Core and variable antimicrobial resistance genes in the gut microbiomes of Chinese and European pigs.

Monitoring the prevalence of antimicrobial resistance genes (ARGs) is vital for addressing the global crisis of antibiotic-resistant bacterial infections. Despite its importance, the characterization of ARGs and microbiome structures, as well as the identification of indicators for routine ARG monitoring in pig farms, are still lacking, particularly concerning variations in antimicrobial exposure in different countries or regions. Here, metagenomics and random forest machine learning were used to elucidate the ARG profiles, microbiome structures, and ARG contamination indicators in pig manure under different antimicrobial pressures between China and Europe. Results showed that Chinese pigs exposed to high-level antimicrobials exhibited higher total and plasmid-mediated ARG abundances compared to those in European pigs ( P<0.05). ANT(6)-Ib, APH(3')-IIIa, and tet(40) were identified as shared core ARGs between the two pig populations. Furthermore, the core ARGs identified in pig populations were correlated with those found in human populations within the same geographical regions. Lactobacillus and Prevotella were identified as the dominant genera in the core microbiomes of Chinese and European pigs, respectively. Forty ARG markers and 43 biomarkers were able to differentiate between the Chinese and European pig manure samples with accuracies of 100% and 98.7%, respectively. Indicators for assessing ARG contamination in Chinese and European pigs also achieved high accuracy ( r=0.72-0.88). Escherichia flexneri in both Chinese and European pig populations carried between 21 and 37 ARGs. The results of this study emphasize the importance of global collaboration in reducing antimicrobial resistance risk and provide validated indicators for evaluating the risk of ARG contamination in pig farms.

Probiotics formulation and cancer nanovaccines show synergistic effect in immunotherapy and prevention of colon cancer.

Probiotics play essential roles in immune modulation. Combining probiotics with cancer vaccines potentially can achieve a synergistic effect. To maximize the efficacy of probiotics, proper probiotics formulation is necessary. Herein, Lactobacillus rhamnosus and Bifidobacterium longum are coated with lipid membrane to achieve the goal of losing less activity and bettering colonization in colon. In the subcutaneous transplanted colon cancer mouse model, probiotics formulation showed potent preventive and therapeutic efficacy, and the efficacy could be further improved by combining with cancer nanovaccines. Probiotics formulation can perform as immune adjuvants to enhance the innate immune response or as in-situ cancer vaccines. In the study of preventing chemical-induced orthotopic colon cancer model, probiotics formulation alone efficiently reduced tumor number in colon and the efficacy is improved by combining with cancer nanovaccines. All in all, the studies demonstrated that probiotics formulation can assist to maximize the efficacy of cancer nanovaccines.

Rethinking Antigen Source: Cancer Vaccines Based on Whole Tumor Cell/tissue Lysate or Whole Tumor Cell.

Cancer immunotherapies have improved human health, and one among the important technologies for cancer immunotherapy is cancer vaccine. Antigens are the most important components in cancer vaccines. Generally, antigens in cancer vaccines can be divided into two categories: pre-defined antigens and unidentified antigens. Although, cancer vaccines loaded with predefined antigens are commonly used, cancer vaccine loaded with mixed unidentified antigens, especially whole cancer cells or cancer cell lysates, is a very promising approach, and such vaccine can obviate some limitations in cancer vaccines. Their advantages include, but are not limited to, the inclusion of pan-spectra (all or most kinds of) antigens, inducing pan-clones specific T cells, and overcoming the heterogeneity of cancer cells. In this review, the recent advances in cancer vaccines based on whole-tumor antigens, either based on whole cancer cells or whole cancer cell lysates, are summarized. In terms of whole cancer cell lysates, the focus is on applying whole water-soluble cell lysates as antigens. Recently, utilizing the whole cancer cell lysates as antigens in cancer vaccines has become feasible. Considering that pre-determined antigen-based cancer vaccines (mainly peptide-based or mRNA-based) have various limitations, developing cancer vaccines based on whole-tumor antigens is a promising alternative.

Lipid Microparticles Show Similar Efficacy With Lipid Nanoparticles in Delivering mRNA and Preventing Cancer.

