Clinical efficacy of intravenous immunoglobulin therapy in critical ill patients with COVID-19: a multicenter retrospective cohort study.

OBJECTIVE: Coronavirus disease 2019 (COVID-19) outbreak is a major challenge all over the world, without acknowledged treatment. Intravenous immunoglobulin (IVIG) has been recommended to treat critical coronavirus disease 2019 (COVID-19) patients in a few reviews, but the clinical study evidence on its efficacy in COVID-19 patients was lacking. METHODS: 325 patients with laboratory-confirmed critical COVID-19 were enrolled from 4 government-designated COVID-19 treatment centres in southern China from December 2019 to March 2020. The primary outcomes were 28- and 60-day mortality, and the secondary outcomes were the total length of in-hospital and the total duration of the disease. Subgroup analysis was carried out according to clinical classification of COVID-19, IVIG dosage and timing. RESULTS: In the enrolled 325 patients, 174 cases used IVIG and 151 cases did not. The 28-day mortality was improved with IVIG after adjusting confounding in overall cohort (P = 0.0014), and the in-hospital and the total duration of disease were longer in the IVIG group (P < 0.001). Subgroup analysis showed that only in patients with critical type, IVIG could significantly reduce the 28-day mortality, decrease the inflammatory response and improve some organ functions (all P < 0.05); the application of IVIG in the early stage (admission ≤ 7 days) with a high dose (> 15 g per day) exhibited significant reduction in 60-day mortality in the critical-type patients. CONCLUSION: Early administration of IVIG with high dose improves the prognosis of critical-type patients with COVID-19. This study provides important information on clinical application of IVIG in the treatment of SARS-CoV-2 infection, including patient selection and administration dosage and timing.

Thymosin α1 therapy in critically ill patients with COVID-19: A multicenter retrospective cohort study.

BACKGROUND: COVID-19 characterized by refractory hypoxemia increases patient mortality because of immunosuppression effects. This study aimed to evaluate the efficacy of immunomodulatory with thymosin α1 for critical COVID-19 patients. METHODS: This multicenter retrospective cohort study was performed in 8 government-designated treatment centers for COVID-19 patients in China from Dec. 2019 to Mar. 2020. Thymosin α1 was administrated with 1.6 mg qd or q12 h for >5 days. The primary outcomes were the 28-day and 60-day mortality, the secondary outcomes were hospital length of stay and the total duration of the disease. Subgroup analysis was carried out according to clinical classification. RESULTS: Of the 334 enrolled COVID-19 patients, 42 (12.6%) died within 28 days, and 55 (16.5%) died within 60 days of hospitalization. There was a significant difference in the 28-day mortality between the thymosin α1 and non-thymosin α1-treated groups in adjusted model (P = 0.016), without obvious differences in the 60-day mortality and survival time in the overall cohort (P > 0.05). In the subgroup analysis, it was found that thymosin α1 therapy significantly reduced 28-day mortality (Hazards Ratios HR, 0.11, 95% confidence interval CI 0.02-0.63, P=0.013) via improvement of Pa02/FiO2 (P = 0.036) and prolonged the hospital length of stay (P = 0.024) as well as the total duration of the disease (P=0.001) in the critical type patients, especially those aged over 64 years, with white blood cell >6.8×109/L, neutrophil >5.3×109/L, lymphocyte < 0.73 × 109/L, PaO2/FiO2 < 196, SOFA > 3, and acute physiology and chronic health evaluation (APACHE) II > 7. CONCLUSION: These results suggest that treatment with thymosin α1 can markedly decrease 28-day mortality and attenuate acute lung injury in critical type COVID-19 patients.

COPS5 and LASP1 synergistically interact to downregulate 14-3-3σ expression and promote colorectal cancer progression via activating PI3K/AKT pathway.

