Efficacy and safety of levosimendan in patients with sepsis: a systematic review and network meta-analysis.

Objective: We conducted a systematic review to assess the advantages and disadvantages of levosimendan in patients with sepsis compared with placebo, milrinone, and dobutamine and to explore the clinical efficacy of different concentrations of levosimendan. Methods: PubMed, Web of Science, Cochrane Library, Embase, CNKI, Wanfang data, VIP, and CBM databases were searched using such keywords as simendan, levosimendan, and sepsis. The search time was from the establishment of the database to July 2023. Two researchers were responsible for literature screening and data collection respectively. After the risk of bias in the included studies was evaluated, network meta-analysis was performed using R software gemtc and rjags package. Results: Thirty-two randomized controlled trials (RCTs) were included in the network meta-analysis. Meta-analysis results showed that while levosimendan significantly improved CI levels at either 0.1 µg/kg/min (mean difference [MD] [95%CrI] = 0.41 [-0.43, 1.4]) or 0.2 µg/kg/min (MD [95%CrI] =0.54 [0.12, 0.99]). Levosimendan, at either 0.075 µg/kg/min (MD [95% CrI] =0.033 [-0.75, 0.82]) or 0.2 µg/kg/min (MD [95% CrI] = -0.014 [-0.26, 0.23]), had no significant advantage in improving Lac levels. Levosimendan, at either 0.1 µg/kg/min (RR [95% CrI] = 0.99 [0.73, 1.3]) or 0.2 µg/kg/min (RR [95% CrI] = 1.0 [0.88, 1.2]), did not have a significant advantage in reducing mortality. Conclusion: The existing evidence suggests that levosimendan can significantly improve CI and lactate levels in patients with sepsis, and levosimendan at 0.1 µg/kg/min might be the optimal dose. Unfortunately, all interventions in this study failed to reduce the 28-day mortality. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42023441220.

Annexin A2 modulates radiation-sensitive transcriptional programming and cell fate.

We previously established annexin A2 as a radioresponsive protein associated with anchorage independent growth in murine epidermal cells. In this study, we demonstrate annexin A2 nuclear translocation in human skin organotypic culture and murine epidermal cells after exposure to X radiation (10-200 cGy), supporting a conserved nuclear function for annexin A2. Whole genome expression profiling in the presence and absence of annexin A2 [shRNA] identified fundamentally altered transcriptional programming that changes the radioresponsive transcriptome. Bioinformatics predicted that silencing AnxA2 may enhance cell death responses to stress in association with reduced activation of pro-survival signals such as nuclear factor kappa B. This prediction was validated by demonstrating a significant increase in sensitivity toward tumor necrosis factor alpha-induced cell death in annexin A2 silenced cells, relative to vector controls, associated with reduced nuclear translocation of RelA (p65) following tumor necrosis factor alpha treatment. These observations implicate an annexin A2 niche in cell fate regulation such that AnxA2 protects cells from radiation-induced apoptosis to maintain cellular homeostasis at low-dose radiation.

Line-scanning detection instrument for photonic crystal enhanced fluorescence.

A laser line-scanning instrument was developed to optimize the near-field enhancement capability of a one-dimensional photonic crystal (PC) for excitation of surface-bound fluorophores. The excitation laser beam is shaped into an 8 µm × 1 mm line that is focused along the direction of the PC grating, while remaining collimated perpendicular to the grating. Such a beam configuration offers high excitation power density while simultaneously providing high resonant coupling efficiency from the laser to the PC surface. Using a panel of 21 immunofluorescence assays on the PC surface in a microarray format, the approach achieves an enhancement factor as high as 90-fold between on-resonance and off-resonance illumination. The instrument provides a capability for sensitive and inexpensive analysis of cancer biomarkers in clinical applications.

Multiplexed cancer biomarker detection using quartz-based photonic crystal surfaces.