PURPOSE: Messenger RNA (mRNA) has shown great promise for vaccine against both infectious diseases and cancer. However, mRNA is unstable and requires a delivery vehicle for efficient cellular uptake and degradation protection. So far, lipid nanoparticles (LNPs) represent the most advanced delivery platform for mRNA delivery. However, no published studies have compared lipid microparticles (LMPs) with lipid nanoparticles (LNPs) in delivering mRNA systematically, therefore, we compared the impact of particle size on delivery efficacy of mRNA vaccine and subsequent immune responses. METHODS: Herein, we prepared 3 different size lipid particles, from nano-sized to micro-sized, and they loaded similar amounts of mRNA. These lipid particles were investigated both in vitro and in vivo, followed by evaluating the impact of particle size on inducing cellular and humoral immune responses. RESULTS: In this study, all mRNA vaccines showed a robust immune response and lipid microparticles (LMPs) show similar efficacy with lipid nanoparticles (LNPs) in delivering mRNA and preventing cancer. In addition, immune adjuvants, either toll like receptors or active molecules from traditional Chinese medicine, can improve the efficacy of mRNA vaccines. CONCLUSIONS: Considering the efficiency of delivery and endocytosis, besides lipid nanoparticles with size smaller than 150 nm, lipid microparticles (LMPs) also have the potential to be an alternative and promising delivery system for mRNA vaccines.

Across-cancer specific immune responses induced by nanovaccines or microvaccines to prevent different cancers and cancer metastasis.

Metastatic cancers and recurrent cancers are diverse, different from primary cancers, and organ-dependent. However, how strong are across-cancer immune responses among different types of cancers remain unclear. Herein, vaccines-encapsulated-whole-components-of-tumor-tissue (VEWCOTT) were applied to demonstrate the across-cancer immune responses, thanks to inducing pan-clones T-cell immune responses. Either lung-cancer-tissue- or melanoma-tissue-based VEWCOTT simultaneously prevented melanoma, lung cancer, hepatoma, and metastatic cancer, which showed that strong across-cancer immune responses were induced. Both nanovaccines and microvaccines showed potent across-cancer prevention efficacy. VEWCOTT induced tumor-specific T cells in peripheral immune organs and major organs, and adjusted the immune-microenvironment of cancer-colonized organs. In addition, the allograft of T cells from VEWCOTT immunized mice to allogeneic naive mice efficiently prevent various cancers. Many neoantigens are shared by melanoma cells and lung cancer cells. Across-cancer immune responses exist among different types of cancers, and thus VEWCOTT has the advantage of simultaneously preventing cancer metastasis and cancers in different organs.

A Multiple-Target Simultaneous Detection Method for Immunosorbent Assay and Immunospot Assay.

Enzyme-linked immunosorbent assay (ELISA) is one of the most common methods in biological studies, and enzyme-linked immunospot (ELISpot) is a method to measure specific cell numbers by detecting protein secretion at a single-cell level. However, these two current methods can only detect one signal at one time and the sensitivity is not high enough to test low-concentration samples, which are major shortcomings in systematically analyzing the samples of interest. Herein, we demonstrated fluorescence-based oligo-linked immunosorbent assay (FOLISA) and fluorescence-based oligo-linked immunospot (FOLISPOT), which utilized DNA-barcoded antibodies to provide a highly multiplexed method with signal amplification. Signal amplification and simultaneous multiple-target detection were achieved by DNA complementary pairing and modular orthogonal DNA concatemers. By comparing FOLISA with traditional ELISA and comparing FOLISPOT with traditional ELISPOT, we found that the detection sensitivities of FOLISA and FOLISPOT are much higher than those of traditional ELISA and ELISPOT. The detection limit of ELISA is around 3 pg/mL, and the detection limit of FOLISA is below 0.06 pg/mL. FOLISPOT can detect more spots than ELISPOT and can detect targets that are undetectable for ELISPOT. Furthermore, FOLISA and FOLISPOT allowed sequential detection of multiple targets by using a single dye or multiple dyes in one round and sequential detection in multiple rounds. Thus, FOLISA and FOLISPOT enabled simultaneous detection of a large number of targets, significantly improved the detection sensitivity, and overcame the shortcomings of ELISA and ELISPOT. Overall, FOLISA and FOLISPOT presented effective and general platforms for rapid and multiplexed detection of antigens or antibodies with high sensitivity, either in laboratory tests or potentially in clinic tests.

Therapeutic effects of cationic liposomes on lupus-prone MRL/lpr mice are mediated via inhibition of TLR4-triggered B-cell activation.

We previously reported that co-delivery of dihydroartemisinin and high mobility group box 1 (HMGB1) siRNAs, using cell penetrating peptide (TAT)-modified cationic liposomes (TAT-CLs-DHA/siRNA), resulted in promising activity for the treatment of inflammatory disease through TLR4 signaling pathway. In the current study, we further investigated the therapeutic effects of TAT-CLs-DHA/siRNA on lupus-prone MRL/lpr mice and explored its effects on B cell responses. In vitro, we found that TAT-CLs-DHA/siRNA suppressed the proliferation and activation of B cells through the TLR4 signaling pathway. Following parenteral administration every 4 days, TAT-CLs-DHA/siRNA significantly reduced proteinuria, glomerulonephritis, serum anti-dsDNA antibody and secretion of interleukin (IL)-6, IL-10, IL-17 and IL-21. Moreover, Western blotting showed that TAT-CLs-DHA/siRNA modulated the B-cell intrinsic pathway by downregulating expression of HMGB1, TLR4, MyD88 and NF-κB. This co-delivery system thus represents a promising treatment option for lupus nephritis, and also highlights a novel target of lupus treatment through B cell TLR4 signal pathway.