Overexpression of LIM and SH3 protein 1 (LASP1) is required for colorectal cancer (CRC) development and progression. Here, C-Jun activation domain-binding protein-1 (Jab1), also known as COP9 signalosome subunit 5 (COPS5), was verified as a new LASP1-interacting protein through yeast two-hybrid assay. The role of COPS5 in LASP1-mediated CRC progression remains unknown. GST pull-down assay indicated that the SH3 domain of LASP1 could directly bind to MPN domain of COPS5. In vitro gain- and loss-of-function analyses revealed the stimulatory role of COPS5 on CRC cell proliferation, migration and invasion. Endogenous overexpression of COPS5 could also enhance the homing capacity of CRC cells in vivo. Further analysis showed that COPS5 and LASP1 synergistically interact to stimulate the ubiquitination and degradation of 14-3-3σ and promote colorectal cancer progression via PI3K/Akt dependent signaling pathway. Clinically, the expression of COPS5 was studied in CRC tissues and it is associated with CRC differentiation, metastasis and poor prognosis. The colocalization of LASP1 and COPS5 was demonstrated in both nonmetastatic and metastatic CRC tissues. A positive correlation was found between the expression of LASP1 and COPS5 while a negative correlation existed between 14-3-3σ and COPS5/LASP1 in most CRC samples. A combination of COPS5 and LASP1 tends to be an independent prognostic indicator for CRC patients, and this is also suitable for CRC without lymph node metastasis. The current research has further advanced our understanding on the complicated molecular mechanism underlying LASP1-mediated CRC progression, which hopefully will contribute to the development of novel diagnostic and therapeutic strategies in CRC.

MicroRNA-105 is involved in TNF-α-related tumor microenvironment enhanced colorectal cancer progression.

TNF-α is a central proinflammatory cytokine contributing to malignant tumor progression in tumor microenvironment. In this study, we found the upregulation of miR-105 in colorectal cancer was associated with aggressive phenotype, and the enhanced expression of miR-105 was required for TNF-α-induced epithelial-mesenchymal transition (EMT). The expression of miR-105 was remarkably stimulated by TNF-α in a time-dependent manner using real-time qPCR analysis. Inhibition of miR-105 remarkably weakened the aggressive effects of TNF-α through preventing the activation of NF-κB signaling and the initiation of EMT. Furthermore, miR-105 was demonstrated directly targeted on the 3'-UTRs of RAP2C, a Rap2 subfamily of small GTP-binding protein. Consistently, suppression of RAP2C stimulated the role of miR-105, which dramatically promoted the invasion and metastasis of CRC cells. Thalidomide, a TNF-α and NF-κB inhibitor, significantly weakened the metastasis and homing capacity of miR-105-overexpressed CRC cells in nude mice. Our investigation initiatively illustrated the modulatory role of miR-105 in TNF-α-induced EMT and further CRC metastasis. We also offer a better understanding of TNFα-induced metastasis and suggest an effective therapeutic strategy against CRC metastasis.

lncRNA uc009yby.1 promotes renal cell proliferation and is associated with poor survival in patients with clear cell renal cell carcinomas.

The expression and function of long non-coding RNAs (lncRNAs) in clear cell renal cell carcinoma (ccRCC) remains unclear. The present study measured the expression profiles of three lncRNAs (uc009yby.1, ENST00000514034, and ENST00000450687) using reverse transcription-quantitative polymerase chain reaction, and assessed their signatures in distinguishing ccRCC from matched normal tissues via analysis of receiver operating characteristic (ROC) curves. The expression of uc009yby.1 was inhibited by transfection of renal cells with small interfering RNA, and then the cell proliferation was evaluated by using a Cell Counting Kit-8. The results showed that the expressions of uc009yby.1 and ENST00000514034 were markedly increased in ccRCC compared with the matched normal tissues (P<0.0001 and P=0.0008, respectively), whereas the ENST00000450687 expression was not significantly altered. ROC curves yielded an area under the curve (AUC) value of 0.7000 for uc009yby.1, with sensitivity of 54.29% and specificity of 82.86%; and an AUC value of 0.6627 for ENST00000514034, with sensitivity of 60.00% and specificity of 67.14%. Furthermore, knockdown of uc009yby.1 suppressed renal cell proliferation (Day 0, P=0.7844; Day 1, P=0.0018; Day 2, P=0.0001; Day 3, P<0.000; Day 4, P<0.0001). Taken together, these findings suggest that the expression profiles of uc009yby.1 and ENST00000514034 may serve as novel biomarkers for ccRCC detection, and that uc009yby.1 is strongly associated with renal cell proliferation.

