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The non-linear voltage-dependent hysteresis (Hys(V)) of voltage-gated ionic currents can be robustly activated by the isosceles-triangular ramp voltage (Vramp) through digital-to-analog conversion. Perturbations on this Hys(V) behavior play a role in regulating membrane excitability in different excitable cells. A variety of small molecules may influence the strength of Hys(V) in different types of ionic currents elicited by long-lasting triangular Vramp. Pirfenidone, an anti-fibrotic drug, decreased the magnitude of Ih's Hys(V) activated by triangular Vramp, while dexmedetomidine, an agonist of α2-adrenoceptors, effectively suppressed Ih as well as diminished the Hys(V) strength of Ih. Oxaliplatin, a platinum-based anti-neoplastic drug, was noted to enhance the Ih's Hys(V) strength, which is thought to be linked to the occurrence of neuropathic pain, while honokiol, a hydroxylated biphenyl compound, decreased Ih's Hys(V). Cell exposure to lutein, a xanthophyll carotenoid, resulted in a reduction of Ih's Hys(V) magnitude. Moreover, with cell exposure to UCL-2077, SM-102, isoplumbagin, or plumbagin, the Hys(V) strength of erg-mediated K+ current activated by triangular Vramp was effectively diminished, whereas the presence of either remdesivir or QO-58 respectively decreased or increased Hys(V) magnitude of M-type K+ current. Zingerone, a methoxyphenol, was found to attenuate Hys(V) (with low- and high-threshold loops) of L-type Ca2+ current induced by long-lasting triangular Vramp. The Hys(V) properties of persistent Na+ current (INa(P)) evoked by triangular Vramp were characterized by a figure-of-eight (i.e., ∞) configuration with two distinct loops (i.e., low- and high-threshold loops). The presence of either tefluthrin, a pyrethroid insecticide, or t-butyl hydroperoxide, an oxidant, enhanced the Hys(V) strength of INa(P). However, further addition of dapagliflozin can reverse their augmenting effects in the Hys(V) magnitude of the current. Furthermore, the addition of esaxerenone, mirogabalin, or dapagliflozin was effective in inhibiting the strength of INa(P). Taken together, the observed perturbations by these small-molecule modulators on Hys(V) strength in different types of ionic currents evoked during triangular Vramp are expected to influence the functional activities (e.g., electrical behaviors) of different excitable cells in vitro or in vivo. [ABSTRACT FROM AUTHOR]

QO-58 (5-(2,6-dichloro-5-fluoropyridin-3-yl)-3-phenyl-2-(trifluoromethyl)-1H-pyrazolol[1,5-a]pyrimidin-7-one) has been regarded to be an activator of KV7 channels with analgesic properties. However, whether and how the presence of this compound can result in any modifications of other types of membrane ion channels in native cells are not thoroughly investigated. In this study, we investigated its perturbations on M-type K+ current (IK(M)), Ca2+-activated K+ current (IK(Ca)), large-conductance Ca2+-activated K+ (BKCa) channels, and erg-mediated K+ current (IK(erg)) identified from pituitary tumor (GH3) cells. Addition of QO-58 can increase the amplitude of IK(M) and IK(Ca) in a concentration-dependent fashion, with effective EC50 of 3.1 and 4.2 μM, respectively. This compound could shift the activation curve of IK(M) toward a leftward direction with being void of changes in the gating charge. The strength in voltage-dependent hysteresis (Vhys) of IK(M) evoked by upright triangular ramp pulse (Vramp) was enhanced by adding QO-58. The probabilities of M-type K+ (KM) channels that will be open increased upon the exposure to QO-58, although no modification in single-channel conductance was seen. Furthermore, GH3-cell exposure to QO-58 effectively increased the amplitude of IK(Ca) as well as enhanced the activity of BKCa channels. Under inside-out configuration, QO-58, applied at the cytosolic leaflet of the channel, activated BKCa-channel activity, and its increase could be attenuated by further addition of verruculogen, but not by linopirdine (10 μM). The application of QO-58 could lead to a leftward shift in the activation curve of BKCa channels with neither change in the gating charge nor in single-channel conductance. Moreover, cell exposure of QO-58 (10 μM) resulted in a minor suppression of IK(erg) amplitude in response to membrane hyperpolarization. The docking results also revealed that there are possible interactions of the QO-58 molecule with the KCNQ or KCa1.1 channel. Overall, dual activation of IK(M) and IK(Ca) caused by the presence of QO-58 eventually may have high impacts on the functional activity (e.g., anti-nociceptive effect) residing in electrically excitable cells. Care must be exercised when interpreting data generated with QO-58 as it is not entirely KCNQ/KV7 selective. [ABSTRACT FROM AUTHOR]

Mirogabalin (MGB, Tarlige®), an inhibitor of the α2δ-1 subunit of voltage-gated Ca2+ (CaV) channels, is used as a way to alleviate peripheral neuropathic pain and diabetic neuropathy. However, to what extent MGB modifies the magnitude, gating, and/or hysteresis of various types of plasmalemmal ionic currents remains largely unexplored. In pituitary tumor (GH3) cells, we found that MGB was effective at suppressing the peak (transient, INa(T)) and sustained (late, INa(L)) components of the voltage-gated Na+ current (INa) in a concentration-dependent manner, with an effective IC50 of 19.5 and 7.3 μM, respectively, while the KD value calculated on the basis of minimum reaction scheme was 8.2 μM. The recovery of INa(T) inactivation slowed in the presence of MGB, although the overall current–voltage relation of INa(T) was unaltered; however, there was a leftward shift in the inactivation curve of the current. The magnitude of the window (INa(W)) or resurgent INa (INa(R)) evoked by the respective ascending or descending ramp pulse (Vramp) was reduced during cell exposure to MGB. MGB-induced attenuation in INa(W) or INa(R) was reversed by the further addition of tefluthrin, a pyrethroid insecticide known to stimulate INa. MGB also effectively lessened the strength of voltage-dependent hysteresis of persistent INa in response to the isosceles triangular Vramp. The cumulative inhibition of INa(T), evoked by pulse train stimulation, was enhanced in its presence. Taken together, in addition to the inhibition of CaV channels, the NaV channel attenuation produced by MGB might have an impact in its analgesic effects occurring in vivo. [ABSTRACT FROM AUTHOR]

This review paper delves into the current body of evidence, offering a thorough analysis of the impact of large-conductance Ca2+-activated K+ (BKCa or BK) channels on the electrical dynamics of the heart. Alterations in the activity of BKCa channels, responsible for the generation of the overall magnitude of Ca2+-activated K+ current at the whole-cell level, occur through allosteric mechanisms. The collaborative interplay between membrane depolarization and heightened intracellular Ca2+ ion concentrations collectively contribute to the activation of BKCa channels. Although fully developed mammalian cardiac cells do not exhibit functional expression of these ion channels, evidence suggests their presence in cardiac fibroblasts that surround and potentially establish close connections with neighboring cardiac cells. When cardiac cells form close associations with fibroblasts, the high single-ion conductance of these channels, approximately ranging from 150 to 250 pS, can result in the random depolarization of the adjacent cardiac cell membranes. While cardiac fibroblasts are typically electrically non-excitable, their prevalence within heart tissue increases, particularly in the context of aging myocardial infarction or atrial fibrillation. This augmented presence of BKCa channels' conductance holds the potential to amplify the excitability of cardiac cell membranes through effective electrical coupling between fibroblasts and cardiomyocytes. In this scenario, this heightened excitability may contribute to the onset of cardiac arrhythmias. Moreover, it is worth noting that the substances influencing the activity of these BKCa channels might influence cardiac electrical activity as well. Taken together, the BKCa channel activity residing in cardiac fibroblasts may contribute to cardiac electrical function occurring in vivo. [ABSTRACT FROM AUTHOR]

Objectives: The β1 integrin (CD29) is a putative marker for cancerous epithelial stem cells. Cancer stem cells are essential to drive tumor growth, recurrence, and metastasis. We investigated the role of β1-integrin expression in the development of malignant phenotypes of oral squamous cell carcinoma (OSCC). Materials and methods: Immunostaining was used to analyze the expression levels of β1 integrins in different types of cell colonies and tumor spheres. The results of cell viability and migration assays with and without siRNA knockdown of β1-integrin expression were compared. Cells expressing β1 integrins were evaluated for their tumorigenicity in mice. The expression of β1 integrins in human specimens of oral cancers at different clinical stages was semiquantified based on immunohistochemical staining of the β1-integrin protein. Results: The expression level of β1 integrins in Meng-1 oral epidermoid carcinoma cells (OECM-1) cells was significantly higher in holoclonal colonies and tumor spheres compared to control cells. The knockdown of β1-integrin expression in OECM-1 cells reduced cell proliferation, migration, and tumor sphere formation. Beta-1 integrin (+) cells were more tumorigenic in the mouse xenograft model than β1 integrin (−) cells. In the human specimens, the expression level of the β1-integrin protein positively correlated with the clinical stage. Conclusion: The expression of β1 integrin in OECM-1 cells is involved in the development of malignant phenotypes of OSCC. Clinical relevance: Inhibitors for β1-integrin signaling may be suitable to become target-specific therapies for OSCC. [ABSTRACT FROM AUTHOR]