A photonic crystal (PC) surface is demonstrated as a high-sensitivity platform for detection of a panel of 21 cancer biomarker antigens using a sandwich enzyme-linked immunosorbent assay (ELISA) microarray format. A quartz-based PC structure fabricated by nanoimprint lithography, selected for its low autofluorescence, supports two independent optical resonances that simultaneously enable enhancement of fluorescence detection of biomarkers and label-free quantification of the density of antibody capture spots. A detection instrument is demonstrated that supports fluorescence and label-free imaging modalities, with the ability to optimize the fluorescence enhancement factor on a pixel-by-pixel basis throughout the microarray using an angle-scanning approach for the excitation laser that automatically compensates for variability in surface chemistry density and capture spot density. Measurements show that the angle-scanning illumination approach reduces the coefficient of variation of replicate assays by 20-99% compared to ordinary fluorescence microscopy, thus supporting reduction in limits of detectable biomarker concentration. Using the PC resonance, biomarkers in mixed samples were detectable at the lowest concentrations tested (2.1-41 pg/mL), resulting in a three-log range of quantitative detection.

Reactive oxygen species alter autocrine and paracrine signaling.

Cytochrome P450 (P450) 3A4 (CYP3A4) is the most abundant P450 protein in human liver and intestine and is highly inducible by a variety of drugs and other compounds. The P450 catalytic cycle is known to uncouple and release reactive oxygen species (ROS), but the effects of ROS from P450 and other enzymes in the endoplasmic reticulum have been poorly studied from the perspective of effects on cell biology. In this study, we expressed low levels of CYP3A4 in HepG2 cells, a human hepatocarcinoma cell line, and examined effects on intracellular levels of ROS and on the secretion of a variety of growth factors that are important in extracellular communication. Using the redox-sensitive dye RedoxSensor red, we demonstrate that CYP3A4 expression increases levels of ROS in viable cells. A custom ELISA microarray platform was employed to demonstrate that expression of CYP3A4 increased secretion of amphiregulin, intracellular adhesion molecule 1, matrix metalloprotease 2, platelet-derived growth factor (PDGF), and vascular endothelial growth factor, but suppressed secretion of CD14. The antioxidant N-acetylcysteine suppressed all P450-dependent changes in protein secretion except for CD14. Quantitative RT-PCR demonstrated that changes in protein secretion were consistently associated with corresponding changes in gene expression. Inhibition of the NF-κB pathway blocked P450 effects on PDGF secretion. CYP3A4 expression also altered protein secretion in human mammary epithelial cells and C10 mouse lung cells. Overall, these results suggest that increased ROS production in the endoplasmic reticulum alters the secretion of proteins that have key roles in paracrine and autocrine signaling.

Reducing heterophilic antibody interference in immunoassays using single-chain antibodies.

Sandwich enzyme-linked immunosorbent assay (ELISA) microarrays can simultaneously quantify the levels of multiple diagnostic targets in a biological sample. However, as with traditional ELISA diagnostics, endogenous antibodies in patient sera can cause interference. We demonstrate here that reducing the diagnostic capture antibody to its minimal functional unit (i.e., a single-chain antibody fragment [scFv]) is an effective strategy for reducing assay interference. Our finding illustrates a source of error introduced by the reliance on immunoglobulin-based capture reagents in sandwich immunoassays with human serum samples. We demonstrate that scFvs can be used in such assays to improve reliability by reducing heterophilic antibody interference, thereby improving biomarker analysis and validation.

Smoking, COPD, and 3-nitrotyrosine levels of plasma proteins.