Renoprotective effects of ursolic acid on ischemia/reperfusion­induced acute kidney injury through oxidative stress, inflammation and the inhibition of STAT3 and NF­κB activities.

Ursolic acid, a pentacyclic triterpene compound with low toxicity and easy availability, has a variety of biological activities, including antitumor, antioxidant, antihepatitis, anti­inflammatory and antibacterial effects. The present study aimed to investigate the renoprotective effects of ursolic acid on ischemia/reperfusion­induced acute kidney injury (I/R­IAKI) in rats associated with its antioxidant and anti­inflammatory effects, as well as interference with the signal transducer and activator of transcription (STAT)3/nuclear factor (NF)­κB signaling pathway. The present study demonstrated that pre­treatment with ursolic acid significantly increased renal functioning and attenuated increases of serum angiotensin II levels in rats subjected to I/R­IAKI. In addition, I/R­IAKI­induced inflammation and oxidative stress were significantly reduced by pre­treatment with ursolic acid. Furthermore, ursolic acid significantly suppressed the upregulation of STAT3, NF­κB and caspase­3 activities in rats following I/R­IAKI. These results indicated that ursolic acid may be a potential drug for reducing I/R­IAKI through suppression of inflammation and oxidative stress damage, as well as modulation of STAT3 and NF­κB activities.

Loss of the 14-3-3σ is essential for LASP1-mediated colorectal cancer progression via activating PI3K/AKT signaling pathway.

LIM and SH3 protein 1 (LASP1) can promote colorectal cancer (CRC) progression and metastasis, but the direct evidence that elucidates the molecular mechanism remains unclear. Here, our proteomic data showed that LASP1 interacted with 14-3-3σ and decreased the expression of 14-3-3σ in CRC. Deletion of 14-3-3σ was required for LASP1-mediated CRC cell aggressiveness. In vitro gain- and loss-of-function assays showed that 14-3-3σ suppressed the ability of cell migration and decreased the phosphorylation of AKT in CRC cells. We further observed clearly co-localization between AKT and 14-3-3σ in CRC cells. Treatment of PI3K inhibitor LY294002 markedly prevented phosphorylation of AKT and subsequently counteract aggressive phenotype mediated by siRNA of 14-3-3σ. Clinically, 14-3-3σ is frequently down-regulated in CRC tissues. Down-regulation of 14-3-3σ is associated with tumor progression and poor prognosis of patients with CRC. Multivariate analysis confirmed low expression of 14-3-3σ as an independent prognostic factor for CRC. A combination of low 14-3-3σ and high LASP1 expression shows a worse trend with overall survival of CRC patients. Our research paves the path to future investigation of the LASP1-14-3-3σ axis as a target for novel anticancer therapies of advanced CRC.

LASP1-S100A11 axis promotes colorectal cancer aggressiveness by modulating TGFß/Smad signaling.