Abstract: The aim of the study was to investigate the toxicity of benzyl alcohol (BA), the preservative in commercial triamcinolone acetonide (TA) suspensions, on retinal pigment epithelial (RPE) cells. Cultured RPE cells from a human cell line (ARPE-19) and from rabbits were exposed to the balanced salt solution (control) or BA (0.0225, 0.225, 0.9, 3 or 9mg/mL) for 5, 30, 60, or 120min. Morphological changes of RPE cells were evaluated by the trypan blue in situ staining. The proportions of dead cells were quantitatively measured by the trypan blue exclusion assay, and those of functional cells were assessed by a mitochondrial dehydrogenase assay. The mechanism of cytotoxicity was determined by the acridine orange/ethidium bromide staining and DNA laddering technique. Furthermore, ultrastructural changes were observed by transmission electron microscopy. The results showed that RPE cell damage was dose- and time-dependent. BA 0.225mg/mL, the clinically relevant concentration in TA following intravitreal injection, caused ultrastructural damage and impaired human RPE cell function at 2h; but BA 0.0225mg/mL did not. BA 9.0mg/mL, the concentration in commercial TA suspensions, was toxic within 5min on each assay for both human and rabbit RPE cells. The major mechanism of cell death was necrosis. In conclusion, BA in commercial TA suspensions injected intravitreally (0.225–9mg/mL) can damage RPE cells. Our in vitro study on benzyl alcohol cytotoxicity has significant clinical implications for intravitreal use of TA. We suggest that, before a commercial TA solution is used intravitreally, the vehicle should be removed to prevent damaging the RPE layer, particularly during macular hole surgery. Commercial development of a preservative-free TA suspension for intraocular use is urged. [Copyright &y& Elsevier]

Abstract: Some anaerobic and facultative anaerobic bacteria have been employed as anticancer agents. Previously, we have demonstrated tumor-targeting and antitumor activities of attenuated Salmonella choleraesuis carrying antiangiogenic genes. Here we exploited S. choleraesuis as a single-agent therapy and as part of a combination therapy with low-dose cisplatin for syngeneic murine lung tumor and hepatoma. Systemically injected S. choleraesuis preferentially accumulated within tumors for at least 4 weeks and the bacteria accumulated preferentially in not only subcutaneous but also orthotopic tumors over livers and spleens at ratios ranging from 1000:1 to 100,000:1. S. choleraesuis was capable of delaying tumor growth and enhancing survival in both subcutaneous tumor and experimental metastasis models. More strikingly, the combination of S. choleraesuis plus cisplatin acted additively to retard tumor growth and extensively prolong the survival time of the mice bearing hepatomas or lung tumors. Such combination treatment also increased infiltrating neutrophils and CD8+ T cells, as well as apoptotic cells, in the tumors, compared with S. choleraesuis or cisplatin treatment alone. These findings suggest that S. choleraesuis in combination with cisplatin, which exerts oncolytic effects and enhances antitumor immune responses, represents a promising strategy for the treatment of primary and metastatic tumors. [Copyright &y& Elsevier]

Female-dominant tendinopathies are musculoskeletal disorders caused by repetitive hand posture and motion; they are considered overuse syndromes. Both external mechanical stress and changes in hormone levels might affect disease progression. We have previously reported that estrogen receptor-β (ER)-β expression was associated with the pathogenesis of de Quervain's disease. To study the underlying mechanisms, a cyclic stretching culture system was applied to tendon tissue from ovariectomized (OVX) rats. Furthermore, a collagenase I-induced rat tendinopathy model was established to examine the association of ER-β with disease progression. Our results showed that ER-β expression and the number of apoptotic cells were higher and associated with disease severity in rats with tendinopathy. Mechanical stress altered the morphology of primary tenocytes and collagen fiber alignment in tendons, and up-regulated the expression of matrix metalloproteinase-9, ER-β, and interleukin-1β, as well as induced apoptosis in tenocytes and tendon tissue from OVX rats. This is the first report on the effects of ER-β and mechanical stress in tendinopathy. We hope these findings contribute to new pharmacological therapies targeting ER-β signaling pathways to treat tendon-related diseases. [ABSTRACT FROM AUTHOR]

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with enhanced NETosis and impaired degradation of neutrophil extracellular traps (NETs). Galectin-3 is a β-galactoside binding protein and is associated with neutrophil functions as well as involved in mediating autoimmune disorders. In this study, we plan to examine the associations of galectin-3 with the pathogenesis of SLE and NETosis. Galectin-3 expression levels were determined in peripheral blood mononuclear cells (PBMCs) of SLE patients for the association with lupus nephritis (LN) or correlation of SLE disease activity index 2000 (SLEDAI-2K). NETosis was observed in human normal and SLE and murine galectin-3 knockout (Gal-3 KO) neutrophils. Gal-3 KO and wild-type (WT) mice induced by pristane were used to evaluate disease signs, including diffuse alveolar haemorrhage (DAH), LN, proteinuria, anti-ribonucleoprotein (RNP) antibody, citrullinated histone 3 (CitH3) levels, and NETosis. Galectin-3 levels are higher in PBMCs of SLE patients compared with normal donors and positively correlated with LN or SLEDAI-2K. Gal-3 KO mice have higher percent survival and lower DAH, LN proteinuria, and anti-RNP antibody levels than WT mice induced by pristane. NETosis and citH3 levels are reduced in Gal-3 KO neutrophils. Furthermore, galectin-3 resides in NETs while human neutrophils undergo NETosis. Galectin-3-associated immune complex deposition can be observed in NETs from spontaneously NETotic cells of SLE patients. In this study, we provide clinical relevance of galectin-3 to the lupus phenotypes and the underlying mechanisms of galectin-3-mediated NETosis for developing novel therapeutic strategies targeting galectin-3 for SLE. [ABSTRACT FROM AUTHOR]

Evodiamine (EVO) exhibits anti-cancer activity through the inhibition of cell proliferation; however, little is known about its underlying mechanism. To determine whether ferroptosis is involved in the therapeutic effects of EVO, we investigated critical factors, such as lipid peroxidation levels and glutathione peroxidase 4 (GPX4) expression, under EVO treatment. Our results showed that EVO inhibited the cell proliferation of poorly differentiated, high-grade bladder cancer TCCSUP cells in a dose- and time-dependent manner. Lipid peroxides were detected by fluorescence microscopy after cancer cell exposure to EVO. GPX4, which catalyzes the conversion of lipid peroxides to prevent cells from undergoing ferroptosis, was decreased dose-dependently by EVO treatment. Given the features of iron dependency and lipid-peroxidation-driven death in ferroptosis, the iron chelator deferoxamine (DFO) was used to suppress EVO-induced ferroptosis. The lipid peroxide level significantly decreased when cells were treated with DFO prior to EVO treatment. DFO also attenuated EVO-induced cell death. Co-treatment with a pan-caspase inhibitor or necroptosis inhibitor with EVO did not alleviate cancer cell death. These results indicate that EVO induces ferroptosis rather than apoptosis or necroptosis. Furthermore, EVO suppressed the migratory ability, decreased the expression of mesenchymal markers, and increased epithelial marker expression, determined by a transwell migration assay and Western blotting. The TCCSUP bladder tumor xenograft tumor model confirmed the effects of EVO on the inhibition of tumor growth and EMT. In conclusion, EVO is a novel inducer for activating the ferroptosis of bladder cancer cells and may be a potential therapeutic agent for bladder cancer. [ABSTRACT FROM AUTHOR]

Background: Influenza is one of the most important viral infections globally. Viral RNA-dependent RNA polymerase (RdRp) consists of the PA, PB1, and PB2 subunits, and the amino acid residues of each subunit are highly conserved among influenza A virus (IAV) strains. Due to the high mutation rate and emergence of drug resistance, new antiviral strategies are needed. Host cell factors are involved in the transcription and replication of influenza virus. Here, we investigated the role of galectin-3, a member of the β-galactoside-binding animal lectin family, in the life cycle of IAV infection in vitro and in mice. Methods: We used galectin-3 knockout and wild-type mice and cells to study the intracellular role of galectin-3 in influenza pathogenesis. Body weight and survival time of IAV-infected mice were analyzed, and viral production in mouse macrophages and lung fibroblasts was examined. Overexpression and knockdown of galectin-3 in A549 human lung epithelial cells were exploited to assess viral entry, viral ribonucleoprotein (vRNP) import/export, transcription, replication, virion production, as well as interactions between galectin-3 and viral proteins by immunoblotting, immunofluorescence, co-immunoprecipitation, RT-qPCR, minireplicon, and plaque assays. We also employed recombinant galectin-3 proteins to identify specific step(s) of the viral life cycle that was affected by exogenously added galectin-3 in A549 cells. Results: Galectin-3 levels were increased in the bronchoalveolar lavage fluid and lungs of IAV-infected mice. There was a positive correlation between galectin-3 levels and viral loads. Notably, galectin-3 knockout mice were resistant to IAV infection. Knockdown of galectin-3 significantly reduced the production of viral proteins and virions in A549 cells. While intracellular galectin-3 did not affect viral entry, it increased vRNP nuclear import, RdRp activity, and viral transcription and replication, which were associated with the interaction of galectin-3 with viral PA subunit. Galectin-3 enhanced the interaction between viral PA and PB1 proteins. Moreover, exogenously added recombinant galectin-3 proteins also enhanced viral adsorption and promoted IAV infection in A549 cells. Conclusion: We demonstrate that galectin-3 enhances viral infection through increases in vRNP nuclear import and RdRp activity, thereby facilitating viral transcription and replication. Our findings also identify galectin-3 as a potential therapeutic target for influenza. [ABSTRACT FROM AUTHOR]

