau, which is sensitive to future climate change. Two climate change scenarios and future hydropower development were combined to produce five scenarios of future changes. Under the impacts of independent extreme climate change or hydropower development, non-native fishes showed greater habitat gain in total, while native fishes shifted their distribution into tributaries and higher elevations, and impacts were stronger in combined scenarios. Habitat overlap between the two groups also increased in future scenarios. Certain fish traits correlated with stressors in habitat change prediction. River basins with hydropower development were shown to face higher risk of non-native fishes invasion under future climate change. As the most biodiverse river basins globally are threatened by hydropower development, our results emphasize the importance of regulating non-native fish introduction in reservoirs. Our approaches are also applicable to other systems globally to better understand how hydropower development and climate change may increase invasion risk, and therefore help conserve native species effectively.

ZBTB46 is a shear-sensitive transcription factor inhibiting endothelial cell proliferation via gene expression regulation of cell cycle proteins.

ZBTB46 is a transcription factor identified in classical dendritic cells and keeps dendritic cells in a quiescent state. Chromatin immunoprecipitation sequencing in dendritic cells has identified over 1300 potential gene targets of ZBTB46, affecting many processes including cell cycle. Endothelial cells (ECs) also express ZBTB46 and are mostly in a quiescent non-proliferative state. While EC proliferation is a critical process in development, dysregulation of EC proliferation as seen in areas of disturbed flow play an important role in many disease processes such as atherosclerosis, pulmonary hypertension, transplant vasculopathy, neointimal hyperplasia, and in-stent restenosis. We studied the role of ZBTB46 in ECs, hypothesizing that it inhibits EC proliferation. Using a model of disturbed flow in mice, we found that ZBTB46 is expressed in murine arterial ECs in vivo, and is downregulated by disturbed flow. In vitro results using HAECs showed that cell confluence and laminar shear stress, both known physiological conditions promoting EC quiescence, led to upregulation of ZBTB46 expression. Adenoviral-mediated overexpression of ZBTB46 in vitro caused reduced EC proliferation, and increased number of cells in the G0/G1 phase of cell cycle, without affecting apoptosis or senescence, while siRNA knockdown of ZBTB46 negated the known inhibitory role of unidirectional laminar shear stress on EC proliferation. ZBTB46 overexpression also led to a broad suppression of genes involved in cell cycle progression including multiple cyclins and cyclin-dependent kinases, but an increase in the CDK inhibitor CDKN1A. Phosphorylation of the retinoblastoma protein was also decreased as assessed by Western blot. Tube formation on Matrigel was reduced, suggesting an inhibitory role for ZBTB46 in angiogenesis. Further research is required to investigate the potential role of ZBTB46 in specific pathologic conditions and whether it can be targeted in a therapeutic manner.

Phylogeographic studies of schizothoracine fishes on the central Qinghai-Tibet Plateau reveal the highest known glacial microrefugia.

Pleistocene climatic oscillations have greatly influenced the evolutionary history and distribution pattern of most extant species. However, their effects on species on the Qinghai-Tibet Plateau (QTP) are not well understood. To investigate the effects of past climatic shifts, particularly the Last Glacial Maximum (LGM), on plateau fish, we analysed the phylogeographic structure and demographic history of five closely related taxa of the subfamily Schizothoracinae, a representative endemic taxon of the QTP, from nine endorheic lakes on the central QTP and three peripheral exorheic rivers using the mitochondrial control region (D-loop) sequence and 12 microsatellite (SSR) markers. Phylogram from D-loop haplotypes revealed two well-supported lineages (North and South) separated by the Tanggula Mountains. The results from the D-loop and SSR revealed that endorheic populations possess high genetic diversity and a unique genetic structure. The most recent demographic expansion occurred post-LGM for most endorheic populations and in the last interglacial period for Siling Co and all exorheic populations. Phylogeographic structure, together with species distribution modelling, supports the scenario of multiple glacial refugia on the QTP during the LGM and suggests that Siling Co (4540 m asl) is a cryptic glacial microrefugia for plateau fish, which would be the highest glacial microrefugia known.

Gap junction blockade induces apoptosis in human endometrial stromal cells.