BACKGROUND: Nitric oxide is a physiological regulator of endothelial function and hemodynamics. Oxidized products of nitric oxide can form nitrotyrosine, which is a marker of nitrative stress. Cigarette smoking decreases exhaled nitric oxide, and the underlying mechanism may be important in the cardiovascular toxicity of smoking. Even so, it is unclear if this effect results from decreased nitric oxide production or increased oxidative degradation of nitric oxide to reactive nitrating species. These two processes would be expected to have opposite effects on nitrotyrosine levels, a marker of nitrative stress. OBJECTIVE: In this study, we evaluated associations of cigarette smoking and chronic obstructive pulmonary disease (COPD) with nitrotyrosine modifications of specific plasma proteins to gain insight into the processes regulating nitrotyrosine formation. METHODS: A custom antibody microarray platform was developed to analyze the levels of 3-nitrotyrosine modifications on 24 proteins in plasma. In a cross-sectional study, plasma samples from 458 individuals were analyzed. RESULTS: Average nitrotyrosine levels in plasma proteins were consistently lower in smokers and former smokers than in never smokers but increased in smokers with COPD compared with smokers who had normal lung-function tests. CONCLUSIONS: Smoking is associated with a broad decrease in 3-nitrotyrosine levels of plasma proteins, consistent with an inhibitory effect of cigarette smoke on endothelial nitric oxide production. In contrast, we observed higher nitrotyrosine levels in smokers with COPD than in smokers without COPD. This finding is consistent with increased nitration associated with inflammatory processes. This study provides insight into a mechanism through which smoking could induce endothelial dysfunction and increase the risk of cardiovascular disease.

Plasma biomarker profiles differ depending on breast cancer subtype but RANTES is consistently increased.

BACKGROUND: Current biomarkers for breast cancer have little potential for detection. We determined whether breast cancer subtypes influence circulating protein biomarkers. METHODS: A sandwich ELISA microarray platform was used to evaluate 23 candidate biomarkers in plasma samples that were obtained from subjects with either benign breast disease or invasive breast cancer. All plasma samples were collected at the time of biopsy, after a referral due to a suspicious screen (e.g., mammography). Cancer samples were evaluated on the basis of breast cancer subtypes, as defined by the HER2 and estrogen receptor statuses. RESULTS: Ten proteins were statistically altered in at least one breast cancer subtype, including four epidermal growth factor receptor ligands, two matrix metalloproteases, two cytokines, and two angiogenic factors. Only one cytokine, RANTES, was significantly increased (P < 0.01 for each analysis) in all four subtypes, with areas under the curve (AUC) for receiver operating characteristic values that ranged from 0.76 to 0.82, depending on cancer subtype. The best AUC values were observed for analyses that combined data from multiple biomarkers, with values ranging from 0.70 to 0.99, depending on the cancer subtype. Although the results for RANTES are consistent with previous publications, the multi-assay results need to be validated in independent sample sets. CONCLUSIONS: Different breast cancer subtypes produce distinct biomarker profiles, and circulating protein biomarkers have potential to differentiate between true- and false-positive screens for breast cancer. IMPACT: Subtype-specific biomarker panels may be useful for detecting breast cancer or as an adjunct assay to improve the accuracy of current screening methods.

Application of photonic crystal enhanced fluorescence to cancer biomarker microarrays.

We report on the use of photonic crystal surfaces as a high-sensitivity platform for detection of a panel of cancer biomarkers in a protein microarray format. The photonic crystal surface is designed to provide an optical resonance at the excitation wavelength of cyanine-5 (Cy5), thus providing an increase in fluorescent intensity for Cy5-labeled analytes measured with a confocal microarray scanner, compared to a glass surface. The sandwich enzyme-linked immunosorbent assay (ELISA) is undertaken on a microarray platform to undertake a simultaneous, multiplex analysis of 24 antigens on a single chip. Our results show that the resonant excitation effect increases the signal-to-noise ratio by 3.8- to 6.6-fold, resulting in a decrease in detection limits of 6-89%, with the exact enhancement dependent upon the antibody-antigen interaction. Dose-response characterization of the photonic crystal antibody microarrays shows the capability to detect common cancer biomarkers in the <2 pg/mL concentration range within a mixed sample.

Cellular recognition and trafficking of amorphous silica nanoparticles by macrophage scavenger receptor A.