Background: Cisplatin-based chemotherapy is the first line of treatment for bladder cancer. However, cisplatin induces muscle wasting associated with NF-κB and cancer cachexia. HOTAIR, an oncogenic long non-coding RNA (lncRNA), promotes cancer progression in different cancers. Crosstalk between HOTAIR and NF-κB is documented. Prothymosin α (ProT) plays important roles in cancer progression and inflammation. However, the potential link between HOTAIR, ProT, and cisplatin-induced cancer cachexia remains unexplored. Here, we investigated the contribution of HOTAIR in cisplatin-induced cancer cachexia and dissected the potential signaling cascade involving the epidermal growth factor receptor (EGFR), ProT, NF-κB, and HOTAIR. Materials and methods: Expression of ProT and HOTAIR transcripts and their correlations in tumor tissues of bladder cancer patients and bladder cancer cell lines were determined by RT-qPCR. Next, levels of phospho-EGFR, EGFR, phospho-NF-κB, and NF-κB were examined by immunoblot analysis in human bladder cancer cells treated with cisplatin. Expression of HOTAIR in cisplatin-treated cells was also assessed by RT-qPCR. Pharmacological inhibitors and overexpression and knockdown approaches were exploited to decipher the signaling pathway. The murine C2C12 myoblasts were used as an in vitro muscle atrophy model. The syngeneic murine MBT-2 bladder tumor was used to investigate the role of mouse Hotair in cisplatin-induced cancer cachexia. Results: Expression of ProT and HOTAIR was higher in bladder tumors than in normal adjacent tissues. There were positive correlations between ProT and HOTAIR expression in clinical bladder tumors and bladder cancer cell lines. Cisplatin treatment increased EGFR and NF-κB activation and upregulated ProT and HOTAIR expression in bladder cancer cells. ProT overexpression increased, whereas ProT knockdown decreased, HOTAIR expression. Notably, cisplatin-induced HOTAIR upregulation was abrogated by EGFR inhibitors or ProT knockdown. ProT-induced HOTAIR overexpression was diminished by NF-κB inhibitors. HOTAIR overexpression enhanced, whereas its knockdown reduced, cell proliferation, cachexia-associated pro-inflammatory cytokine expression, and muscle atrophy. Cachexia-associated symptoms were ameliorated in mice bearing Hotair-knockdown bladder tumors undergoing cisplatin treatment. Conclusions: We demonstrate for the first time a critical role for HOTAIR and identify the involvement of the EGFR-ProT-NF-κB-HOTAIR signaling axis in cisplatin-induced cachexia in bladder cancer and likely other cancers. Our findings also provide therapeutic targets for this disease. [ABSTRACT FROM AUTHOR]

Deltamethrin (DLT) is a type-II pyrethroid ester insecticide used in agricultural and domestic applications as well as in public health. However, transmembrane ionic channels perturbed by this compound remain largely unclear, although the agent is thought to alter the gating characteristics of voltage-gated Na+ (NaV) channel current. In this study, we reappraised whether and how it and other related compounds can make any further modifications on voltage-gated Na+ current (INa) in pituitary tumor (GH3) cells. Cell exposure to DLT produced a differential and dose-dependent stimulation of peak (transient, INa(T)) or sustained (late, INa(L)) INa; consequently, the EC50 value required for DLT-stimulated INa(T) or INa(L) was determined to be 11.2 or 2.5 μM, respectively. However, neither the fast nor slow component in the inactivation time constant of INa(T) activated by short depolarizing pulse was changed with the DLT presence; conversely, tefluthrin (Tef), a type-I pyrethroid insecticide, can accentuate INa with a slowing in inactivation time course of the current. The INa(L) augmented by DLT was attenuated by further application of either dapagliflozin (Dapa) or amiloride, but not by chlorotoxin. During pulse train (PT) stimulation, with the Tef or DLT presence, the cumulative inhibition of INa(T) became slowed; moreover, following PT stimuli, a large tail current with a slowly recovering process was observed. Alternatively, during rapid depolarizing pulse, the amplitude of INa(L) and tail INa (INa(Tail)) for each depolarizing pulse became progressively increased by adding DLT, not by Tef. The recovery time constant following PT stimulation with continued presence of Tef or DLT was shortened by further addition of Dapa. The voltage-dependent hysteresis (Hys(V)) of persistent INa was differentially augmented by Tef or DLT. Taken together, the magnitude, gating, frequency dependence, as well as Hys(V) behavior of INa exerted by the presence of DLT or Tef might exert a synergistic impact on varying functional activities of excitable cells in culture or in vivo. [ABSTRACT FROM AUTHOR]

Objective Synovial fibroblasts (SFs) with aberrant expression of microRNAs (miRNAs) are critical pathogenic regulators in rheumatoid arthritis (RA), and studies analyzing the effect of overexpressing or silencing miRNA expression in arthritis models can contribute to the development of miRNA-based therapeutic strategies. This study was undertaken to examine the hypothesis that miRNAs 140-3p and 140-5p are involved in the pathogenesis of RA, and to determine whether targeting SFs through the intraarticular (IA) delivery of these molecules could ameliorate autoimmune arthritis in mice. Methods Synovial tissue samples were obtained from patients with RA. In addition, 2 experimental models in mice were used, collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA). Overexpression of miRNAs 140-3p and 140-5p in SFs and synovial tissue was induced using lentivirus (LV)-mediated transfer of pre-miR-140 precursor molecules. Results Lower expression levels of miR-140-3p and miR-140-5p were detected in synovial tissue and SFs from patients with RA and from mice in both arthritis models. In mice with CIA and mice with CAIA, the LV-mediated IA transfer of miR-140-3p and miR-140-5p ameliorated arthritis, as determined by clinical examination and histopathologic evaluations showing a decrease in SF densities. Overexpression of miRNAs 140-3p and 140-5p caused a reduction in expression, with correlated kinetic patterns, of their corresponding target molecules sirtuin 1 and stromal cell-derived factor 1 in the SFs and joints of mice. Transfection of miR-140-3p and miR-140-5p into SFs increased cell apoptosis, reduced proliferation responses and migration abilities, and verified the concept that miR-140 expression is regulated by proinflammatory cytokines. Conclusion These results demonstrate that targeting SFs by IA delivery of miRNAs 140-3p and 140-5p can ameliorate autoimmune arthritis. These findings might facilitate the pharmacologic development of molecular-based therapies in RA. [ABSTRACT FROM AUTHOR]

Interleukin-23 (IL-23) plays a pivotal role in rheumatoid arthritis (RA). IL-23 and microRNA-223 (miR-223) are both up-regulated and mediate osteoclastogenesis in mice with collagen-induced arthritis (CIA). The aim of this study was to examine the association between IL-23 and miR-223 in contributing to osteoclastogenesis and arthritis. Levels of IL-23p19 in joints of mice with CIA were determined. Lentiviral vectors expressing short hairpin RNA (shRNA) targeting IL-23p19 and lisofylline (LSF) were injected intraperitoneally into arthritic mice. Bone marrow-derived macrophages (BMMs) were treated with signal transducers and activators of transcription 4 (STAT4) specific shRNA and miR-223 sponge carried by lentiviral vectors in response to IL-23 stimulation. Treatment responses were determined by evaluating arthritis scores and histopathology in vivo, and detecting osteoclast differentiation and miR-223 levels in vitro. The binding of STAT4 to the promoter region of primary miR-223 (pri-miR-223) was determined in the Raw264.7 cell line. IL-23p19 expression was increased in the synovium of mice with CIA. Silencing IL-23p19 and inhibiting STAT4 activity ameliorates arthritis by reducing miR-223 expression. BMMs from mice in which STAT4 and miR-223 were silenced showed decreased osteoclast differentiation in response to IL-23 stimulation. IL-23 treatment increased the expression of miR-223 and enhanced the binding of STAT4 to the promoter of pri-miR-223. This study is the first to demonstrate that IL-23 promotes osteoclastogenesis by transcriptional regulation of miR-223 in murine macrophages and mice with CIA. Furthermore, our data indicate that LSF, a selective inhibitor of STAT4, should be an ideal therapeutic agent for treating RA through down-regulating miR-223-associated osteoclastogenesis. [ABSTRACT FROM AUTHOR]

Lutein (β,ε-carotene-3,3′-diol), a xanthophyll carotenoid, is found in high concentrations in the macula of the human retina. It has been recognized to exert potential effectiveness in antioxidative and anti-inflammatory properties. However, whether and how its modifications on varying types of plasmalemmal ionic currents occur in electrically excitable cells remain incompletely answered. The current hypothesis is that lutein produces any direct adjustments on ionic currents (e.g., hyperpolarization-activated cation current, Ih [or funny current, If]). In the present study, GH3-cell exposure to lutein resulted in a time-, state- and concentration-dependent reduction in Ih amplitude with an IC50 value of 4.1 μM. There was a hyperpolarizing shift along the voltage axis in the steady-state activation curve of Ih in the presence of this compound, despite being void of changes in the gating charge of the curve. Under continued exposure to lutein (3 μM), further addition of oxaliplatin (10 μM) or ivabradine (3 μM) could be effective at either reversing or further decreasing lutein-induced suppression of hyperpolarization-evoked Ih, respectively. The voltage-dependent anti-clockwise hysteresis of Ih responding to long-lasting inverted isosceles-triangular ramp concentration-dependently became diminished by adding this compound. However, the addition of 10 μM lutein caused a mild but significant suppression in the amplitude of erg-mediated or A-type K+ currents. Under current-clamp potential recordings, the sag potential evoked by long-lasting hyperpolarizing current stimulus was reduced under cell exposure to lutein. Altogether, findings from the current observations enabled us to reflect that during cell exposure to lutein used at pharmacologically achievable concentrations, lutein-perturbed inhibition of Ih would be an ionic mechanism underlying its changes in membrane excitability. [ABSTRACT FROM AUTHOR]

