Alectinib in Resected ALK-Positive Non-Small-Cell Lung Cancer.

BACKGROUND: Platinum-based chemotherapy is the recommended adjuvant treatment for patients with resectable, ALK-positive non-small-cell lung cancer (NSCLC). Data on the efficacy and safety of adjuvant alectinib as compared with chemotherapy in patients with resected ALK-positive NSCLC are lacking. METHODS: We conducted a global, phase 3, open-label, randomized trial in which patients with completely resected, ALK-positive NSCLC of stage IB (tumors ≥4 cm), II, or IIIA (as classified according to the seventh edition of the Cancer Staging Manual of the American Joint Committee on Cancer and Union for International Cancer Control) were randomly assigned in a 1:1 ratio to receive oral alectinib (600 mg twice daily) for 24 months or intravenous platinum-based chemotherapy in four 21-day cycles. The primary end point was disease-free survival, tested hierarchically among patients with stage II or IIIA disease and then in the intention-to-treat population. Other end points included central nervous system (CNS) disease-free survival, overall survival, and safety. RESULTS: In total, 257 patients were randomly assigned to receive alectinib (130 patients) or chemotherapy (127 patients). The percentage of patients alive and disease-free at 2 years was 93.8% in the alectinib group and 63.0% in the chemotherapy group among patients with stage II or IIIA disease (hazard ratio for disease recurrence or death, 0.24; 95% confidence interval [CI], 0.13 to 0.45; P<0.001) and 93.6% and 63.7%, respectively, in the intention-to-treat population (hazard ratio, 0.24; 95% CI, 0.13 to 0.43; P<0.001). Alectinib was associated with a clinically meaningful benefit with respect to CNS disease-free survival as compared with chemotherapy (hazard ratio for CNS disease recurrence or death, 0.22; 95% CI, 0.08 to 0.58). Data for overall survival were immature. No unexpected safety findings were observed. CONCLUSIONS: Among patients with resected ALK-positive NSCLC of stage IB, II, or IIIA, adjuvant alectinib significantly improved disease-free survival as compared with platinum-based chemotherapy. (Funded by F. Hoffmann-La Roche; ALINA ClinicalTrials.gov number, NCT03456076.).

Perioperative Toripalimab Plus Chemotherapy for Patients With Resectable Non-Small Cell Lung Cancer: The Neotorch Randomized Clinical Trial.

Importance: Adjuvant and neoadjuvant immunotherapy have improved clinical outcomes for patients with early-stage non-small cell lung cancer (NSCLC). However, the optimal combination of checkpoint inhibition with chemotherapy remains unknown. Objective: To determine whether toripalimab in combination with platinum-based chemotherapy will improve event-free survival and major pathological response in patients with stage II or III resectable NSCLC compared with chemotherapy alone. Design, Setting, and Participants: This randomized clinical trial enrolled patients with stage II or III resectable NSCLC (without EGFR or ALK alterations for nonsquamous NSCLC) from March 12, 2020, to June 19, 2023, at 50 participating hospitals in China. The data cutoff date for this interim analysis was November 30, 2022. Interventions: Patients were randomized in a 1:1 ratio to receive 240 mg of toripalimab or placebo once every 3 weeks combined with platinum-based chemotherapy for 3 cycles before surgery and 1 cycle after surgery, followed by toripalimab only (240 mg) or placebo once every 3 weeks for up to 13 cycles. Main Outcomes and Measures: The primary outcomes were event-free survival (assessed by the investigators) and the major pathological response rate (assessed by blinded, independent pathological review). The secondary outcomes included the pathological complete response rate (assessed by blinded, independent pathological review) and adverse events. Results: Of the 501 patients randomized, 404 had stage III NSCLC (202 in the toripalimab + chemotherapy group and 202 in the placebo + chemotherapy group) and 97 had stage II NSCLC and were excluded from this interim analysis. The median age was 62 years (IQR, 56-65 years), 92% of patients were male, and the median follow-up was 18.3 months (IQR, 12.7-22.5 months). For the primary outcome of event-free survival, the median length was not estimable (95% CI, 24.4 months-not estimable) in the toripalimab group compared with 15.1 months (95% CI, 10.6-21.9 months) in the placebo group (hazard ratio, 0.40 [95% CI, 0.28-0.57], P < .001). The major pathological response rate (another primary outcome) was 48.5% (95% CI, 41.4%-55.6%) in the toripalimab group compared with 8.4% (95% CI, 5.0%-13.1%) in the placebo group (between-group difference, 40.2% [95% CI, 32.2%-48.1%], P < .001). The pathological complete response rate (secondary outcome) was 24.8% (95% CI, 19.0%-31.3%) in the toripalimab group compared with 1.0% (95% CI, 0.1%-3.5%) in the placebo group (between-group difference, 23.7% [95% CI, 17.6%-29.8%]). The incidence of immune-related adverse events occurred more frequently in the toripalimab group. No unexpected treatment-related toxic effects were identified. The incidence of grade 3 or higher adverse events, fatal adverse events, and adverse events leading to discontinuation of treatment were comparable between the groups. Conclusions and Relevance: The addition of toripalimab to perioperative chemotherapy led to a significant improvement in event-free survival for patients with resectable stage III NSCLC and this treatment strategy had a manageable safety profile. Trial Registration: ClinicalTrials.gov Identifier: NCT04158440.