The cellular uptake of engineered nanoparticles (ENPs) is known to involve active transport mechanisms, yet the biological molecules involved are poorly understood. We demonstrate that the uptake of amorphous silica ENPs by macrophage cells, and the secretion of proinflammatory cytokines, is strongly inhibited by silencing expression of scavenger receptor A (SR-A). Conversely, ENP uptake is augmented by introducing SR-A expression into human cells that are normally non-phagocytic. Confocal microscopy analyses show that the majority of single or small clusters of silica ENPs co-localize with SR-A and are internalized through a pathway characteristic of clathrin-dependent endocytosis. In contrast, larger silica ENP agglomerates (>500 nm) are poorly co-localized with the receptor, suggesting that the physical agglomeration state of an ENP influences its cellular trafficking. As SR-A is expressed in macrophages throughout the reticulo-endothelial system, this pathway is likely an important determinant of the biological response to ENPs.

Cellular dichotomy between anchorage-independent growth responses to bFGF and TPA reflects molecular switch in commitment to carcinogenesis.

We have investigated gene expression patterns underlying reversible and irreversible anchorage-independent growth (AIG) phenotypes to identify more sensitive markers of cell transformation for studies directed at interrogating carcinogenesis responses. In JB6 mouse epidermal cells, basic fibroblast growth factor (bFGF) induces an unusually efficient and reversible AIG response, relative to 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced AIG which is irreversible. The reversible and irreversible AIG phenotypes are characterized by largely nonoverlapping global gene expression profiles. However, a subset of differentially expressed genes were identified as common to reversible and irreversible AIG phenotypes, including genes regulated in a reciprocal fashion. Hepatic leukemia factor (HLF) and D-site albumin promoter-binding protein (DBP) were increased in both bFGF and TPA soft agar colonies and selected for functional validation. Ectopic expression of human HLF and DBP in JB6 cells resulted in a marked increase in TPA- and bFGF-regulated AIG responses. HLF and DBP expression were increased in soft agar colonies arising from JB6 cells exposed to gamma radiation and in a human basal cell carcinoma tumor tissue, relative to paired nontumor tissue. Subsequent biological network analysis suggests that many of the differentially expressed genes that are common to bFGF- and TPA-dependent AIG are regulated by c-Myc, SP-1, and HNF-4 transcription factors. Collectively, we have identified a potential molecular switch that mediates the transition from reversible to irreversible AIG.

Clinical outcome of HIV-infected antiretroviral-naive patients with discordant immunologic and virologic responses to highly active antiretroviral therapy.

BACKGROUND: The prognostic significance of a response to highly active antiretroviral therapy (HAART) that is immunologically and virologically discordant is not well understood. METHODS: Four hundred four antiretroviral-naive patients initiating HAART at an urban HIV outpatient clinic in 1995 to 2004 were analyzed. The association of treatment responses at 3 to 9 months after HAART initiation with time to development of an opportunistic infection (OI) or death was determined using Cox proportional hazards modeling. Logistic regression modeling was used to examine the association between discordant responses and patient characteristics. RESULTS: Of 404 patients, 70.5% experienced favorable concordant responses (CD4 cell count [CD4]+/viral load [VL]+: increase in CD4 count of >or=50 cells/microL and achievement of undetectable plasma HIV RNA level), 15.8% an immunologic response only (CD4+/VL(-)), 8.7% a virologic response only (CD4(-)/VL+), and 5.0% a concordant unfavorable response (CD4(-)/VL(-)). Both types of discordant responses (CD4+/VL(-) and CD4(-)/VL+), nonresponse (CD4(-)/VL(-)), and baseline CD4 cell count were significantly associated with earlier development of an OI or death (relative hazard [RH] = 2.81, 95% confidence interval [CI]: 1.31 to 3.97; RH = 4.83, 95% CI: 2.10 to 11.12; and RH = 0.93, 95% CI: 0.88 to 0.99, respectively). CD4+/VL(-) and CD4(-)/VL(-) were associated with nonwhite race in multivariate logistic regression models (adjusted OR = 2.83, 95% CI: 1.46 to 5.47 and adjusted OR = 6.50, 95% CI: 1.65 to 25.69, respectively). CONCLUSION: Discordant immunologic and virologic responses at 3 to 9 months after HAART initiation play important roles in predicting long-term clinical outcomes in treatment-naive patients.