LIM and SH3 protein 1(LASP1) can promote colorectal cancer (CRC) progression and metastasis, but the mechanism remains unclear. Here, we show that LASP1 interacts with S100 calcium binding protein A11(S100A11) and enhances its expression in CRC. LASP1-S100A11 axis is essential for TGFß-mediated epithelial-mesenchymal transition (EMT) and cell aggressive phenotype. Clinically, S100A11 is overexpressed in CRC tissues and localized in both the cytoplasm and the nucleus of CRC cells. Overexpression of S100A11 in cytoplasmic and nuclear subcellular compartments is associated with tumor metastasis and poor prognosis of CRC patients. Introduction of cytoplasmic and nuclear S100A11 promotes aggressive phenotypes of CRC cells in vitro as well as growth and metastasis of CRC xenografts, whereas suppressing S100A11 abrogates these effects. Furthermore, we identify flotillin-1 (FLOT1) and histone H1 as downstream factors for cytoplasmic and nuclear pathway of S100A11, which are required for LASP1-S100A11 axis-mediated EMT and CRC progression. These findings indicate S100A11, combined with LASP1, plays a critical role in promoting CRC metastasis via its subcellular effectors, FLOT1 and histone H1.

Epigenetic silencing of miR-490-3p promotes development of an aggressive colorectal cancer phenotype through activation of the Wnt/ß-catenin signaling pathway.

The Wnt/ß-catenin pathway is known to contribute to colorectal cancer (CRC) progression, although little is known about the contribution of ß-catenin on this process. We investigated the role of miR-490-3p, which was recently reported to suppress tumorigenesis through its effect on Wnt/ß-catenin signaling. We found that hypermethylation of the miR-490-3p promoter down-regulates miR-490-3p expression in CRC tissue. Gain- and loss-of-function assays in vitro and in vivo reveal that miR-490-3p suppresses cancer cell proliferation by inducing apoptosis and inhibits cell invasiveness by repressing the initiation of epithelial-to-mesenchymal transition (EMT), a key mechanism in cancer cell invasiveness and metastasis. The frequently rearranged in advanced T-cell lymphomas (FRAT1) protein was identified as a direct target of miR-490-3p and contributes to its tumor-suppressing effects. miR-490-3p appears to have an inhibitory effect on ß-catenin expression in nuclear fractions of CRC cells, whereas FRAT1 expression is associated with the accumulation of ß-catenin in the nucleus of cells, which could be weakened by transfection with miR-490-3p. Our findings suggest that the miR-490-3p/FRAT1/ß-catenin axis is important in CRC progression and provides new insight into the molecular mechanisms underlying CRC. They may help to confirm the pathway driving CRC aggressiveness and serve for the development of a novel miRNA-targeting anticancer therapy.

MicroRNA-187, a downstream effector of TGFß pathway, suppresses Smad-mediated epithelial-mesenchymal transition in colorectal cancer.

Constitutive overactivation of TGFß signaling is a common event in human cancer progression and acts as a major inducer of epithelial-mesenchymal transition (EMT). In pre-metastatic colorectal cancer (CRC) cells, however, this cascade is tightly controlled and the underlying mechanism in TGFß stimulated hyperactivation of downstream Smad pathway remains elusive. In this study, expression of miR-187 was downregulated in colorectal cancer (CRC) compared with adjacent normal tissues. miR-187 could suppress the formation of aggressive phenotype in CRC and inactivate Smad pathway, thus preventing EMT. TGFß stimulation significantly suppressed the expression of miR-187, and overexpressed miR-187 counteracted the influence of TGFß on cell phenotype and downstream pathway. Furthermore, we found that miR-187 directly suppressed the expression of SOX4, NT5E and PTK6, which were identified as essential upstream effectors of Smad pathway. Together with the fact that high SOX4 or NT5E levels were associated with poor prognosis, we also demonstrated that downregulation of miR-187 was closely related to tumor metastasis and poor prognosis in CRC. These findings revealed a plausible mechanism for sustained TGFß activation in cancer progression and might have suggested a novel miR-187-based clinical intervention target for patients with advanced CRC.

LIM and SH3 protein 1 induces TGFß-mediated epithelial-mesenchymal transition in human colorectal cancer by regulating S100A4 expression.

