A Convenient and Effective Preoxygenation Technique for Prolonging Deep Inspiration Breath-Hold Duration With a Venturi Mask With a 50% Oxygen Concentration.

PURPOSE: Voluntary deep inspiration breath-hold (DIBH) is commonly used in radiation therapy (RT), but the short duration of a single breath-hold, estimated to be around 20 to 40 seconds, is a limitation. This prospective study aimed to assess the feasibility and safety of using a simple preoxygenation technique with a Venturi mask to prolong voluntary DIBH. METHODS AND MATERIALS: The study included 33 healthy volunteers and 21 RT patients. Preoxygenation was performed using a Venturi mask with a 50% oxygen concentration. Paired t tests compared the duration of a single DIBH in room air and after 5, 15, and 30 minutes of preoxygenation in healthy volunteers. Sustainability of breath-hold and tolerability of heart rate and blood pressure were assessed for multiple DIBH durations in both volunteers and patients. RESULTS: In healthy volunteers, a 15-minute preoxygenation significantly prolonged the duration of a single DIBH by 24.95 seconds compared with 5-minute preoxygenation (89 ± 27.76 vs 113.95 ± 30.63 seconds; P < .001); although there was a statistically significant increase in DIBH duration after 30-minute preoxygenation, it was only extended by 4.95 seconds compared with 15-minute preoxygenation (113.95 ± 30.63 vs 118.9 ± 29.77 seconds; P < .01). After 15-minute preoxygenation, a single DIBH lasted over 100 seconds in healthy volunteers and over 80 seconds in RT patients, with no significant differences among 6 consecutive cycles of DIBH. Furthermore, there were no significant differences in heart rate or blood pressure after DIBHs, including DIBH in room air and 6 consecutive DIBHs after 15-minute preoxygenation (all P > .05). CONCLUSIONS: Preoxygenation with a 50% oxygen concentration for 15 minutes effectively prolongs the duration of 6 cycles of DIBH both in healthy volunteers and RT patients. The utilization of a Venturi mask to deliver 50% oxygen concentration provides a solution characterized by its convenience, good tolerability, and effectiveness.

Polysaccharide Isolated From Tetrastigma hemsleyanum Activates TLR4 in Macrophage Cell Lines and Enhances Immune Responses in OVA-Immunized and LLC-Bearing Mouse Models.

Tetrastigma hemsleyanum Diels et Gilg is a valuable Chinese medicinal herb with a long history of clinical application. Our previous study isolated and characterized a purified polysaccharide from the aerial part of Tetrastigma hemsleyanum (SYQP) and found it having antipyretic and antitumor effects in mice. A preliminary mechanistic study suggests these effects may be related to the binding of toll-like receptor (TLR4). The objective of this study is to further explore the detailed stimulating characteristics of SYQP on TLR4 signaling pathway and its in vivo immune regulating effect. We use HEK-BLUE hTLR4, mouse and human macrophage cell lines, as research tools. In vitro results show SYQP activated HEK-BLUE hTLR4 instead of HEK-BLUE Null cells. The secretion and the mRNA expression of cytokines related to TLR4 signaling significantly increased after SYQP treatment in both PMA-induced THP-1 and RAW264.7 macrophage cell lines. The TLR4 antagonist TAK-242 can almost completely abolish this activation. Furthermore, molecules such as IRAK1, NF-κB, MAPKs, and IRF3 in both the MyD88 and TRIF branches were all activated without pathway selection. In vivo results show SYQP enhanced antigen-specific spleen lymphocyte proliferation and serum IgG levels in OVA-immunized C57BL/6 mice. Orally administered 200 mg/kg SYQP induced obvious tumor regression, spleen weight increase, and the upregulation of the mRNA expression of TLR4-related cytokines in Lewis lung carcinoma-bearing mice. These results indicate SYQP can act as both a human and mouse TLR4 agonist and enhance immune responses in mice (p < 0.05). This study provides a basis for the development and utilization of SYQP as a new type of TLR4 agonist in the future.

MPL Adjuvant Contains Competitive Antagonists of Human TLR4.