One of the most dynamic adult human tissues is the endometrium. Through coordinated, cyclical proliferation, differentiation, leukocyte recruitment, apoptosis, and desquamation, the uterine lining is expanded and shed monthly, unless pregnancy is established. Errors in these steps potentially cause endometrial dysfunction, abnormal uterine bleeding, failed embryonic implantation, infertility, or endometrial carcinoma. Our prior studies showed that gap junctions comprised of Gap junction alpha-1 (GJA1) protein, also known as connexin 43 (CX43), subunits are critical to endometrial stromal cell differentiation. The current studies were undertaken to explore the mechanism of endometrial dysfunction when gap junction intercellular communication (GJIC) is disrupted. Gap junction blockade by two distinct GJIC inhibitors, 18α-glycyrrhetinic acid (AGA) and octanol (OcOH), suppressed proliferation and induced apoptosis in endometrial stromal cells, as manifested by reduced biomarkers of cell viability, increased TUNEL staining, caspase-3 activation, sub-G1 chromosomal DNA complement, as well as shortened telomere length. Unexpectedly, we also observed that the chemical inhibitors blocked CX43 gene expression. Moreover, when endometrial stromal cells were induced to undergo hormonal decidualization, following a 7-day exposure to 10 nM 17ß-estradiol + 100 nM progesterone + 0.5 mM dibutyryl cAMP, characteristic epithelioid changes in cell shape and secretion of prolactin were blunted in the presence of AGA or OcOH, recapitulating effects of RNA interference of CX43. Our findings indicate that endometrial stromal cell proliferation and maintenance of decidualized endometrial function are GJIC-dependent, and that disruption of gap junctions induces endometrial stromal cell apoptosis. These observations may have important implications for several common clinical endometrial pathologies.

Distinct contributions of MSL complex subunits to the transcriptional enhancement responsible for dosage compensation in Drosophila.

The regulatory mechanism of dosage compensation is the paramount example of epigenetic regulation at the chromosomal level. In Drosophila, this mechanism, designed to compensate for the difference in the dosage of X-linked genes between the sexes, depends on the MSL complex that enhances the transcription of the single dose of these genes in males. We have investigated the function of various subunits of the complex in mediating dosage compensation. Our results confirm that the highly enriched specific acetylation of histone H4 at lysine 16 of compensated genes by the histone acetyl transferase subunit MOF induces a more disorganized state of their chromatin. We have determined that the association of the MSL complex reduces the level of negative supercoiling of the deoxyribonucleic acid of compensated genes, and we have defined the role that the other subunits of the complex play in this topological modification. Lastly, we have analyzed the potential contribution of ISWI-containing remodeling complexes to the architecture of compensated chromatin, and we suggest a role for this remodeling factor in dosage compensation.

Postconditioning attenuates cardiomyocyte apoptosis via inhibition of JNK and p38 mitogen-activated protein kinase signaling pathways.

A sequence of intermittent interruptions of oxygen supply (i.e., postconditioning, Postcon) at reoxygenation reduces oxidant-induced cardiomyocyte loss. This study tested the hypothesis that prevention of cardiomyocyte apoptosis by Postcon is mediated by mitogen-activated protein kinases pathways. Primary cultured neonatal rat cardiomyocytes were exposed to 3 h hypoxia followed by 6 h of reoxygenation. Cardiomyocytes were postconditioned by three cycles each of 5 min reoxygenation and 5 min hypoxia after prolonged hypoxia. Relative to hypoxia alone, reoxygenation stimulated expression of JNKs and p38 kinases, corresponding to increased activity of JNKs (phospho-c-Jun) and p38 (phospho-ATF2). The level of TNFalpha in cell lysates, activity of cytosolic caspases-8, -3, expression of Bax and the number of apoptotic cardiomyocytes were increased while expression of Bcl-2 was decreased with reoxygenation. Consistent with an attenuation in generation of superoxide anions detected by lucigenin-enhanced chemiluminescence at early period of reoxygenation, treatment of cardiomyocytes with Postcon further reduced expression and activity of JNKs and p38 kinases, level of TNFalpha, the frequency of apoptotic cells and expression of Bax. However, the inhibitory effects of Postcon on these changes were lost when its application was delayed by 5 min after the start of reoxygenation. Addition of a JNK/p38 stimulator, anisomycin into cardiomyocytes at the beginning of reoxygenation eliminated protection by Postcon. These data suggest that 1) hypoxia/reoxygenation elicits cardiomyocyte apoptosis in conjunction with expression and activation of JNK and p38 kinases, release of TNFalpha, activation of caspases, and an increase in imbalance of pro-/anti-apoptotic proteins; 2) Postcon attenuates cardiomyocyte apoptosis, potentially mediated by inhibiting JNKs/p-38 signaling pathways and reducing TNFalpha release and caspase expression.