PURPOSE: The expression of LIM and SH3 protein 1 (LASP1) was upregulated in colorectal cancer cases, thereby contributing to the aggressive phenotypes of colorectal cancer cells. However, we still cannot decipher the underlying molecular mechanism associated with colorectal cancer metastasis. EXPERIMENTAL DESIGN: In this study, IHC was performed to investigate the expression of proteins in human colorectal cancer tissues. Western blot analysis was used to assess the LASP1-induced signal pathway. Two-dimensional difference gel electrophoresis was performed to screen LASP1-modulated proteins and uncover the molecular mechanism of LASP1. TGFß was used to induce an epithelial-mesenchymal transition (EMT). RESULTS: LASP1 expression was correlated with the mesenchymal marker vimentin and was inversely correlated with epithelial markers, namely, E-cadherin and ß-catenin, in clinical colorectal cancer samples. The gain- and loss-of-function assay showed that LASP1 induces EMT-like phenotypes in vitro and in vivo. S100A4, identified as a LASP1-modulated protein, was upregulated by LASP1. Moreover, it is frequently coexpressed with LASP1 in colorectal cancer. S100A4 was required for EMT, and an increased cell invasiveness of colorectal cancer cell is induced by LASP1. Furthermore, the stimulation of TGFß resulted in an activated Smad pathway that increased the expression of LASP1 and S100A4. The depletion of LASP1 or S100A4 expression inhibited the TGFß signaling pathway. Moreover, it significantly weakened the proinvasive effects of TGFß on colorectal cancer cells. CONCLUSION: These findings elucidate the central role of LASP1 in the TGFß-mediated EMT process and suggest a potential target for the clinical intervention in patients with advanced colorectal cancer.

[Construction of a recombinant adenovirus vector for interferon-gamma-inducible protein 10 and the adenovirus preparation].

OBJECTIVE: To construct a recombinant adenovirus vector for expressing interferon-gamma-inducible protein 10 (IP-10) by homogenous bacterial recombination. METHODS: IP-10 gene was cloned into the shuttle plasmid pAdTrack-CMV that contained the coding sequence of enhanced green fluorescent protein (EGFP). The shuttle plasmid was then transformed into E. coli BJ5183 with pAdEasy-1 vector by chemical transformation. The recombinant adenovirus vector pAd/IP-10 was identified by enzyme digestion with Pac I and the linearized plasmid was transfected into HEK293 cells. RESULTS: The positive clones were identified with enzyme digestion and polymerase chain reaction (PCR) and were further verified by DNA sequencing. The recombinant adenovirus of high titration was obtained after transfection and packaging in HEK293 cells. CONCLUSION: A recombinant adenovirus vector for expression of IP-10 has been constructed successfully and high-titer active adenovirus is obtained for functional study of IP-10 protein.

[Cloning, expression and purification of His-tagged mouse mitochondrial transcription factor A and preparation of its polyclonal antibody].

OBJECTIVE: To express and purify the fusion protein of His-tagged mouse mitochondrial transcription factor A (mtTFA) in prokaryocytes and prepare rabbit anti-mtTFA polyclonal antibody. METHODS: The total RNA was extracted from mouse liver cells, and the coding sequence of mtTFA without signal peptides was amplified with reverse transcriptional (RT)-PCR. The PCR product was then cloned into the prokaryotic expression vector pET14b. After enzyme digestion and DNA sequencing, the plasmid was transformed into BL21(DE3) competent cells, and IPTG was used to induce the expression of His-tagged mtTFA. The expressed fusion protein was purified with Ni(2+)-NTA His-bind resin and quantified with Bradford method. RESULTS: The amplified product was confirmed as the coding sequence of mtTFA without signal peptides by DNA sequencing, and the recombinant expression vector could express His-tagged mtTFA in E.coil following the induction by IPTG. The fusion protein of high purity and rabbit anti-mtTFA serum with high specificity were obtained. CONCLUSIONS: The prokaryotic expression vector for mtTFA is successfully constructed and His-tagged mtTFA protein and specific polyclonal antibody are obtained, which can be helpful for further investigation of the biological function and signaling pathways involved in the regulation of functional cooperation between the nucleus and mitochondria.