Crassolide, a cembranoid diterpene extracted from the soft coral Lobophytum crissum, has been proven to possess antioxidant and immunomodulatory properties. In the present study, we assessed the anticancer effects of crassolide on human H460 non-small-cell lung cancer (NSCLC) cells. We found that crassolide exerted cytotoxic effects on H460 cancer cells in vitro, inducing G2/M phase arrest and apoptosis. In addition, in H460 cells exposed to crassolide, the expression of the autophagy-related proteins LC3-II and beclin was increased, while the expression of p62 was decreased. Moreover, inhibiting autophagy with chloroquine (CQ) suppressed the crassolide-induced G2/M arrest and apoptosis of H460 cells. Moreover, we also found that crassolide induced endoplasmic reticulum (ER) stress in lung cancer cells by increasing the expression of ER stress marker proteins and that the crassolide-induced G2/M arrest, apoptosis, and autophagy were markedly attenuated by the ER stress inhibitor 4-phenylbutyric acid (4-PBA). Furthermore, we found that crassolide promoted reactive oxygen species (ROS) production by H460 cells and that the ROS inhibitor N-acetylcysteine (NAC) decreased the crassolide-induced ER stress, G2/M arrest, apoptosis, and autophagy. In conclusion, our findings show that crassolide inhibits NSCLC cell malignant biological behaviors for the first time, suggesting that this effect may be mechanistically achieved by inducing G2/M arrest, apoptosis, and autophagy through ROS accumulation, which activates the ER stress pathway. As a result of our findings, we now have a better understanding of the molecular mechanism underlying the anticancer effect of crassolide, and we believe crassolide might be a candidate for targeted cancer therapy. [ABSTRACT FROM AUTHOR]

Prothymosin α (ProT), a highly acidic nuclear protein with multiple cellular functions, has shown potential neuroprotective properties attributed to its anti-necrotic and anti-apoptotic activities. The present study aimed to investigate the beneficial effect of ProT on neuroplasticity after ischemia-reperfusion injury and elucidate its underlying mechanism of action. Primary cortical neurons were either treated with ProT or overexpressing ProT by gene transfection and exposed to oxygen-glucose deprivation for 2 h in vitro. Immunofluorescence staining for ProT and MAP-2 was performed to quantify ProT protein expression and assess neuronal arborization. Mice treated with vehicle or ProT (100 µg/kg) and ProT overexpression in transgenic mice received middle cerebral artery occlusion for 50 min to evaluate the effect of ProT on neuroplasticity-associated protein following ischemia-reperfusion injury. The results demonstrated that in cultured neurons ProT significantly increased neurite lengths and the number of branches, accompanied by an upregulation mRNA level of brain-derived neurotrophic factor. Furthermore, ProT administration improved the protein expressions of synaptosomal-associated protein, 25 kDa and postsynaptic density protein 95 after ischemic-reperfusion injury in vivo. These findings suggested that ProT can potentially induce neuroplasticity effects following ischemia-reperfusion injury. [ABSTRACT FROM AUTHOR]

Systemic administration of Salmonella to tumor-bearing mice leads to the preferential accumulation within tumor sites and retardation of the tumor growth. Host factors including innate and adaptive immune responses influence Salmonella-induced antitumor activity. Antitumor activities of Salmonella are not only determined by the tumor regression but also by the host immune response. Herein, we demonstrated that B cells play an important role in the antitumor activity mediated by Salmonella. Body weight and survival of B cell-deficient mice were decreased compared with wild-type, CD8 cell-deficient, or CD4 cell-deficient mice after Salmonella administration. Although Salmonella accumulated within the tumors in B cell-deficient mice, the bacterial loads of healthy organs were higher than those in wild-type mice. The inflammation cytokine and bacteremia were found in B cell-deficient mice after Salmonella treatment. When Salmonella accumulated within the tumor, B cells inhibited the dissemination of Salmonella to other healthy organs. The depletion of host B cells resulted in a noticeably higher total number of Salmonella in the tumor and inhibited tumor growth. Meanwhile, B cell-depletive and B cell-adoptive transfer of serum experiments demonstrated that the natural antibody produced by B cell takes part in the control of Salmonella dissemination in tumor-bearing mice. In this study, we want to address the mechanisms of incorporating host immunoresponse as a way to augment the antitumor activities of Salmonella. [ABSTRACT FROM AUTHOR]

Chronic exercise has been reported to improve cognitive function. However, whether and how different types of exercise affect various learning and memory tasks remain uncertain. To address this issue, male BALB/c mice were trained for 4 weeks under two different exercise protocols: moderate treadmill running or voluntary wheel running. After exercise training, their spatial memory and aversive memory were evaluated by a Morris water maze and by one-trial passive avoidance (PA), respectively. Levels of neural plasticity-related proteins, i.e. brain-derived neurotrophic factor (BDNF), tropomyosin-related kinase B (TrkB) and synaptotagmin I (Syt I), in hippocampus and amygdala were determined by ELISA or immunoblotting. Finally, the functional roles of these proteins in the basolateral amygdala were verified by locally blocking them with K252a (a TrkB kinase inhibitor), or lentivirus expressing Syt I shRNA. We found that (1) although both moderate treadmill running and wheel running improved the Morris water maze performance, only the former improved PA performance; (2) likewise, both exercise protocols upregulated the BDNF–TrkB pathway and Syt I in the hippocampus, whereas only treadmill exercise upregulated their expression levels in the amygdala; (3) local injection of K252a abolished the treadmill exercise-facilitated PA performance and upregulation of amygdalar TrkB and Syt I; and (4) local administration of Syt I shRNA abolished the treadmill exercise-facilitated PA performance and upregulation of amygdalar Syt I. Therefore, our results support the notion that different forms of exercise induce neuroplasticity changes in different brain regions, and thus exert diverse effects on various forms of learning and memory. [ABSTRACT FROM AUTHOR]

Psoriasis is an immune-mediated inflammatory disease that affects 2% to 3% of the world population. Alantolactone, a sesquiterpene lactone, was isolated from Inula helenium and Radix inulae and has several biological effects, including antifungal, anthelmintic, antimicrobial, anti-inflammatory, antitrypanosomal, and anticancer properties. This study aimed to evaluate the antipsoriatic potential of alantolactone in vitro and in vivo and to explore its underlying mechanisms. These results showed that alantolactone significantly attenuated IL-17A, IL-22, oncostatin M, IL-1α, and TNF-α (M5) cytokine-induced hyperproliferation in HaCaT keratinocytes. Moreover, M5 cytokines significantly upregulated the mRNA levels of TNF-α, IL-6, IL-1β, and IL-8. However, alantolactone attenuated the upregulation of these inflammatory cytokines. In addition, alantolactone was found to inhibit STAT3 phosphorylation and NF-κB p65 nuclear translocation in HaCaT keratinocytes. Furthermore, alantolactone treatment in mice significantly alleviated the severity of skin lesions (erythema, scaling and epidermal thickness, and inflammatory cell infiltration) and decreased the mRNA expression of inflammatory cytokines (e.g., TNF-α, IL-6, IL-1β, IL-8, IL-17A, and IL-23) in an IMQ-induced-like mouse model. Therefore, our new findings revealed that alantolactone alleviates psoriatic skin lesions by inhibiting inflammation, making it an attractive candidate for future development as an antipsoriatic agent. [ABSTRACT FROM AUTHOR]

Image, graphical abstract ProT is a cell survival gene, which modulates oxidative stress and transforming growth factor (TGF)-β signaling. We hypothesized that the delivery of the ProT cDNA gene in rats could protect against right heart dysfunction secondary to pulmonary hypertension (PH) induced by left-to-right shunt. A 2-hit rat model of flow-induced PH was used, and a single intravenous injection of adenoviral vectors (2 billion plaque-forming unit) carrying ProT or Luc gene was administered. The animals were euthanized 21 days after gene delivery to assess cardiopulmonary function, serum biochemistry, pulmonary artery (PA), and vasomotor reactivity. Immunohistology and immunoblotting of PA tissues were also performed. ProT transduction significantly reduced PA pressure, right ventricle muscle mass, and wall stress, thereby improving the overall survival of the treated rat. Increased production of ProT through gene therapy preserved both the smooth muscle myosin heavy chain-II and α-smooth muscle actin while counteracting the abundance of TGF-β in PA. Protein abundances of phosphorylated p47-phox, heme oxygenase-1, caspase-3, inducible nitric oxide synthase, cyclo-oxygenase 2, and monocyte chemoattractant protein-1 in PA tissues were reduced. ProT also preserved microRNA-223, thereby suppressing the abundance of PARP-1, which is independent of hypoxia-inducible factor-1α signaling. ProT gene transduction improved PA function by reducing oxidative stress, attenuating inflammation, and preserving the contractile phenotype of vascular smooth muscle cells. The modification of microRNA-223–associated downstream signaling through ProT transduction may play an important role in mitigating cardiopulmonary remodeling in flow-induced PH. [ABSTRACT FROM AUTHOR]