Microarray analysis of Mu transposition in Salmonella enterica, serovar Typhimurium: transposon exclusion by high-density DNA binding proteins.

All organisms contain transposons with the potential to disrupt and rearrange genes. Despite the presence of these destabilizing sequences, some genomes show remarkable stability over evolutionary time. Do bacteria defend the genome against disruption by transposons? Phage Mu replicates by transposition and virtually all genes are potential insertion targets. To test whether bacteria limit Mu transposition to specific parts of the chromosome, DNA arrays of Salmonella enterica were used to quantitatively measure target site preference and compare the data with Escherichia coli. Essential genes were as susceptible to transposon disruption as non-essential ones in both organisms, but the correlation of transposition hot spots among homologous genes was poor. Genes in highly transcribed operons were insulated from transposon mutagenesis in both organisms. A 10 kb cold spot on the pSLT plasmid was near parS, a site to which the ParB protein binds and spreads along DNA. Deleting ParB erased the plasmid cold spot, and an ectopic parS site placed in the Salmonella chromosome created a new cold spot in the presence of ParB. Our data show that competition between cellular proteins and transposition proteins on plasmids and the chromosome is a dominant factor controlling the genetic footprint of transposons in living cells.

DNA microarray analysis reveals a role for lysophosphatidic acid in the regulation of anti-inflammatory genes in MC3T3-E1 cells.

Lysophosphatidic acid (LPA) is a bioactive lipid with functional properties that overlap those of growth factors and cytokines. LPA production in vivo is linked to platelet degranulation and the biological activities of this lipid are associated with wound healing. Osteoblasts and their progenitor cells are exposed to high levels of this lipid factor in regions adjacent to bone fractures and we postulate a role for LPA in skeletal healing. The regeneration of bone injuries requires a complex array of changes in gene expression, but the effects of LPA on mRNA levels in bone cells have not been investigated. We performed a genome-wide expression analysis in LPA-treated MC3T3-E1 pre-osteoblastic cells using Affymetrix GeneChip arrays. Cells exposed to LPA for 6 h exhibited 513 regulated genes, whereas changes in the levels of 54 transcripts were detected after a 24-h LPA treatment. Gene ontology analysis linked LPA-regulated gene products to biological processes that are known to govern bone healing, including cell proliferation, response to stress, organ development, chemotaxis/motility, and response to stimuli. Among the gene products most highly up-regulated by LPA were transcripts encoding the anti-inflammatory proteins sST2, ST2L, and heat-shock protein 25 (HSP25). RT-PCR analysis confirmed that these mRNAs were increased significantly in MC3T3-E1 cells and primary osteoblasts exposed to LPA. The response of cells to LPA is mediated by G-protein-coupled receptors, and the stimulation of anti-inflammatory gene expression in MC3T3-E1 cells was blocked by Ki16425, an inhibitor of LPA(1) and LPA(3) receptor forms. Pertussis toxin impaired only the LPA-induced expression of sST2. LPA-stimulated levels of sST2, ST2L and HSP25 mRNAs persisted if the cytosolic Ca(2+) elevations elicited by this lipid were blocked with BAPTA. In contrast to the stimulatory effect of LPA, exposure of MC3T3-E1 cells to fluid shear reduced the transcript levels of all three anti-inflammatory genes. The induction of sST2, ST2L and HSP25 expression by LPA suggests a role for this lipid factor in the regulation of osteoblastic cell function during periods of inflammation.