Hypoxic postconditioning reduces cardiomyocyte loss by inhibiting ROS generation and intracellular Ca2+ overload.

We have shown that intermittent interruption of immediate reflow at reperfusion (i.e., postconditioning) reduces infarct size in in vivo models after ischemia. Cardioprotection of postconditioning has been associated with attenuation of neutrophil-related events. However, it is unknown whether postconditioning before reoxygenation after hypoxia in cultured cardiomyocytes in the absence of neutrophils confers protection. This study tested the hypothesis that prevention of cardiomyocyte damage by hypoxic postconditioning (Postcon) is associated with a reduction in the generation of reactive oxygen species (ROS) and intracellular Ca(2+) overload. Primary cultured neonatal rat cardiomyocytes were exposed to 3 h of hypoxia followed by 6 h of reoxygenation. Cardiomyocytes were postconditioned after the 3-h index hypoxia by three cycles of 5 min of reoxygenation and 5 min of rehypoxia applied before 6 h of reoxygenation. Relative to sham control and hypoxia alone, the generation of ROS (increased lucigenin-enhanced chemiluminescence, SOD-inhibitable cytochrome c reduction, and generation of hydrogen peroxide) was significantly augmented after immediate reoxygenation as was the production of malondialdehyde, a product of lipid peroxidation. Concomitant with these changes, intracellular and mitochondrial Ca(2+) concentrations, which were detected by fluorescent fluo-4 AM and X-rhod-1 AM staining, respectively, were elevated. Cell viability assessed by propidium iodide staining was decreased consistent with increased levels of lactate dehydrogenase after reoxygenation. Postcon treatment at the onset of reoxygenation reduced ROS generation and malondialdehyde concentration in media and attenuated cardiomyocyte death assessed by propidium iodide and lactate dehydrogenase. Postcon treatment was associated with a decrease in intracellular and mitochondrial Ca(2+) concentrations. These data suggest that Postcon treatment reduces reoxygenation-induced injury in cardiomyocytes and is potentially mediated by attenuation of ROS generation, lipid peroxidation, and intracellular and mitochondrial Ca(2+) overload.

Myocardial protection with postconditioning is not enhanced by ischemic preconditioning.

BACKGROUND: Ischemic preconditioning (IPC) has been used in off-pump coronary artery bypass surgery (OPCAB) to reduce potential injury secondary to ligation of the target vessel. Previous studies have shown that a brief period of repetitive coronary occlusion applied at the onset of reperfusion, postconditioning (postcon), attenuates myocardial injury. This study tested the hypothesis that coincident application of IPC and postcon would provide more cardioprotection than either intervention alone by inhibiting oxidant-mediated injury after ischemia and reperfusion. METHODS: Four groups of open-chest canines endured 60 minutes coronary occlusion followed by 3 hours reperfusion: control (n = 10), no intervention; IPC (n = 9), 5 minutes left anterior descending coronary artery occlusion preceded 10 minutes of reperfusion before prolonged occlusion; postcon (n = 10), 3 cycles of 30 seconds reperfusion-30 seconds reocclusion were imposed immediately upon reperfusion; IPC+postcon (n = 8), IPC and postcon algorithms were combined. RESULTS: Collateral blood flow during ischemia was similar in all groups. Compared to control (24% +/- 2%), infarct size was comparably reduced in IPC (13% +/- 2%* [* denotes p less than 0.05 compared with control]), and postcon (10% +/- 1%*), consistent with a reduction in plasma creative kinase activity in these groups; infarct size was not further reduced by IPC+postcon (12% +/- 3%*). Tissue water content in ischemic myocardium was comparably reduced in IPC, postcon, and IPC+postcon compared to control. Superoxide anion generation detected by dihydroethidium staining in area at risk myocardium was comparably reduced in all intervention groups relative to control. Plasma malondialdehyde (microM), a lipid peroxidation byproduct of oxidant injury, was less at 1 hour of reperfusion in IPC (2.2 +/- 0.2*), postcon (2.1 +/- 0.2*), and IPC+postcon (2.5 +/- 0.2*) relative to control (3.3 +/- 0.2). Ventricular fibrillation occurred less often in all intervention groups. CONCLUSIONS: No additive cardioprotective effects by IPC and postcon were observed in a canine model of regional ischemia and reperfusion. The potent attenuation of myocardial injury by postcon may suggest a clinically applicable strategy during some surgical revascularization procedures (ie, OPCAB).