Background: Expression of Oct4 maintains cancer stem cell (CSC)-like properties in lung cancer cells and is correlated with poor prognosis of lung adenocarcinoma. M2-type tumor-associated macrophages (TAMs) promote cancer cell migration and metastasis. Tumor microenvironments promote monocyte differentiation into M2 TAMs via a complex cytokine-based connection. We explored the role of Oct4 in cytokine secretion in lung cancer and its impact on M2 TAM polarization. Methods: Monocytes co-cultured with the conditioned medium from Oct4-overexpressing lung cancer cells were used to investigate M2 TAM differentiation. The inflammatory factors in the conditioned medium of Oct4-overexpressing A549 cells were examined using human inflammation antibody arrays. The correlations of Oct4, macrophage colony-stimulating factor (M-CSF), and M2 TAMs were validated in lung cancer cells, syngeneic mouse lung tumor models, and clinical samples of non-small cell lung cancer (NSCLC). Results: Oct4-overexpressing A549 cells expressed elevated levels of M-CSF, which contributed to increased M2 macrophages and enhanced tumor migration. Overexpression of Oct4 enhanced tumor growth and reduced the survival of lung tumor-bearing mice, which was correlated with increased number of M2 macrophages in lung cancer. Notably, NSCLC patients with high expression levels of Oct4, M-CSF, and M2 TAMs had the poorest recurrence-free survival. A positive correlation between Oct4, M-CSF, and M2 TAMs was observed in the tumor tissue of NSCLC patient. Treatment with all-trans retinoic acid exerted anti-tumor effects and reduced M2 TAMs in tumor-bearing mice. Conclusions: Our results indicate that Oct4 expressed by lung cancer cells promotes M2 macrophage polarization through upregulation of M-CSF secretion, leading to cancer growth and metastasis. Our findings also implicate that the Oct4/M-CSF axis in M2 macrophage polarization may be potential therapeutic targets for lung cancer. [ABSTRACT FROM AUTHOR]

Molnupiravir (MOL) is a ribonucleoside prodrug for oral treatment of COVID-19. Common adverse effects of MOL are headache, diarrhea, and nausea, which may be associated with altered sodium channel function. Here, we investigated the effect of MOL on voltage-gated Na+ current (I Na) in pituitary GH 3 cells. We show that MOL had distinct effects on transient and late I Na , in combination with decreased time constant in the slow component of I Na inactivation. The 50% inhibitory concentration (IC 50) values of MOL for suppressing transient and late I Na were 26.1 and 6.3 μM, respectively. The overall steady-state current-voltage relationship of I Na remained unchanged upon MOL exposure. MOL-induced alteration of I Na may lead to changes in physiological function through sodium channels. Apart from its effect on inhibiting RNA virus replication, MOL exerts inhibitory effects on plasmalemma I Na , which might constitute an additional yet crucial underlying mechanism of its pharmacological activity or adverse events. • MOL inactivates voltage-gated sodium channels, resulting in repressing transient and late sodium currents. • The IC 50 values of MOL to suppress transient and late sodium currents are 26.1 and 6.3 μM, respectively. • The IC 50 value of MOL to decrease sodium currents is similar to the concentration for inhibiting SARS-CoV-2 replication. • The IC 50 value of MOL is comparable to the maximum observed blood concentration in persons who receive 800 mg of oral MOL. • Inhibitory effects of MOL on voltage-gated sodium channels may contribute to its adverse events. [ABSTRACT FROM AUTHOR]

Prothymosin α (ProTα) is a nuclear protein that serves a role in oncogenesis, by promoting proliferation and inhibiting apoptosis in various malignancies. The present study was designed to investigate ProTα expression in resected human non-small cell lung cancer to define the clinicopathological associations of ProTα-positive lung cancer. Immunohistochemical staining of ProTα was performed using tumor sample slides from 149 patients with non-small cell lung cancer, who underwent surgical resection. Association between the expression of ProTα and the following clinicopathological parameters was accessed: Age, sex, stage, lymph node involvement, pathological subtype, recurrence and cigarette smoking. A total of 85 tumors (57%) were classified as ProTα-positive lung cancer by staining intensity and 73 tumors (49%) were regarded as ProTα-positive by scoring index. The majority of patients with ProTα-positive tumors were younger (P=0.05) and had squamous cell carcinoma (P<0.01) compared with older and adenocarcinoma. Positive expression of ProTα by staining intensity was associated with a higher incidence rate of cancer recurrence (P=0.05) compared with negative ProTα expression. ProTα was also associated with cigarette smoking, particularly in the group with squamous cell carcinoma. Therefore, the present data suggested that ProTα-positive non-small cell lung cancer was associated with younger patients, squamous cell carcinoma, cigarette smoking and a higher incidence recurrence rate, subsequently indicating a subtype consisting of patients with smoking-associated inferior outcomes. [ABSTRACT FROM AUTHOR]

Objective The transcription factor Snail is involved in various biologic functions. We hypothesized that this molecule regulates tumor necrosis factor α (TNFα)-mediated synovial fibroblast activation in the rheumatoid joint. The aim of this study was to examine the role of Snail in the expression of cadherin-11 (Cad-11) and myofibroblast markers, interleukin-6 (IL-6) production, and the invasive ability of cells. Methods Synovium samples were obtained from patients with rheumatoid arthritis (RA) and from rats with collagen-induced arthritis (CIA). Synovial fibroblasts were treated with TNFα or a Wnt signaling inducer, and the joints of rats with CIA were injected with a TNFα antagonist. Modulation of Snail expression in the synovial fibroblasts and joints was performed by lentiviral vector-mediated transfer of complementary DNA or short hairpin RNA. Results The expression of Snail and Cad-11 was higher in synovium and synovial fibroblasts from patients with RA compared with patients with osteoarthritis and was increased in rats with CIA. TNFα stimulation or activation of Wnt signaling up-regulated the expression of Snail, Cad-11, and α-smooth muscle actin (α-SMA) in synovial fibroblasts, and anti-TNFα therapy down-regulated the expression of Snail, Cad-11, and α-SMA in the joints of rats with CIA. Although synovial fibroblast transfectants in which Snail was overexpressed showed increased expression of Cad-11 and α-SMA and enhanced TNFα-mediated invasive capacity and IL-6 production, synovial fibroblast transfectants from rats with CIA in which Snail was silenced showed decreased expression and had the opposite effect on these functions. Normal joints in which Snail was overexpressed had hyperplastic synovium, with increased expression of Cad-11, α-SMA, and IL-6. Silencing Snail expression ameliorated arthritis, with reduced Cad-11 expression and reduced levels of extracellular matrix deposition in the joints of rats with CIA, whereas overexpression of Snail exacerbated arthritis, with increased Cad-11 expression and increased levels of extracellular matrix deposition. Conclusion Our results demonstrate that Snail regulates TNFα-mediated activation of synovial fibroblasts in the rheumatoid joint. These findings may contribute to the pharmacologic development of therapeutics targeting synovial fibroblasts in patients with RA. [ABSTRACT FROM AUTHOR]

Fruiting bodies of Taiwanofungus camphoratus have been widely used as an antidote for food poisoning and considered to be a precious folk medicine for anti-inflammation and hepatoprotection. Zhankuic acid A (ZAA) is its major pharmacologically active compound. Janus kinase 2 (JAK2), whose activation is involved in cytokine signaling, plays critical roles in the development and biology of the hematopoietic system. JAK2 has been implicated as a therapeutic target in inflammatory diseases. The HotLig modeling approach was used to generate the binding model for ZAA with JAK2, showing that ZAA could bind to the ATP-binding pocket of JAK2 exclusively via the H-bond. The interaction between ZAA and JAK2 was verified by antibody competition assay. Binding of ZAA to JAK2 reduced antibody recognition of native JAK2. The expressions of phosphorylated JAK2 and STATs were analyzed by immuno-blotting. ZAA reduced the phosphorylation and downstream signaling of JAK2, and inhibited the interferon (IFN)-γ/signal transducer and activator of transcription (STAT) 1/interferon regulatory factor (IRF)-1 pathway. The protective effect of ZAA on liver injury was evaluated in mice by Con-A-induced acute hepatitis. Pre-treatment with ZAA also significantly ameliorated acute liver injury in mice. Therefore, ZAA can inhibit JAK2 phosphorylation and protect against liver injury during acute hepatitis in mice. In this study, we present data that ZAA exerts anti-inflammatory effects through the JAK2 signaling pathway. As such, ZAA may be a potential therapeutic agent for the treatment of inflammatory diseases. [ABSTRACT FROM AUTHOR]