Monophosphoryl lipid A (MPL®) is the first non-alum vaccine adjuvant to achieve widespread clinical and market acceptance, a remarkable achievement given that it is manufactured from a Salmonella enterica endotoxin. To understand how MPL® successfully balanced the dual mandate of vaccine design-low reactogenicity with high efficacy-clinical- and research-grade MPL was evaluated in human and mouse cell systems. Stimulatory dose response curves revealed that most preparations of MPL are much more active in mouse than in human cell systems, and that the limited efficacy observed in human cells correlated with TLR4 inhibitory activity that resulted in a partial agonist profile. Further analysis of the major components of MPL® adjuvant prepared synthetically identified two structural variants that functioned as competitive antagonists of human TLR4. A partial agonist profile could be recapitulated and manipulated by spiking synthetic agonists with synthetic antagonists to achieve a broad dose range over which TLR4 stimulation could be constrained below a desired threshold. This report thus identifies mixed agonist-antagonist activity as an additional mechanism by which MPL® adjuvant is detoxified, relative to its parental LPS, to render it safe for use in prophylactic vaccines.

Polysaccharide from Phellinus Igniarius activates TLR4-mediated signaling pathways in macrophages and shows immune adjuvant activity in mice.

Polysaccharide from Phellinus igniarius (PPI) is known for its immune-regulating effect with low toxicity. Toll like receptor 4 (TLR4) is important in both innate and adaptive immune responses and considered to be a promising target for new immune adjuvants. In this study, PPI was investigated for its effect on activating TLR4 in RAW264.7 and peritoneal macrophages. The adjuvant potential of PPI was evaluated in OVA-immunized mice. The results showed PPI treatment significantly increased the secretion and the mRNA expression of both MyD88 dependent and TRIF dependent cytokines. IRAK-1, a key molecule on the downstream of MyD88, was polyubiquitinated while IRF-3, another key molecule on the downstream of TRIF, was phosphorylated obviously after the treatment of PPI. The phosphorylation of molecules involved in both NF-κB pathway and MAPK pathway were significantly up-regulated after PPI treatment. In addition, the effects of PPI on the macrophages almost completely disappeared after treating the cells with the TLR4 antagonist TAK-242. Further in vivo results showed PPI significantly increased the serum OVA-specific antibody and the OVA-specific spleen cell proliferation. Taken together, PPI can specifically stimulate TLR4 and activate both MyD88 and TRIF pathways. PPI has immune adjuvant activity and may become a new potential immune adjuvant.

The Loss of a Sugar Chain at C(3) Position Enhances Stemucronatoside K-Induced Apoptosis, Cell Cycle Arrest, and Hedgehog Pathway Inhibition in HT-29 Cells.

Stemucronatoside K (SMK) and its aglycone stephanthraniline A (STA) are the most representative of a series of novel C21 steriodal compounds that we have previously isolated from Asclepiadaceae plants. The objectives of this study were to investigate the antitumor activity of SMK and STA, and clarify the effect of the sugar chain at the C(3) position. Our results showed that both SMK and STA decreased the growth of HT-29 cells in a dose- and time-dependent manner. Meanwhile, STA showed much stronger inhibitory effect than SMK. Treatment of HT-29 cells with STA increased the apoptotic cell numbers and the protein expression of cleaved caspase 3 and cleaved-PARP. G1 phase cell cycle arrest and decreased expression of cyclin D1 and cyclin-dependent kinases 4 were also observed after STA treatment. Furthermore, STA reduced the mRNA levels of four Hedgehog pathway components (GLI1, GLI2, GLI3, and PTCH1) and suppressed Shh-induced Hedgehog pathway activation in a concentration-dependent manner. These results indicated that SMK and STA could inhibit the growth of HT-29 cells by inducing apoptosis, cell cycle arrest, and hedgehog pathway inhibition. The loss of sugar chain at C(3) position could enhance SMK's activity. This study is beneficial to understand the use of natural C21 steroids as antitumor lead compounds.

[Rapid Detection of Adenovirus in Fecal Samples by Capillary Electrophoresis-laser Induced Fluorescence and Microchip Capillary Electrophoresis-laser Induced Fluorescence].