Immunotherapy and Prevention of Cancer by Nanovaccines Loaded with Whole-Cell Components of Tumor Tissues or Cells.

Tumor tissues/cells are the best sources of antigens to prepare cancer vaccines. However, due to the difficulty of solubilization and delivery of water-insoluble antigens in tumor tissues/cells, including water-insoluble antigens into cancer vaccines and delivering such vaccines efficiently to antigen-presenting cells (APCs) remain challenging. To solve these problems, herein, water-insoluble components of tumor tissues/cells are solubilized by 8 m urea and thus whole components of micrometer-sized tumor cells are reasssembled into nanosized nanovaccines. To induce maximized immunization efficacy, various antigens are loaded both inside and on the surface of nanovaccines. By encapsulating both water-insoluble and water-soluble components of tumor tissues/cells into nanovaccines, the nanovaccines are efficiently phagocytosed by APCs and showed better therapeutic efficacy than the nanovaccine loaded with only water-soluble components in melanoma and breast cancer. Anti-PD-1 antibody and metformin can improve the efficacy of nanovaccines. In addition, the nanovaccines can prevent lung cancer (100%) and melanoma (70%) efficiently in mice. T cell analysis and tumor microenvironment analysis indicate that tumor-specific T cells are induced by nanovaccines and both adaptive and innate immune responses against cancer cells are activated by nanovaccines. Overall, this study demonstrates a universal method to make tumor-cell-based nanovaccines for cancer immunotherapy and prevention.

[Comparison of pulse oxygen saturation/fraction of inhaled oxygen and arterial partial pressure of oxygen/fraction of inhaled oxygen in the assessment of oxygenation in acute respiratory distress syndrome patients at different high altitudes in Yunnan Province].

OBJECTIVE: To investigate and evaluate if pulse oxygen saturation/fraction of inhaled oxygen (SpO2/FiO2) can be used, as replacement of arterial partial pressure of oxygen/fraction of inhaled oxygen (PaO2/FiO2), to assess oxygenation in acute respiratory distress syndrome (ARDS) patients at different high altitudes in Yunnan Province, and to find a rapid and non-invasive method for the diagnosis of ARDS at different altitudes. METHODS: Patients with ARDS at different high altitudes in Yunnan Province from January 2019 to December 2020 were enrolled. The patients were divided into three groups according to different altitudes, and received different oxygen therapies according to their respective medical conditions. Group 1 consisted of patients with moderate to severe ARDS from the department of critical care medicine of the First Affiliated Hospital of Kunming Medical University (average altitude approximately 1 800 m), and received mechanical ventilation to maintain SpO2 of 0.90-0.96 with a low FiO2 for more than 30 minutes, and SpO2, FiO2, PaO2 were recorded. Group 2 consisted of patients with moderate to severe ARDS at the department of critical care medicine of People's Hospital of Diqing Tibetan Autonomous Prefecture (mean altitude about 3 200 m), and received oxygen with an attached reservoir mask to maintain SpO2 of 0.90-0.96 for 10 minutes, and then SpO2, FiO2, and PaO2 were recorded. Group 3 consisted of patients with mild to moderate-severe ARDS who admitted to the emergency department of the People's Hospital of Lijiang (average altitude approximately 2 200 m); when SpO2 < 0.90, patients received oxygen with the oxygen storage mask, and the FiO2 required to maintain SpO2 ≥ 0.90 was recorded, and SpO2, FiO2, PaO2 were recorded after oxygen inhalation for 10 minutes. Spearman coefficient was used to analyze the correlation between SpO2/FiO2 and PaO2/FiO2 in each group. Linear analysis was used to derive the linear equation between SpO2/FiO2 and PaO2/FiO2, and to evaluate arterial pH, arterial partial pressure of carbon dioxide (PaCO2), FiO2, tidal volume (VT), positive end-expiratory pressure (PEEP) and other related factors which would change the correlation between SpO2/FiO2 and PaO2/FiO2. The receiver operator characteristic curve (ROC curve) was plotted to calculate the sensitivity and specificity of using SpO2/FiO2 instead of PaO2/FiO2 to assess oxygenation of ARDS patients. RESULTS: Group 1 consisted of 24 ARDS patients from whom 271 blood gas analysis results were collected; group 2 consisted of 14 ARDS patients from whom a total of 47 blood gas analysis results were collected; group 3 consisted of 76 ARDS patients, and a total of 76 blood gas analysis results were collected. The PaO2/FiO2 (mmHg, 1 mmHg = 0.133 kPa) in groups 1, 2 and 3 were 103 (79, 130), 168 (98, 195) and 232 (146, 271) respectively, while SpO2/FiO2 were 157 (128, 190), 419 (190, 445) and 319 (228, 446) respectively. Among the three groups, patients in group 1 had the lowest PaO2/FiO2 and SpO2/FiO2, while patients in group 3 had the highest. Spearman correlation analysis showed that PaO2/FiO2 was highly correlated with SpO2/FiO2 in groups 1, 2 and 3 (r values were 0.830, 0.951, 0.828, all P < 0.05). Regression equation was fitted according to linear analysis: in group 1 SpO2/FiO2 = 58+0.97×PaO2/FiO2 (R2 = 0.548, P < 0.001); in group 2 SpO2/FiO2 = 6+2.13×PaO2/FiO2 (R2 = 0.938, P < 0.001); in group 3 SpO2/FiO2 = 53+1.33×PaO2/FiO2 (R2 = 0.828, P < 0.001). Further analysis revealed that PEEP, FiO2, and arterial blood pH could affect the correlation between SpO2/FiO2 and PaO2/FiO2. ROC curve analysis showed that the area under ROC curve (AUC) was 0.848 and 0.916 in group 1 with moderate to severe ARDS; based on the regression equation, the corresponding SpO2/FiO2 cut-off values at a PaO2/FiO2 of 100 mmHg and 200 mmHg were 155, 252 with a sensitivity of 84.9% and 100%, specificity of 87.2% and 70.6%, respectively. Patients with moderate to severe ARDS in group 2 (AUC was 0.945 and 0.977), the corresponding SpO2/FiO2 cut-off values at PaO2/FiO2 of 100 mmHg and 200 mmHg were 219 and 432 with the sensitivity of 100% and 85.2%, specificity of 82.5% and 100%, respectively. Patients with mild to moderate-severe ARDS in group 3 (AUC was 0.903 and 0.936), the corresponding SpO2/FiO2 cut-off values at a PaO2/FiO2 of 200 mmHg and 300 mmHg were 319 and 452 with the sensitivity of 100% and 100%, specificity of 80.9% and 86.2%, respectively. CONCLUSIONS: SpO2/FiO2 and PaO2/FiO2 in ARDS patients at different high altitudes in Yunnan Province have a good correlation, and non-invasive SpO2/FiO2 can be used to replace PaO2/FiO2 to assess the oxygenation in ARDS patients.