Postconditioning attenuates myocardial ischemia-reperfusion injury by inhibiting events in the early minutes of reperfusion.

OBJECTIVE: We previously showed that brief intermittent ischemia applied during the onset of reperfusion (i.e., postconditioning) is cardioprotective in a canine model of ischemia-reperfusion. This study tested the hypothesis that the early minutes of reperfusion (R) during which postconditioning (Post-con) is applied are critical to its cardioprotection. METHODS: In anesthetized open-chest rats, the left coronary artery (LCA) was occluded for 30 min and reperfused for 3 h. All rats were randomly divided into six groups: Control (n=8): no intervention at R; Ischemic preconditioning (IPC) (n=8): the LCA was occluded for 5 min followed by 10 min of R before the index occlusion; Post-con 1 (n=8): after LCA occlusion, three cycles of 10 s R followed by 10 s LCA re-occlusion were applied during the first minute of R; Post-con 2 (n=8): Six cycles of 10 s R and 10 s re-occlusion were applied during the first 2 min of R; Delayed Post-con (n=8): the ligature was loosened for full reflow for the first minute of R, after which the three-cycle Post-con algorithm was applied; Sham (n=6): the surgical procedure was identical to other groups, but the LCA ligature was not ligated. RESULTS: Infarct size (TTC staining) was 23% smaller in Post-con 1 (40+/-2%*) than in Control (52+/-3%), confirmed by plasma creatine kinase activity (18+/-2* vs. 46+/-6 IU/g protein). There was no further reduction in infarct size with 6 cycles of Post-con (40+/-2.9%, p>0.05 vs. Post-con 1). Meanwhile, infarct size reduction was significantly greater in the IPC group (17+/-3%) than in Post-con1 (p<0.01). The plasma lipid peroxidation product malondialdehyde (MDA, microM/ml) was less after R in IPC and Post-con 1 (0.8+/-0.07* and 0.8+/-0.06*) vs. Control (1.21+/-0.08), consistent with a visual decrease in superoxide anion generation (dihydroethidium staining) in the AAR myocardium after 3 h of reperfusion. Neutrophil accumulation (myeloperoxidase activity, MPO, U/100 g tissue) in the AAR was less in IPC (1.4+/-0.3*) and Post-con 1 (2.5+/-0.3*) vs. Control (5.5+/-0.6). The reductions in infarct size, creatine kinase, MDA and DHE staining were lost with delayed Post-con, while MPO activity remained lower than in Control (3.2+/-0.4*). CONCLUSIONS: (1) Post-con at onset of R reduces myocardial injury; (2) cardioprotection may be mediated, in part, by inhibiting oxidant generation and oxidant mediated injury; (3) the first minute of R in the rat model is critical to cardioprotection by Post-con; and (4) cardioprotection by Post-con may be independent of neutrophil accumulation in AAR. *p<0.05 Post-con vs. Control.

Involvement of Na+/H+ exchanger in hypoxia/re-oxygenation-induced neonatal rat cardiomyocyte apoptosis.

Although increased Na(+)/H(+) exchanger type-1 (NHE-1) activity has been implicated in the pathogenesis of myocardial infarction, the role of NHE-1 in induction of apoptosis, and the potential mechanisms involved have not been fully characterized. This study tested the hypothesis that NHE-1 activity is involved in hypoxia (H)/re-oxygenation (Re)-induced cardiomyocyte apoptosis by increasing mitochondrial Ca(2+) ([Ca(2+)]m). Primary cultured neonatal rat cardiomyocytes were subjected to 4.5 h of H followed by 12 h of Re. Relative to H alone, the level of X-rhod-1 acetoxymethyl (AM)-labeled [Ca(2+)]m was increased, and the frequency of cell death (propidium iodide (PI) staining) and apoptotic cells (terminal deoxynucleotidyl transferase (TdT)-mediated-UTP nick end labeling [TUNEL]), confirmed by Annexin-V, were augmented at the end of Re, along with appearance of cytosolic cytochrome c, activation of caspase-3, and increased ratio of Bax and Bcl-2. Addition of cariporide (20 micromol/l), a well-known NHE-1 inhibitor, to cultured cells before H significantly reduced [Ca(2+)]m, the number of PI and TUNEL positive cells relative to the levels at end of Re, but did not completely eliminate these changes compared to Sham control. There was a strong trend for attenuation in increased levels of [Ca(2+)]m, and the number of PI and TUNEL positive cells when same dose of cariporide was added only at Re, but the difference in these variables did not reach significance. In contrast, the levels of [Ca(2+)]m and the number of PI and TUNEL positive cells were significantly reduced to a level comparable to Sham control when cariporide (20 micromol/l) was administered before H and during Re, respectively, associated with a reduction in cytosolic cytochrome c, caspase-3 activity and ratio of Bax and Bcl-2. In conclusion, these data suggest that NHE-1 is involved in induction of cardiomyocyte apoptosis during both H and Re through a [Ca(2+)]m-dependent manner, thereby resulting in activation of cytochrome c-caspase-3 signaling pathways.