OBJECTIVE: To establish a rapid and sensitive method based on polymerase chain reaction (PCR) combined with capillary electrophoresis-laser induced fluorescence (CE-LIF) and microchip capillary electrophoresis-laser induced fluorescence (MCE-LIF) for detecting adenoviruses in fecal samples. METHODS: The DNA of adenovirus in fecal samples were extracted by the commercial kits and the conserved region of hexon gene was selected as the target gene and amplified by PCR reaction. After labeling highly sensitive nucleic acid fluorescent dye SYBR Gold and SYBR Orange respectively, PCR amplification products were separated by CE and MCE under the optimized condition and detected by LIF detector. RESULTS: PCR amplification products could be detected within 9 min by CE-LIF and 6 min by MCE-LIF under the optimized separation condition. The sequenced PCR product showed good specificity in comparison with the prototype sequences from NCBI. The intraday and inter-day relative standard deviation (RSD) of the size (bp) of the target DNA was in the range of 1.14%-1.34% and 1.27%- 2.76%, respectively, for CE-LIF, and 1.18%-1.48% and 2.85%-4.06%, respectively, for MCE-LIF. The detection limits was 2.33 x 10(2) copies/mL for CE-LIF and 2.33 x 10(3) copies/mL for MCE-LIF. The two proposed methods were applied to detect fecal samples, both showing high accuracy. CONCLUSION: The two proposed methods of PCR-CE-LIF and PCR-MCE-LIF can detect adenovirus in fecal samples rapidly, sensitively and specifically.

Andrographolide sodium bisulfate-induced apoptosis and autophagy in human proximal tubular endothelial cells is a ROS-mediated pathway.

BACKGROUND AND AIMS: The nephrotoxic mechanisms of andrographolide sodium bisulfate (ASB) remain largely unknown. This study attempted to explore the mechanism of ASB-induced nephrotoxicity using human proximal tubular endothelial cells (HK-2). METHODS: For this study HK-2 cells were treated with rising concentrations of ASB. Their survival rate was detected using MTT assay and ultrastructure was observed with electron microscopy. L-Lactate dehydrogenase (LDH) assay was followed by examination of mitochondrial membrane potential (MMP). Reactive oxygen species (ROS) was detected using different methods and apoptosis/autophage related proteins were detected using immunoblotting. RESULTS: We found that ASB inhibited HK-2 cell proliferation and decreased cell survival rate in a time and dose-dependent manner (P<0.05, P<0.01, respectively). With increasing ASB concentration, cell structure was variably damaged and evidence of apoptosis and autophagy were observed. MMP gradually decreased and ROS was induced. The expression of JNK and Beclin-1 increased and activation of the JNK signaling pathway were seen. Apoptosis was induced via the mitochondrial-dependent caspase-3 and caspase-9 pathway, and autophagy related protein Beclin-1 was enhanced by ASB. CONCLUSION: The data show that ASB induces high levels of ROS generation in HK-2 cells and activates JNK signaling. Furthermore, ASB induces cell apoptosis via the caspase-dependent mitochondrial pathway, and induces cellular autophagy, in part by enhancing Beclin-1 protein expression.

Two new cytotoxic iridoid esters from the rhizomes and roots of Patrinia heterophylla Bunge.

Two new iridoid esters, named patriheterdoid B, C, have been isolated from the rhizomes and roots of Patrinia heterophylla Bunge. Their structures were elucidated by extensive spectroscopic technologies. Together with patriheterdoid B, C, two known analogues have been isolated and identified by means of mass spectrometry and (1)H and (13)C NMR spectrometry. These compounds showed cytotoxic activity against SGC-7901, PC3 cell lines.

Cytotoxic and apoptosis-inducing properties of a C21-steroidal glycoside isolated from the roots of Cynanchum auriculatum.

The present study aimed to investigate the anti-cancer effect of a C21-steroidal glycoside (CG) isolated from the roots of Cynanchum auriculatum. CG was able to inhibit the growth of human cancer cells (SGC-7901 cells) in a concentration and time-dependent manner in vitro. SGC-7901 cells exposed to CG (10.8 and 21.6 µM) exhibited typical morphological apoptosis characteristics, such as nuclear-chromatin condensation and apoptotic body formation. Flow cytometric analysis showed that after treatment with CG at 10.8 and 21.6 µM for 24 h, the percentage of apoptotic cells increased to 30.4 and 43.2%, respectively, while the number of cells in the G0/G1, S and G2/M phases of the cell cycle decreased (P<0.05). Furthermore, treatment with CG at a concentration of 21.6 µM for 24 h significantly increased the expression of caspase-3 and the activity of caspase-3 was increased ∼3-fold in SGC-7901 cells. These results suggest that CG is the active anticancer component of the total C21-glycosides of the roots of Cynanchum auriculatum which is able to inhibit the growth of cancer cells and induce cancer cell apoptosis through caspase-3-dependent pathways.