[Feasibility of pulse oxygen saturation for the condition evaluation of acute respiratory distress syndrome patients in the Lijiang region].

OBJECTIVE: To explore the feasibility of using pulse oxygen saturation (SpO2) to evaluate the condition of patients with acute respiratory distress syndrome (ARDS) in the Lijiang region. METHODS: Patients with ARDS who visited the department of emergency of People's Hospital of Lijiang from August to December 2020 were selected as study subjects. Patients were divided by severity into mild ARDS group [200 mmHg (1 mmHg = 0.133 kPa) ≤ oxygenation index (PaO2/FiO2, P/F) ≤ 300 mmHg] and moderate to severe ARDS group (P/F ≤ 200 mmHg). The general condition, clinical diagnosis, arterial blood gas analysis results of the patients were recorded, and the differences of the above indexes between the two groups of ARDS were compared. Spearman correlation analysis was used to analyze the correlation between SpO2 and arterial oxygen saturation (SaO2). SpO2 was carried into the Ellis equation and the Rice equation to calculate the derived P/F and analyze the correlation between the derived P/F and the P/F measured in arterial blood gas analysis; receiver operator characteristic curve (ROC curves) were plotted, the sensitivity and specificity of SpO2/fraction of inspiration oxygen (SpO2/FiO2, S/F) instead of P/F to assess oxygenation in patients with ARDS was calculated. To evaluate the feasibility of SpO2 for the condition evaluation of patients with ARDS in the Lijiang region. RESULTS: Compared with the mild ARDS group, the arterial partial pressure of oxygen (PaO2), SaO2 and hemoglobin (Hb) were significantly decreased in the moderate to severe ARDS group [PaO2 (mmHg): 50.5 (39.3, 56.5) vs. 60.0 (55.0, 67.5), SaO2: 0.86 (0.73, 0.91) vs. 0. 93 (0.90, 0.96), Hb (g/L): 142±27 vs. 156±24, respectively, all P < 0.05]. Correlation analysis revealed a significant positive correlation between SpO2 and SaO2 in ARDS patients residing at high altitude (R = 0.650, P = 0.000). The P/F derived by the Rice formula was significantly and positively correlated with the P/F derived from arterial blood gas analysis (R = 0.802, P = 0.000). The deduced P/F in mild and moderate to severe ARDS groups were all significantly correlated with the measured P/F (R values were 0.562, 0.647, both P = 0.000). The P/F derived using the Ellis formula showed a significant positive correlation with the P/F derived from arterial blood gas analysis (R = 0.822, P = 0.000). The deduced P/F of mild ARDS group and moderate to severe ARDS group were all positively correlated with the measured P/F (R values were 0.556, 0.589, P values were 0.000, 0.010). There was a significant positive correlation between S/F and P/F in ARDS patients (R = 0.828, P = 0.000), and the regression equation was S/F = 1.33 P/F+52.41. ROC curve analysis showed that S/F had some predictive value for patients with mild and moderate to severe ARDS, and area under ROC curve (AUC) and 95% confidence interval (95%CI) were 0.903 (0.829-0.977), 0.936 (0.870-1.000), both P = 0.000. When the cut-off value was 452 mmHg, S/F had a sensitivity of 100% and a specificity of 80.9% for predicting mild ARDS. When the cut-off value was 319 mmHg, S/F predicted moderate to severe ARDS with 95.1% sensitivity and 86.2% specificity. CONCLUSIONS: At high altitude, SpO2 and SaO2 have been correlated in patients with ARDS, and P/F derived using SpO2 and measured P/F were significantly correlated in patients with ARDS, especially in those with moderate to severe ARDS. SpO2 may be useful in the assessment of severity of illness in patients with ARDS at high altitude.