Inhibition of myocardial apoptosis reduces infarct size and improves regional contractile dysfunction during reperfusion.

OBJECTIVE: Myocardial apoptosis is primarily triggered during reperfusion (R) through various mechanisms that may involve endonuclease to cleavage genomic DNA in the internucleosomal linker regions. However, the relative contribution of myocardial apoptosis to development of myocardial injury during R remains unknown. In the present study, we examined whether inhibition of apoptosis with aurintricarboxylic acid (ATA), an endonuclease inhibitor, during R reduces infarct size and improves regional contractile function. METHODS AND RESULTS: In two groups of chronically-instrumented dogs, 1 h of left anterior descending (LAD) coronary occlusion was followed by 24 h of R with infusion of saline (control, n=8) or ATA (1 mg/kg/h, n=8) into the left atrium starting 5 min before R and continuing for 2 h. ATA significantly reduced apoptotic cells (TUNEL staining) in the peri-necrotic myocardium (12+/-1%* vs. 36+/-4%), consistent with the absence of DNA laddering. To confirm inhibition of apoptosis with ATA, densitometrically, Bcl-2 (% of normal myocardium) was significantly increased vs. control (102+/-12* vs. 68+/-9) and Bax as well as the activated caspase-3 were significantly reduced vs. control (108+/-17* vs. 194+/-42 and -29+/-4* vs. 174+/-43, respectively). ATA significantly improved segmental shortening (3.3+/-1.2* vs. -1.8+/-0.7%) and segmental work (79.3+/-11.3* vs. 7.1+/-5.8 mmHg/mm) in area at risk myocardium, and reduced infarct size (TTC staining, 27+/-0.2* vs. 37+/-0.5%), confirmed by lower plasma creatine kinase activity. In addition, myocardial blood flow (0.9+/-0.1* vs. 0.4+/-0.1 ml/min/g) and endothelial-dependent maximal vascular relaxation (119+/-6* vs. 49+/-8%) were significantly improved. Myeloperoxidase activity in area at risk myocardium, a marker for neutrophil accumulation, was also significantly reduced (17+/-4* vs. 138+/-28 Delta Abs/min). CONCLUSIONS: These data suggest that the inhibition of apoptosis during R is associated with a reduction in infarction, improvement in regional contractile and vascular endothelial functions as well as augmentation in myocardial blood flow. *P<0.05 vs. control group.

Dibenzoylmethane, a natural dietary compound, induces HIF-1 alpha and increases expression of VEGF.

Hypoxia-inducible factor 1 (HIF-1) is the major transcription factor activated during hypoxia. It is composed of HIF-1 alpha and HIF-1 beta subunits. While HIF-1 beta is constitutively expressed, HIF-1 alpha is targeted to proteasome degradation under normoxic conditions. Under hypoxia, HIF-1 alpha is stabilized and heterodimerizes with HIF-1 beta. Iron chelators have also been reported to stabilize HIF-1 alpha protein and activate HIF-1. In this study, we investigated the effects of dibenzoylmethane (DBM), a natural dietary compound and an iron chelator, on HIF-1 pathway. We found that DBM increases HIF-1 alpha protein levels in a dose- and time-dependent manner. This induction was accompanied with activation of HIF-1, measured by reporter gene assay and increased production of its downstream target, the vascular endothelial growth factor. Mechanistically, HIF-1 alpha was stabilized by DBM at a step prior to ubiquitination. The effect of DBM on HIF-1 and its low toxicity profile might be therapeutically beneficial in ischemic diseases.