Abstract: Background: Gene therapy of proopiomelanocortin, the precursor of α-melanocyte-stimulating hormone (α-MSH), suppresses the neovascularization in tumors. However, the roles of α-MSH in angiogenesis remain unclear. Methods: The influence of α-MSH on angiogenesis was evaluated by ex vivo rat aorta and in vivo, including transgenic zebrafish and chicken chorioallantoic membrane (CAM) assays. The effect of α-MSH on proliferation, matrix metalloproteinase (MMP) secretion, migration and tube formation was examined using human umbilical vein endothelial cells (HUVECs). The expression of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) was investigated by quantitative RT-PCR, immunoblot and immunofluorescent analysis. Antibodies' neutralization was employed to dissect the receptor(s) transmitting α-MSH signaling. Results: Application of α-MSH potently suppressed the microvessels sprouting in organotypic aortic rings. Besides, α-MSH perturbed the vessels development in zebrafish and chicken embryos. α-MSH (0.01–10nM) inhibited the MMP-2 secretion, migration and tube formation of HUVECs without affecting proliferation. Mechanistic studies unveiled α-MSH decreased the VEGF expression and release in HUVECs. Besides, α-MSH downregulated the VEGFR2 expression at transcriptional and translational levels. Importantly, α-MSH attenuated the Akt phosphorylation, but enhanced the expression of PTEN, endogenous antagonist of PI3K/Akt signaling. Expression analysis and antibody neutralization revealed that MC1-R and MC2-R participated in α-MSH-induced blockage of migration and VEGF/VEGFR2/Akt signaling. However, VEGF supply failed to reverse the anti-angiogenic function of α-MSH. Conclusions: α-MSH inhibits the physiological angiogenesis by attenuating VEGF/VEGFR2/Akt signaling in endothelial cells. General significance: α-MSH is a potent angiogenesis inhibitor targeting at endothelial VEGF/VEGFR2 signaling, which may have potential for therapeutic application. [Copyright &y& Elsevier]

Background & Aims: While non-alcoholic fatty liver disease (NAFLD) is the most common risk factor of chronic liver disease, the mechanisms that initiate its development are obscure. Hepassocin (HPS) is a hepatokine that has been reported to be involved in liver regeneration. In addition to the mitogenic activity of HPS, HPS expression is decreased in patients with hepatoma. However, the role of HPS in NAFLD is still unknown. Methods: A total of 393 subjects with (n=194) or without (n=199) NAFLD were enrolled to evaluate the serum HPS concentration. In order to clarify the causal inference between HPS and NAFLD, we used experimental animal and cell models. Hepatic overexpression or silencing of HPS was achieved by lentiviral vector delivery in mice and lipofectamine transfection in HepG2 cells. Lipogenesis related proteins were detected by Western blots. The expression of inflammatory factors was determined by real-time polymerase chain reaction. Results: Subjects with NAFLD had a higher serum HPS concentration than those without it. Overexpression of HPS increased hepatic lipid accumulation and NAFLD activity scores (NAS), whereas deletion of HPS improved high fat diet-induced hepatic steatosis and decreased NAS in mice. Additionally, oleic acid, a steatogenic reagent, increased HPS expression in hepatocytes. Furthermore, overexpression of HPS in HepG2 cells induced lipid accumulation through an extracellular signal-regulated kinase 1/2 (ERK1/2)-dependent pathway, whereas deletion of HPS decreased oleic acid-induced lipid accumulation. Conclusions: The present study provides evidence that HPS plays an important role in NAFLD and induces hepatic lipid accumulation through an ERK1/2-dependent pathway. [Copyright &y& Elsevier]

Objectives: To determine the plasma levels of thymosin-α1 (TA1) and prothymosin-α (PTMA) proteins in renal cell carcinoma (RCC) or urothelial carcinoma (UC) patients, and explore the potential of these 2 molecules as biomarkers.Materials and Methods: Blood samples were taken from 50 consecutive patients with RCC, 97 with UC, and 55 with benign urologic diseases before surgery. Their clinical characteristics were obtained from medical record review. Plasma TA1 and PTMA levels were measured using enzyme-linked immunosorbent assay and their correlation with tumor grade, pathologic stage, and survival were explored.Results: Plasma TA1 levels were significantly lower in RCC patients than in UC or benign patients, particularly in UC of the renal pelvis patients (P < 0.0001). Plasma PTMA levels were also significantly lower in UC patients compared with RCC patients and benign patients (P < 0.05). Plasma TA1 levels inversely correlated with pathologic stage both in bladder cancer and RCC patients (P = 0.03 and 0.02, respectively). Both plasma TA1 and PTMA did not correlate with tumor grade. Plasma TA1 was a prognostic indicator for progression-free and disease-specific overall survival in bladder cancer patients (P = 0.008 and 0.04, respectively).Conclusions: Plasma TA1 level may be a biomarker for differentiating between UC and RCC. It may also be a prognostic factor for disease progression and disease-specific survival in bladder cancer patients. These findings warrant more studies for validation. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: To determine the plasma levels of thymosin-α1 (TA1) and prothymosin-α (PTMA) proteins in renal cell carcinoma (RCC) or urothelial carcinoma (UC) patients, and explore the potential of these 2 molecules as biomarkers. Materials and methods: Blood samples were taken from 50 consecutive patients with RCC, 97 with UC, and 55 with benign urologic diseases before surgery. Their clinical characteristics were obtained from medical record review. Plasma TA1 and PTMA levels were measured using enzyme-linked immunosorbent assay and their correlation with tumor grade, pathologic stage, and survival were explored. Results: Plasma TA1 levels were significantly lower in RCC patients than in UC or benign patients, particularly in UC of the renal pelvis patients (P < 0.0001). Plasma PTMA levels were also significantly lower in UC patients compared with RCC patients and benign patients (P < 0.05). Plasma TA1 levels inversely correlated with pathologic stage both in bladder cancer and RCC patients (P = 0.03 and 0.02, respectively). Both plasma TA1 and PTMA did not correlate with tumor grade. Plasma TA1 was a prognostic indicator for progression-free and disease-specific overall survival in bladder cancer patients (P = 0.008 and 0.04, respectively). Conclusions: Plasma TA1 level may be a biomarker for differentiating between UC and RCC. It may also be a prognostic factor for disease progression and disease-specific survival in bladder cancer patients. These findings warrant more studies for validation. [Copyright &y& Elsevier]

Abstract: A surface-enhanced Raman scattering (SERS) substrate was fabricated as well ordered Au/Ag multilayered nanorod arrays (fibAu/Ag_R n , n =0–4) via the focused ion beam technique for the detection of the influenza A virus strain. Since the shape, thickness, space between nanorods, and dimensions affect the performance of SERS, these factors were optimized before the fabrication of the substrate by varying the thickness of the Au layer (4 variables), Ag layer (3 variables), and their corresponding Ag/Au repeated layers. Au/Ag multilayers with the optimal thickness were used to fabricate the designed SERS substrate. The results indicate that Ag layer plays an important role in the improvement of SERS mechanism by inducing the electromagnetic effect at the Au surface. The as-prepared fibAu/Ag_R n substrate serves as an excellent SERS substrate, even verified at a very low concentration using rhodamine 6G as a reference, e.g., it exhibited an enhancement factor ranging from 2.62×106 to 1.74×107 using the proposed SERS substrate. Furthermore, the influenza A virus strains (A/WSN/33 (H1N1), A/England/12/64 (H2N2), and A/Philippine/2/82 (H3N2)) could be well distinguished at a low concentration of 106 PFU/ml. [Copyright &y& Elsevier]

Objective MicroRNA (miRNA) plays a role in autoimmune diseases. MiRNA-223 (miR-223) is up-regulated in patients with rheumatoid arthritis (RA) and is involved in osteoclastogenesis, which contributes to erosive disease. The aim of this study was to test the feasibility of using lentiviral vectors expressing the miR-223 target sequence (miR-223T) to suppress miR-223 activity as a therapeutic strategy in a mouse model of collagen-induced arthritis (CIA). Methods Levels of miR-223 in the synovial tissue of patients with RA or osteoarthritis (OA), as well as in the ankle joints of mice with CIA, were determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Lentiviral vectors expressing miR-223T (LVmiR-223T) or luciferase short hairpin RNA (LVshLuc) as a control vector were injected intraperitoneally into mice with CIA. Treatment responses and disease-related bone mineral density were monitored. Levels of nuclear factor 1A (NF-1A), a direct target of miR-223, and macrophage colony-stimulating factor receptor (M-CSFR), which is critical for osteoclastogenesis, were measured by immunohistochemistry and quantitative RT-PCR. Osteoclasts were assessed by tartrate-resistant acid phosphatase staining. Results MiR-223 expression was significantly higher in the synovium of RA patients and in the ankle joints of mice with CIA as compared to OA patients and normal mice. LVmiR-223T treatment reduced the arthritis score, histologic score, miR-223 expression, osteoclastogenesis, and bone erosion in mice with CIA. Down-regulation of miR-223 with concomitant increases in NF-1A levels and decreases in M-CSFR levels was detected in the synovium of LVmiR-223T-treated mice. Conclusion This study is the first to demonstrate that lentivirus-mediated silencing of miR-223 can reduce disease severity of experimental arthritis. Furthermore, our results indicate that inhibition of miR-223 activity should be further explored as a therapeutic strategy in RA. [ABSTRACT FROM AUTHOR]

Abstract: Chemotherapy is one major approach for treating non-small cell lung carcinoma (NSCLC). However, the progression-free survival rate depends on whether there is tumor metastasis after drug treatment. The biological behavior for its characteristics remains to be clarified. Here, we treated A549 and H1299 NSCLC cell lines with cisplatin, doxorubicin and gemcitabine at the IC50 dose. Most attached cells were surviving cells (A549-A and H1299-A), whereas only a small portion of detached cells survived and reattached to tissue culture plates (A549-R and H1299-R) for further growth. Using cisplatin, a series of H1299 sublines (H1299-R2∼H1299-R5) were also generated by the same selection procedure. Drug treatment increased the migratory ability of A549-R and H1299-R cells. A serial selection could enhance the invasiveness of cells. Cisplatin treatment inhibited the adhesion ability of H1299-R cells compared with their H1299 and H1299-A counterparts. H1299-R cells exhibited increased drug resistance to cisplatin and increased expression of ABCG2, CD133 and CD44. Compared with mice subcutaneously injected with H1299 cells, mice subcutaneously injected with H1299-R cells showed an increase in the number of metastatic lung nodules. We conclude that H1299-R cells selected by suboptimal doses of cisplatin following detachment from and reattachment to the tissue culture plate acquire an enhanced malignant phenotype. Therefore, they provide a more faithful lung cancer model associated with biological aggressiveness for studying clinically recurrent cancers after chemotherapy. [Copyright &y& Elsevier]