pH-Sensitive Nanoparticles Codelivering Docetaxel and Dihydroartemisinin Effectively Treat Breast Cancer by Enhancing Reactive Oxidative Species-Mediated Mitochondrial Apoptosis.

Tumor growth and metastasis are the major causes of high mortality in breast cancer. We previously constructed pH-sensitive nanoparticles (D/D NPs) for the codelivery of docetaxel (DTX) and dihydroartemisinin (DHA) and demonstrated that D/D NPs showed anticancer activity in breast cancer cells in vitro. The present study further investigated the therapeutic effect of D/D NPs on orthotopic breast cancer in vivo and examined the antitumor mechanism of D/D NPs. D/D NPs significantly increased the apoptosis of 4T1 cells with a synergistic effect of DTX and DHA. D/D NPs increased reactive oxygen species, reduced mitochondrial membrane potential, increased the expression of p53, and induced cytochrome c release into the cytoplasm to activate caspase-3. In an orthotopic metastatic breast cancer mouse model derived from 4T1 cells, D/D NPs inhibited tumor growth and prevented lung metastasis due to the synergistic effect of DTX and DHA. No distinct changes were observed in the histology of major organs. These results indicate that pH-sensitive D/D NP-based combination therapy may be a promising strategy for the treatment of metastatic breast cancers via the ROS-mediated mitochondrial apoptosis pathway.

Injectable Chitosan-Based Thermosensitive Hydrogel/Nanoparticle-Loaded System for Local Delivery of Vancomycin in the Treatment of Osteomyelitis.

PURPOSE: Osteomyelitis, particularly chronic osteomyelitis, remains a major challenge for orthopedic surgeons. The traditional treatment for osteomyelitis, which involves antibiotics and debridement, does not provide a complete solution for infection and bone repair. Antibiotics such as vancomycin (VCM) are commonly used to treat osteomyelitis in clinical settings. VCM use is limited by a lack of effective delivery methods that provide sustained, high doses to entirely fill irregular bone tissue to treat infections. METHODS: We engineered a chitosan (CS)-based thermosensitive hydrogel to produce a VCM-nanoparticle (NPs)/Gel local drug delivery system. The VCM-NPs were formed with quaternary ammonium chitosan and carboxylated chitosan nanoparticles (VCM-NPs) by positive and negative charge adsorption to enhance the encapsulation efficiency and drug loading of VCM, with the aim of simultaneously preventing infection and repairing broken bones. This hydrogel was evaluated in a rabbit osteomyelitis model. RESULTS: The VCM-NPs had high encapsulation efficiency and drug loading, with values of 60.1±2.1% and 24.1±0.84%, respectively. When embedded in CS-Gel, the VCM-NPs maintained their particle size and morphology, and the injectability and thermosensitivity of the hydrogel, which were evaluated by injectability test and rheological measurement, were retained. The VCM-NPs/Gel exhibited sustained release of VCM over 26 days. In vitro tests revealed that the VCM-NPs/Gel promoted osteoblast proliferation and activity against Staphylococcus aureus. In vivo, VCM-NPs/Gel (with 10 mg vancomycin per rabbit) was used to treat rabbits with osteomyelitis. The VCM-NPs/Gel showed excellent anti-infection properties and accelerating bone repair under osteomyelitis conditions. CONCLUSION: The reported multifunctional NPs hydrogel system for local antibiotic delivery (VCM-NPs/Gel) showed bone regeneration promotion and anti-infection properties, demonstrating significant potential as a scaffold for effective treatment of osteomyelitis.