Induction of cytotoxicity and apoptosis in human gastric cancer cell SGC-7901 by isovaltrate acetoxyhydrin isolated from Patrinia heterophylla bunge involves a mitochondrial pathway and G2/M phase cell cycle arrest.

BACKGROUND: Our previous study demonstrated cytotoxicity of a crude extract from Patrinia heterophylla Bunge (PHEB). In the present study, we aimed to investigate the effects of isovaltrate acetoxyhydrin (IA) isolated from PHEB on the gastric cancer cell SGC-7901, in order to explore a potential treatment for gastric cancer. METHODS: MTT assays were employed to determine the effects of IA on cell vitality and proliferation, with monitoring of cell morphology changes and examination of apoptosis with Annexin V-PI staining. Flow cytometry was used to assess cell cycle progression and mitochondrial membrane potential. The activity of caspase 3, 9 was evaluated by spectrophotometry, and the protein levels of Bax, Bcl2 and Cyclin B1 were analyzed with Western blotting of total proteins extracted from cultured cells. RESULTS: The results demonstrated direct toxicity of IA towards SGC-7901 cells. Evidence of apoptosis included blebbing and chromatin condensation. Annexin V-PI assays revealed early apoptosis, involving rapid depolarization of mitochondrial membranes and activity of caspase 3, 9 signaling pathways. Western blotting showed that Bcl2 and Bax proteins was down- and up-regulated, respectively, and cyclin B1 was up-regulated. Cell cycle analysis further indicated that IA could induce G2/M phase arrest in SGC-7901 cells. CONCLUSIONS: In conclusion, we believe that IA induces apoptosis of SGC-7901 cells, therefore providing a potential therapeutic agent for treatment of gastric cancer.

A C 21 -Steroidal Glycoside Isolated from the Roots of Cynanchum auriculatum Induces Cell Cycle Arrest and Apoptosis in Human Gastric Cancer SGC-7901 Cells.

Caudatin 3-O-ß-D-cymaropyranosyl-(1 → 4)-ß-D-oleandropyranosyl-(1 → 4)-ß-D-cymaropyranosyl-(1 → 4)-ß-D-cymaropyranoside (CGII) is one of the C21-steroidal glycosides isolated from the roots of Cynanchum auriculatum ROYLE ex WIGHT. This study aimed to determine the cell growth, cell proliferation, and apoptotic cell death of human gastric cancer cells after CGII treatment. MTT assay was used to determine cell growth; fluorescence-activated cell sorting analysis was used to evaluate cell cycle distribution and apoptotic cell death. Immunoblotting was applied for measuring the expression of proteins involved in the cell cycle progression. The activities of caspase-3, -8, and -9 were detected by colorimetric caspase activity assays. CGII inhibited cell growth of human gastric cancer SGC-7901 cells in a concentration- and time-dependent manner. Treatment of SGC-7901 cells with CGII resulted in G1 phase cell cycle arrest, accompanied with decreased expression of cyclin D1 and cyclin-dependent kinases 4 and 6. CGII induced cell apoptosis and activated caspase-3, caspase-8, and caspase-9. In contrast, pan-caspase inhibitor z-VAD-fmk partially abolished the CGII-induced growth inhibition of SGC-7901 cells. In conclusion, CGII inhibits cell growth of human gastric cancer cells by inducing G1 phase cell cycle arrest and caspase-dependent apoptosis cascades.

[Inhibitive effect of C-21 steroidal glycosides of Cynanchum auriculatum on rat glioma cells in vitro].

OBJECTIVE: To evaluate the inhibitive effect of C-21 steroidal glycosides from the root of Cynanchum auriculatum (CGB) on rat glioma C6 cells. METHODS: C6 cells were treated with CGB for 24, 48,72 h at concentration of 30, 60, 120 mg/L, respectively. MTT assay was used for evaluating cell viability; fluorescence-activated cell sorting analysis after Annexin V/propidium iodide staining or single propidium iodide staining was used to test cell apoptosis and cell cycle. RESULTS: CGB at 30, 60, 120 mg/L concentration-dependently decreased C6 cell viability (P<0.001). CGB at 60 and 120 mg/L induced C6 cell apoptosis and cell cycle arrest. The fraction of G0/G1 cells was increased (P<0.05) and that of S phase cells was decreased (P<0.01). CONCLUSION: CGB can inhibit the growth of rat glioma C6 cells, and induce apoptosis and G0/G1 cell cycle arrest.