The focused ion beam (FIB) technique was employed to precisely fabricate hexagon-like Au nano-rods (fibAu_h) arrays as a surface enhanced Raman scattering – active substrate. A “ring diameter” (D R) was created by the convergence of three fibAu_h with respect to the dimension of the target viruses (D T), such as adenovirus (Adeno), encephalomyocarditis virus (EMCV), influenza virus (H1N1) with different sizes. Three influenza A virus strains were also compared. The results indicate that as that with a D R/D T ratio of around 1, the discrimination ability for detecting the target viruses and SERS mechanism become obvious. The enhanced lightning rod effect surrounding the seized target virus is anticipated if its size and dimension is suitably embraced within three fibAu_h. Hence the as-designed fibAu_h sample with a target-size embracing dimension provides good discrimination ability for distinguishing virus of various sizes or virus strains. [ABSTRACT FROM AUTHOR]

A novel mode of tumor suppressor protein p53 regulation, mediated by recruitment of the linker histone H1 to the promoters of p53 target genes leading to specific repression of p53-dependent transcription, has recently been uncovered. Yet, how this repression could be relieved is not clear. Previously, a histone-binding nuclear protein prothymosin alpha (ProTa) was shown to trigger a p53 response. The histone-binding region of ProTa was found to be essential for this effect, allowing for the possibility that ProTa stimulates p53-dependent transcription by dissociating the p53-histone H1 repressive complex. In support of this model, here we show that ProTa interacts with the same C-terminal domain of histone H1 as p53 does and, therefore, ProTa and p53 could compete for binding to histone H1. Furthermore, ProTa, when competent for histone H1 binding, is able to liberate p53 from the histone H1-p53 complex in vitro. In vivo, stimulation of p53-dependent transcription by ProTa correlates with the ability of ProTa to interact with histone H1. Ectopic expression of histone H1 or its C-terminal ProTa-binding domain specifically suppresses the stimulating effect of ProTa on transcription of the p53-responsive reporter gene in cultured cells. These results are consistent with the model that ProTa may enhance p53 transcription activity by displacement of histone H1 from p53-H1 repressive complex. [ABSTRACT FROM AUTHOR]

In osteoarthritis, angiogenesis, which occurs in the osteochondral junction and synovium, may accelerate inflammation and contribute to the severity of the disease. We used anterior cruciate ligament-transection (ACLT) to investigate the therapeutic effect of an angiogenesis inhibitor, thrombospondin-1 (TSP-1), in a rat model of osteoarthritis. Osteoarthritis was induced in Wistar rats in the knee of one hind leg. After ACLT, AdTSP-1 (adenoviral vector encoding mouse TSP-1) was intraarticularly injected into the knee joints. Transgene expression, angiogenesis, and inflammatory responses in the knee joints were examined. They were also assessed morphologically, radiographically, and histologically for manifestations of disease. The levels of TSP-1 peaked on day 3 and were substantially maintained for at least 9 days after AdTSP-1 infection. Adenovirus-mediated gene expression was detected in the synovial membrane and chondrocytes. TSP-1 gene transfer induced transforming growth factor-β (TGF-β) production, but it reduced microvessel density, macrophage infiltration, and interleukin-1β (IL-1β) levels. Gross morphological and histopathological examinations revealed that rats treated with AdTSP-1 had less severe osteoarthritis than controls. In vivo adenovirus-mediated TSP-1 gene transfer significantly reduced microvessel density, inflammation, and suppressed the progression of osteoarthritis. This study provides potential applications of TSP-1 gene delivery for treating osteoarthritis. Published by Wiley Periodicals, Inc. J Orthop Res 28:1300-1306, 2010 [ABSTRACT FROM AUTHOR]

Objectives: To investigate the prognostic role of prothymosin-α (PTMA) expression in human upper urinary tract transitional cell carcinoma (UUT-TCC). Methods: Paraffin-embedded tissues were collected from 91 patients with UUT-TCC and from 15 paired normal renal cortex and 13 paired urothelial walls. The primary antibody for PTMA (2F11) used was validated in 4 human urothelial cancer cell lines before assessing the surgical specimen. Immunohistochemistry was then conducted to determine the expression intensity of PTMA, the calculation of immunostaining density using imaging analysis, and for immunostaining localization. The correlates with clinicopathologic characteristics and patient survival were explored. Results: The expression intensity of PTMA demonstrated a significant enhancement of PTMA expression in UUT-TCCs compared with both paired normal tissues (P = .0002 and P = .0004 for UUT-TCC vs the urothelial wall and vs the renal cortex, respectively). As for the localization of PTMA immunoreactivity, of the 91 tumor specimens, 33 (36.3%) were cytoplasmic PTMA-expressing, 51 (56.0%) were nuclear PTMA-expressing, and 7 (7.7%) were PTMA-negative tumors. On univariate and multivariate analyses, PTMA expression localization was the sole independent prognostic indicator for recurrence-free survival (hazard ratio 4.90, 95% confidence interval 1.73-13.9; P = .003), although pathologic staging was an independent prognostic indicator for both progression-free survival (hazard ratio 22.6, 95% confidence interval 2.56-198; P = .005) and disease-specific overall survival (hazard ratio 5.60, 95% confidence interval 1.48-21.2; P = .011). The limitations of our study included small patient numbers and short follow-up. Conclusions: The results of our study have shown that PTMA is overexpressed in UUT-TCCs and that cytoplasmic PTMA expression can provide significant prognostic information for subsequent tumor recurrence in the residual urinary tract after nephroureterectomy. [Copyright &y& Elsevier]

Abstract: Cellular mechanisms involving the enhancement of interferon (IFN) signaling by ribavirin remain poorly understood. Here, we identified a novel role of ribavirin in the communication between p53 and the mammalian target of rapamycin (mTOR) signaling. Ribavirin activates p53 by stimulating mTOR and promoting the interaction between mTOR and p53. Activated p53 stimulates the transcription of IFN regulatory factor 9 and subsequently enhances IFN signaling. Furthermore, ribavirin-induced activation of mTOR and p53 enhances IFN-dependent signaling for the IFN-α/ribavirin combined treatment. We conclude that ribavirin enhances activities of mTOR and p53, which may account for its antiviral and antitumor effects. Structured summary:: MINT-7222801: mTOR (uniprotkb:P42345) physically interacts (MI:0914) with raptor (uniprotkb:Q8N122) by anti bait coimmunoprecipitation (MI:0006) MINT-7222793: mTOR (uniprotkb:P42345) physically interacts (MI:0914) with RICTOR (uniprotkb:Q8N6M7) by anti bait coimmunoprecipitation (MI:0006) MINT-7222784: mTOR (uniprotkb:P42345) physically interacts (MI:0914) with P53 (uniprotkb:P04637) by anti bait coimmunoprecipitation (MI:0006) [Copyright &y& Elsevier]

Abstract: Interleukin-12 (IL-12), despite exerting antitumor activity, has limited therapeutic uses due to its systemic toxicity. Since HER2 (also known as ErbB-2, neu, and HER2/neu) is frequently overexpressed on cancer cells, HER2-targeted delivery of IL-12 to tumors may be a promising strategy for enhancing antitumor immunity. Here we showed that intramuscular electrogene transfer of an expression vector encoding a fusion protein antiHER2scFv-IL12, which consists of antiHER2 single-chain variable fragment (scFv) and single-chain IL-12, significantly retarded tumor growth and prolonged the survival in a syngeneic bladder tumor model. Elevated IL-12 and interferon-γ (IFN-γ) levels, increased infiltration of CD4+ and CD8+ T cells, and reduced vascular endothelial growth factor (VEGF) expression in the tumors, as well as enhanced cytolytic activity of splenocytes were noted in the treated mice. Our results suggest that this approach may be effective for the treatment of HER2-overexpressing tumors. [Copyright &y& Elsevier]

Objectives: To investigate the prothymosin-alpha (PTMA) expression in human bladder cancer using tissue microarrays. Methods: Two tissue microarray slides of 50 bladder tumors and 42 paired normal adjacent tissues were investigated using immunohistochemical staining. The staining distribution was categorized as negative, nuclear, cytoplasmic, and mixed expression. Quantitative immunoreactivity was measured using image analysis, as represented by the integrated optical density for each tissue core. Results: In 36 of 42 normal adjacent tissues, positive PTMA immunoreactivity could be seen in some nuclei of the normal urothelial cells, but not, or only minimally, in the cytoplasm and underlying submucosal tissues. A statistically significant enhancement of PTMA expression was found in bladder tumors of each grade compared with the normal adjacent tissue (P < .0001 for normal adjacent tissues vs grade 1, 2, or 3 tumors, paired t test). Of 48 transitional cell carcinoma specimens, only 4 (8.3%) were graded as negative and 44 (91.7%) were positive for PTMA expression, including nuclear (n = 8), cytoplasmic (n = 12), and mixed expression (n = 24) patterns. A statistically significant correlation was found between high grade and mixed expression (P = 0.0020, χ2 test). Conclusions: Increased PTMA expression was found in human bladder cancers compared with the paired normal adjacent bladder tissue. The distribution of PTMA expression was changed in high-grade tumors. The clinical significance of such an aberrant PTMA expression in bladder cancer is worthy of additional investigation. [Copyright &y& Elsevier]