[Clinical analysis of 32 adult patients with infection-associated hemophagocytic syndrome].

OBJECTIVE: To analyze the clinical feature of adult patients with infection-associated hemophagocytic syndrome (IAHS), and to improve the ability of clinicians to diagnose and treat IAHS. METHODS: A retrospectively study was performed. The clinical data of 32 adult patients with IAHS admitted to the intensive care unit (ICU) of the First Affiliated Hospital of Kunming Medical University from July 2014 to November 2019 were analyzed. The general data, clinical manifestations, laboratory results, imaging findings, pathogen and clinical outcomes were collected, and the patients were divided into survival group and death group according to the 28-day prognosis. The clinical data between the two groups were compared, and multivariate Logistic regression analysis was used to analyze the variables with statistical significance in univariate analysis. The receiver operating characteristic (ROC) curve was drawn to analyze the predictive value of variables with statistical significance in univariate analysis for 28-day prognosis of adult patients with IAHS. RESULTS: Among 32 adult patients with IAHS, there were 17 males (53.1%) and 15 females (46.9%). Eighteen patients were bacterial infection, most of which were Acinetobacter baumannii and Escherichia coli; 14 patients were viral infection, mainly EB virus; and the overall 28-day mortality was 62.5% (20/32). (1) Compared with the survival group (n = 12), the levels of white blood cell (WBC), neutrocyte (NEU), lymphocyte (LYM), platelet (PLT) and oxygenation index (OI) in the death group (n = 20) were lower, while the levels of aspartate aminotransferase (AST), K+, serum ferritin (SF) and lactate dehydrogenase (LDH) were higher [WBC (×109/L): 3.90±3.36 vs. 9.57±6.48, NEU (×109/L): 2.69±2.09 vs. 7.01±6.34, LYM (×109/L): 0.36 (0.23, 0.84) vs. 1.24 (0.61, 2.36), PLT (×109/L): 51.15±27.60 vs. 108.42±80.26, OI (mmHg, 1 mmHg = 0.133 kPa): 134.0 (77.5, 192.0) vs. 292.0 (187.0, 329.0), AST (U/L): 254.00 (67.80, 452.50) vs. 85.50 (38.38, 111.25), K+ (mmol/L): 4.06 (3.65, 4.51) vs. 3.52 (3.26, 3.76), SF (µg/L): 6 290.0 (1 851.0, 13 904.8) vs. 1 777.1 (1 228.5, 3 486.3), LDH (µmol×s-1×L-1): 19.3 (11.9, 27.0) vs. 9.8 (6.9, 11.1), all P < 0.05]. In death group, duration of having a fever after admission was prolonged [days: 13.5 (9.0, 17.2) vs. 6.0 (2.5, 8.0), P < 0.05] and the incidence of cyanosis was higher (40.0% vs. 0%, P < 0.05). There was no significant difference in other indicators between the two groups. (2) Multivariate Logistic regression analysis showed that low OI combined with high LDH were risk factors for 28-day mortality of adult patients with IAHS [odds ratio (OR) was 0.967 and 1.007, respectively, both P < 0.05]. (3) It was shown by ROC curve analysis that WBC, NEU, AST, SF, LDH and OI had predictive value for 28-day prognosis of adult patients with IAHS (both P < 0.05), and the area under ROC curve (AUC) of OI and LDH was higher, that was both 0.847. When the best cut-off of OI was 145.5 mmHg, the sensitivity was 63.2%, and the specificity was 100%. When the best cut-off of LDH was 13.4 µmol×s-1×L-1, the sensitivity was 72.2%, and the specificity was 91.7%. CONCLUSIONS: OI < 145.5 mmHg, and LDH > 13.4 µmol×s-1×L-1 were significant predictors for poor 28-day prognosis of adult patients with IAHS.

Co-Delivery Of Dihydroartemisinin And HMGB1 siRNA By TAT-Modified Cationic Liposomes Through The TLR4 Signaling Pathway For Treatment Of Lupus Nephritis.

BACKGROUND AND PURPOSE: Systemic lupus erythematous (SLE) is an autoimmune disease caused by many factors. Lupus nephritis (LN) is a common complication of SLE and represents a major cause of morbidity and mortality. Previous studies have shown the advantages of multi-targeted therapy for LN and that TLR4 signaling is a target of anti-LN drugs. High-mobility group box 1 (HMGB1), a nuclear protein with a proinflammatory cytokine activity, binds specifically to TLR4 to induce inflammation. We aimed to develop PEGylated TAT peptide-cationic liposomes (TAT-CLs) to deliver anti-HMGB1 siRNA and dihydroartemisinin (DHA) to increase LN therapeutic efficiency and explore their treatment mechanism. METHODS: We constructed the TAT-CLs-DHA/siRNA delivery system using the thin film hydration method. The uptake and localization of Cy3-labeled siRNA were detected by confocal microscopy and flow cytometry. MTT assays were used to detect glomerular mesangial cell proliferation. Real-time PCR, Western blot analysis, and ELISA evaluated the anti-inflammatory mechanism of TAT-CLs-DHA/siRNA. RESULTS: We constructed the TAT-CLs-DHA/siRNA delivery system measuring approximately 140 nm with superior storage and serum stabilities. In vitro, it showed significantly greater uptake compared with unmodified liposomes and significant inhibition of glomerular mesangial cell proliferation. TAT-CLs-DHA/siRNA inhibited NF-κB activation in a concentration-dependent manner. Real-time PCR and Western blot analysis showed that TAT-CLs-DHA/siRNA downregulated expression of HMGB1 mRNA and protein. TAT-CLs-DHA/siRNA markedly diminished Toll-like receptor 4 (TLR4) expression and subsequent activation of MyD88, IRAK4, and NF-κB. CONCLUSION: TAT-CLs-DHA/siRNA may have the potential for treatment of inflammatory diseases such as LN mediated by the TLR4 signaling pathway.

CD44-targeted hyaluronic acid-curcumin reverses chemotherapeutics resistance by inhibiting P-gp and anti-apoptotic pathways.

Chemotherapeutic drug resistance poses a great challenge in cancer therapy. Drug efflux and anti-apoptotic processes are the two most common mechanisms leading to chemotherapy resistance. In this study, we focused on the applicability of curcumin (CUR) as a sensitizer for chemotherapeutics (doxorubicin [DOX] as the model drug) modified with hyaluronic acid (HA) as an effective therapeutic strategy against multidrug resistance (MDR) in cancer cells. We constructed an HA-CUR/DOX delivery system measuring approximately 180 nm with superior encapsulation efficacy and serum stabilities. In vitro, we found that HA modification could facilitate the efficient delivery of chemotherapeutics through CD44 receptor-mediated targeted delivery. MTT assay results confirmed that the combination of CUR and DOX/paclitaxel (PTX) had a significant synergistic effect and significantly reversed MDR. Further experiments including real-time polymerase chain reaction and western blotting proved that the main mechanisms by which CUR reversed MDR in tumor cells were inhibiting the expression and activity of P-glycoprotein (P-gp) and inducing apoptosis through mitochondrial pathway. Taken together, our new engineered tumor-targeting nanoparticle delivery system may have the potential for overcoming MDR in cancer.

Co-delivery of dihydroartemisinin and docetaxel in pH-sensitive nanoparticles for treating metastatic breast cancer via the NF-κB/MMP-2 signal pathway.

Metastasis is a major barrier in cancer chemotherapy. Prolonged circulation and rapid, specific intracellular drug release are two main goals in the development of nanoscale drug delivery systems to treat metastatic breast cancer. In this study, we investigated the anti-metastasis effect of docetaxel (DTX) in combination with dihydroartemisinin (DHA) in metastatic breast cancer 4T1 cells. We synthesized a pH-sensitive material 4-arm-PEG-DTX with a hydrazone bond and used it to construct nanoparticles that co-deliver DTX and DHA (D/D NPs). The D/D NPs had a mean size of 142.5 nm and approximately neutral zeta potential. The pH-sensitive nanoparticles allowed acid-triggered drug release at the tumor site, showing excellent cytotoxicity (IC50 = 7.0 µg mL-1), cell cycle arrest and suppression of cell migration and invasion. The mechanisms underlying the anti-metastasis effect of the D/D NPs involved downregulation of the expression of p-AKT, NF-κB and MMP-2. Therefore, D/D NPs represent a new nanoscale drug delivery system for treating metastatic breast cancer, responding to the acidic tumor microenvironment to release the chemotherapeutic drugs.

Combined functional MRI and diffusion tensor imaging analysis of visual motion pathways.