The embryonic stem cell marker Oct4 is expressed in several human cancers and is positively correlated with a poor outcome in cancer patients. However, its physiological role in cancer progression remains poorly understood. Tumor cells block apoptosis to escape cell death so that they can proliferate indefinitely, leading to ineffective therapy for cancer patients. In this study, we investigated whether Oct4 regulates the apoptosis pathway and contributes to poor prognosis in patients with lung adenocarcinoma. Our results revealed that Oct4 expression is correlated with Stat1 expression in lung adenocarcinoma patients and Oct4 is directly bound to the Stat1 promoter to transactivate Stat1 in lung adenocarcinoma cells. Expression of the Stat1 downstream gene Mcl-1 increased in Oct4-overexpressing cancer cells, while Stat1 knockdown in Oct4-overexpressing cancer cells sensitized them to cisplatin-induced apoptosis. Furthermore, Oct4 promoted Stat1 expression and tumor growth, whereas silencing of Stat1 reduced Oct4-induced tumor growth in human lung tumor xenograft models. Taken together, we demonstrate that Oct4 is a pro-survival factor by inducing Stat1 expression and that the Oct4/Stat1/Mcl-1 axis may be a potential therapeutic target for lung adenocarcinoma. [ABSTRACT FROM AUTHOR]

Abstract: In this study, we exploited a crp (cAMP receptor protein) gene-deleted, virulence plasmid-cured Salmonella choleraesuis mutant with decreased carbon source utilization, designated S.C.-Δcrp/vpl−, as a live vaccine strain. Normal weight gain with no clinical signs was observed in pigs immunized with high doses of S.C.-Δcrp/vpl− live vaccine. Vaccination in pregnant sows induced high maternal antibodies, which could prevent piglets from Salmonella infection. Moreover, serial transmission of the vaccine strain in piglets produced no evidence of reversion to virulence. Furthermore, the peripheral blood mononuclear cells from immunized piglets also developed Salmonella specific T-cell proliferative response in vitro. Our results indicate that immunogenic antigens in S.C.-Δcrp/vpl− can induce adequate immunity to protect pigs against challenge with a heterologous virulent strain. Thus, this mutant holds promise for the development of a new live S. choleraesuis vaccine. [Copyright &y& Elsevier]

Purpose: To investigate the cytotoxicity of triamcinolone acetonide (TA) suspensions to corneal endothelial cells (CECs). Setting: Department of Ophthalmology, Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan. Methods: New Zealand white rabbit CECs were exposed for 1 minute to balanced salt solution (BSS); commercial TA suspension (cTA); vehicle-removed TA (–vTA); pure vehicle (V); 1/10 dilutions of cTA, –vTA, or V in BSS; or benzyl alcohol (BA) (cTA preservative) 9 mg/mL. Corneal endothelial cell toxicity was assessed by light microscopy (trypan blue staining) and transmission electron microscopy. The effects of 3-, 10-, or 30-minute exposures to 1/10 cTA, 1/10 –vTA, or V were also investigated. Results: One-minute exposures to –vTA or 1/10 –vTA did not damage CECs; however, cTA, V, or 1/10 dilutions of cTA or V caused damage and cells exposed to BA showed severe ultrastructural damage/lysis. A 30-minute exposure to 1/10 –vTA did not cause significant cell damage, whereas 3- to 30-minute exposures to 1/10 cTA or V showed significant time-dependent cytotoxicity. Conclusions: Commercial TA suspension was cytotoxic to cultured rabbit CECs because of the preservative, BA, in the vehicle. Because 1/10 –vTA appeared to be safe for up to 30 minutes of exposure, use of 1/10 dilutions of vehicle-removed TA is suggested to help surgeons visualize prolapsed vitreous during anterior vitrectomy in complicated cataract surgeries. [Copyright &y& Elsevier]

Abstract: Objectives: To establish the normal range of urine prothymosin-alpha in humans and to investigate its role as a specific tumor marker for the detection and follow-up of transitional cell carcinoma of the urinary bladder before and after curative treatment. Methods: Urine samples were obtained from 151 healthy volunteers, 60 patients with urinary tract infection, 238 patients with bladder transitional cell carcinoma (96 with tumor and 142 tumor free), and 22 patients with non-transitional cell carcinoma tumors. The urine prothymosin-alpha levels were quantified by enzyme-linked immunosorbent assay and then appropriately analyzed and compared among the different study groups. Results: The mean value of urine prothymosin-alpha in healthy volunteers was 0.68 ± 0.13 ng/mL. Regardless of the presence of urinary tract infection, the urine prothymosin-alpha level in patients with newly diagnosed, as yet untreated, bladder cancer was significantly greater than that in those who were tumor free after curative treatment (P = 0.050 and P = 0.026 for the presence and absence of urinary tract infection, respectively). At follow-up, the urine prothymosin-alpha level was constantly elevated when residual or recurrent tumor was present after treatment. Although the urine prothymosin-alpha level in patients with non-transitional cell carcinoma tumors was not significantly different from that of healthy volunteers, it was definitely lower than the level in patients with bladder tumors (P = 0.003). Conclusions: Our findings have revealed that urine prothymosin-alpha has the potential of being a useful tumor marker for the detection and follow-up of bladder cancer. [Copyright &y& Elsevier]

Abstract: In the current study, amine surface modified iron-oxide nanoparticles of 6nm diameter without polymer coating were fabricated in an aqueous solution by organic acid modification as an adherent following chemical coprecipitation. Structure and the superparamagnetic property of magnetite nanoparticles were characterized by selected area electron diffraction (SAED) and superconducting quantum interference measurement device (SQUID). X-ray photoelectron spectrometer (XPS) and zeta potential measurements revealed cationic surface mostly decorated with terminal –NH3 +. This feature enables them to function as a magnetic carrier for nucleotides via electrostatic interaction. In addition, Fe3O4/trypsin conjugates with well-preserved functional activity was demonstrated. The nanoparticles displayed excellent in vitro biocompatibility. The NMR and the in vitro MRI measurements showed significantly reduced water proton relaxation times of both T 1 and T 2. Significantly reduced T 2 and T 2*-weighted signal intensity were observed in a 1.5T clinical MR imager. In vivo imaging contrast effect showed a fast and prolonged inverse contrast effect in the liver that lasted for more than 1 week. In addition, it was found that the spherical Fe3O4 assembled as rod-like configuration through an aging process in aqueous solution at room temperature. Interestingly, TEM observation of the liver tissue revealed the rod-like shape but not the spherical-type nanoparticles being taken up by the Kupffer cells 120h after tail vein infusion. Combining these results, we have demonstrated the potential applications of the newly synthesized magnetite nanoparticles in a broad spectrum of biomedical applications. [Copyright &y& Elsevier]

Abstract: Objectives: To investigate the frequency and clinicopathologic correlates of ErbB1, ErbB2, and ErbB3 receptor expression in patients with upper urothelial carcinoma. Methods: Immunohistochemical staining for ErbB1, ErbB2, and ErbB3 was done with serial sections from archival specimens of 94 patients who underwent nephroureterectomy plus bladder cuff resection for urothelial carcinoma of the renal pelvis and ureter (median follow-up 40 months, range 1 to 177). The correlates between ErbB receptor expression and clinical outcome in terms of recurrence, progression, disease-free survival, and overall survival were analyzed by Kaplan-Meier plots, with the log-rank test and Cox proportional hazards model. Results: ErbB1, ErbB2, and ErbB3 expression was present in 9 (9.5%), 13 (13.8%), and 26 (27.7%) tumors, respectively. Thirty-seven patients (39.4 %) had at least one receptor expressed and eight (8.5%) had coexpression of two or three receptors. ErbB2 expression was significantly associated with tumor invasiveness (P = 0.03), and ErbB1 and ErbB3 expression was not. The incidence of subsequent tumor recurrence in the urinary bladder correlated significantly with ureteral tumor involvement and ErbB2 expression (P = 0.04 and P = 0.04, respectively). On univariate and multivariate analyses, tumor staging and ErbB2 expression were independent predictors of disease progression (P = 0.01 and P = 0.01, respectively), disease-free survival (P = 0.02 and P = 0.002, respectively), and overall survival (P = 0.01 and P = 0.02, respectively). Conclusions: ErbB2 expression predicts for disease progression and disease-related survival in upper urothelial carcinoma. [Copyright &y& Elsevier]

Abstract: Previously, we showed that murine prothymosin α (ProT) enhances the efficacy of a pseudorabies DNA vaccine delivered by bacterial vectors. In this study, we cloned and sequenced the cDNA for porcine ProT. The deduced amino acid sequence of porcine ProT exhibited high homology to ProT from other mammals. Oral Salmonella choleraesuis vaccine carrying the ProT eukaryotic expression plasmid protected mice against virulent S. choleraesuis challenge. The adjuvant effect of ProT on humoral and cellular immune responses enhanced protective efficacy of the vaccine. Furthermore, both humoral and cellular immune responses played roles in the protective immune responses induced by the vaccine. Collectively, our results show that delivery of the ProT gene carried by attenuated S. choleraesuis augmented the immunogenicity of oral S. choleraesuis vaccine. [Copyright &y& Elsevier]