BACKGROUND: Motion perception may be preserved after damage to striate cortex (primary visual cortex, area V1). Awareness and normal discrimination of fast-moving stimuli have been observed even in the complete absence of V1. These facts suggest that motion-sensitive cortex (the V5/MT complex or V5/MT+) may be activated by direct thalamic or collicular inputs that bypass V1. Such projections have been identified previously in monkeys but have not been shown in humans using neuroimaging techniques. METHODS: We used diffusion tensor imaging (DTI) tractography to visualize white matter fiber tracts connecting with V5/MT+ in 10 healthy volunteers. V5/MT+ was localized for each subject using functional MRI (fMRI). Functional activity maps were overlaid on high-resolution anatomical images and registered with the diffusion-weighted images to define V5/MT+ as the region of interest (ROI) for DTI tractography analysis. Fibers connecting to V1 were excluded from the analysis. RESULTS: Using conservative tractography parameters, we found connections between the V5/MT+ region and the posterior thalamus and/or superior colliculus in 4 of 10 subjects. CONCLUSIONS: Connections between the V5/MT+ region and the posterior thalamus and/or superior colliculus may explain visual motion awareness in the absence of a functioning V1.

[Expression and mutation of Fas-associated death domain (FADD) gene in non-small cell lung cancer].

BACKGROUND & OBJECTIVE: Abnormal expression of Fas-associated death domain (FADD) protein, an important adapter in cell apoptosis signal conduction, may closely relate with tumorigenesis. This study was to detect expression and mutation of FADD gene in non-small cell lung cancer (NSCLC), evaluate its effect on development of NSCLC, and explore the mechanism. METHODS: Polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) was used to detect FADD gene mutation in 62 specimens of NSCLC tissues and 13 specimens of adjacent non-cancerous lung tissues. Immunohistochemistry was used to detect its protein expression. In situ hybridization (ISH) was used to detect FADD mRNA expression in 30 of the 62 specimens of NSCLC tissues. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick labeling (TUNEL) was used to detect apoptotic cells in NSCLC tissues. RESULTS: Of the 62 specimens of NSCLC tissues, 4 cases of stage N2 showed FADD gene mutation. Positive rate of FADD protein in NSCLC tissues was 80.6% (50/62), its protein level positively correlated with differentiation of NSCLC (rs=0.411, P<0.01). Protein level of FADD in NSCLC tissue was significantly higher than that in non-cancerous tissue (P<0.05). Positive rate of FADD mRNA in NSCLC tissue was 80.0%, its concordant rate with positive rate of FADD protein was 88.6% (P>0.05). Apoptotic cells were observed in all specimens of NSCLC, apoptosis indexes of the 4 cases with FADD gene mutation were lower than the mean level, although they showed positive expression of FADD protein. Protein level of FADD was positively related with cell apoptosis of NSCLC (rs=0.599, P<0.001). CONCLUSIONS: FADD gene mutation exists in NSCLC, its mutation and abnormal expression might play a crucial role in carcinogenesis of NSCLC. Protein level of FADD closely correlates with cell apoptosis of NSCLC.

Expression levels and significance of hypoxia inducible factor-1 alpha and vascular endothelial growth factor in human colorectal adenocarcinoma.

BACKGROUND: Hypoxia-inducible factor 1 (HIF-1), a transcription factor, is overexpressed in common human cancers and their metastases. This study aimed at determining the expression levels of HIF-1alpha and vascular endothelial growth factor (VEGF) in SW480 cells and in colorectal adenocarcinoma tissue and ascertaining whether HIF-1alpha and VEGF play important roles in tumor angiogenesis. METHODS: HIF-1alpha mRNA expression was analyzed using in situ hybridization and RT-PCR. HIF-1alpha and VEGF protein were detected in SW480 cells and colorectal adenomas and adenocarcinomas by immunohistochemistry using streptavidin/peroxidase (SP). Western blot was used to detect HIF-1alpha protein extracted from SW480 cells. Microvessel density (MVD) in colorectal carcinomas was determined by anti-CD34 immunostaining in colorectal carcinomas. RESULTS: Optical density values representing HIF-1alpha mRNA expression levels were found to be significantly higher in SW480 cells in hypoxic conditions than in cells under normoxic conditions (P < 0.05) or in hypoxic conditions but treated with genistein (P < 0.05). The levels of HIF-1alpha and VEGF protein expression in SW480 cells were significantly higher in the hypoxia group than in the normoxia group (P < 0.01, P < 0.05, respectively) and hypoxia/genistein group (P < 0.01, P < 0.05, respectively). The positive expression rates of HIF-1alpha mRNA changed dramatically when comparing colorectal adenomas with adenocarcinomas of different Dukes' stages (P < 0.05). HIF-1alpha mRNA was also expressed at higher levels in adenocarcinomas than that in adenomas (P < 0.01). HIF-1alpha protein expression correlated significantly with VEGF protein expression and MVD. CONCLUSIONS: Hypoxia induces the expression of HIF-1alpha and VEGF in colorectal adenocarcinomas. HIF-1alpha may play an important role in angiogenesis and tumor progression by regulating the expression of VEGF in human colorectal carcinomas.