Sintilimab plus chemotherapy for patients with EGFR-mutated non-squamous non-small-cell lung cancer with disease progression after EGFR tyrosine-kinase inhibitor therapy (ORIENT-31): second interim analysis from a double-blind, randomised, placebo-controlled, phase 3 trial.

BACKGROUND: In the first interim analysis of the ORIENT-31 trial, compared with chemotherapy alone, sintilimab plus bevacizumab biosimilar IBI305 plus chemotherapy (pemetrexed and cisplatin) significantly improved progression-free survival in patients with EGFR-mutated non-squamous non-small-cell lung cancer (NSCLC) who progressed on EGFR tyrosine-kinase inhibitor treatment. However, the benefit of anti-PD-1 or PD-L1 antibody added to chemotherapy in this patient population remains unclear, with no prospective evidence from phase 3 trials globally. We report the results from the prespecified second interim analysis of progression-free survival between sintilimab plus chemotherapy and chemotherapy alone, the updated results of sintilimab plus IBI305 plus chemotherapy, and preliminary overall survival results. METHODS: This double-blind, randomised, placebo-controlled, phase 3 trial was done at 52 centres across China and included patients aged 18-75 years with locally advanced or metastatic (stage IIIB, IIIC, or IV according to the American Joint Committee on Cancer, eighth edition) EGFR-mutated non-squamous NSCLC, disease progression after EGFR tyrosine-kinase inhibitor treatment (according to the Response Evaluation Criteria in Solid Tumours version 1.1 [RECIST 1.1]), and at least one measurable lesion (according to RECIST 1.1). Patients were randomly assigned (1:1:1), using an interactive web response system, to receive sintilimab (200 mg) plus IBI305 (15 mg/kg) plus pemetrexed (500 mg/m2) and cisplatin (75 mg/m2), sintilimab plus chemotherapy, or chemotherapy alone on day 1 of each 3-week cycle for four cycles, followed by maintenance therapy of sintilimab, IBI305, and pemetrexed. All study drugs were administered intravenously. The primary endpoint was progression-free survival in the intention-to-treat population assessed by an independent radiographic review committee. Data cutoff was March 31, 2022, unless otherwise specified. The study is registered at ClinicalTrials.gov, NCT03802240 (ongoing). FINDINGS: Between July 11, 2019, and March 31, 2022, 1011 patients were screened and 476 were randomly assigned (158 to the sintilimab plus IBI305 plus chemotherapy group, 158 to the sintilimab plus chemotherapy group, and 160 to the chemotherapy alone group). The median follow-up duration for progression-free survival was 12·9 months (IQR 8·2-17·8) in the sintilimab plus IBI305 plus chemotherapy group, 15·1 months (8·0-19·5) in the sintilimab plus chemotherapy group, and 14·4 months (9·8-23·8) in the chemotherapy alone group. Sintilimab plus chemotherapy significantly improved progression-free survival compared with chemotherapy alone (median 5·5 months [95% CI 4·5-6·1] vs 4·3 months [4·1-5·3]; hazard ratio [HR] 0·72 [95% CI 0·55-0·94]; two-sided p=0·016). Significant progression-free survival benefit was sustained with sintilimab plus IBI305 plus chemotherapy compared with chemotherapy alone (median 7·2 months [95% CI 6·6-9·3]; HR: 0·51 [0·39-0·67]; two-sided p<0·0001). As of data cutoff (July 4, 2022), the median overall survival was 21·1 months (95% CI 17·5-23·9) for sintilimab plus IBI305 plus chemotherapy (HR 0·98 [0·72-1·34]) and 20·5 months (15·8-25·3) for sintilimab plus chemotherapy group (HR 0·97 [0·71-1·32]) versus 19·2 months (15·8-22·4) for chemotherapy alone; after adjusting for crossover, the HR for sintilimab plus IBI305 plus chemotherapy to chemotherapy alone ranged from 0·79 (0·57-1·09) to 0·84 (0·61-1·15) and the HR for sintilimab plus chemotherapy to chemotherapy alone ranged from 0·78 (0·57-1·08) to 0·84 (0·61-1·16). The safety results were generally consistent with those in the first interim analysis; in particular, treatment-related adverse events of grade 3 or worse occurred in 88 (56%) of 158 patients in the sintilimab plus IBI305 plus chemotherapy group, 64 (41%) of 156 patients in the sintilimab plus chemotherapy group, and 79 (49%) of 160 patients in the chemotherapy alone group. INTERPRETATION: This is the first prospective phase 3 trial to show the benefit of anti-PD-1 antibody plus chemotherapy in patients with EGFR-mutated NSCLC who progressed on treatment with tyrosine-kinase inhibitors. Compared with chemotherapy alone, sintilimab combined with pemetrexed and cisplatin showed significant and clinically meaningful improvement of progression-free survival with an optimal safety profile. Sintilimab plus IBI305 plus chemotherapy continued to show progression-free survival benefit compared with chemotherapy alone in this second interim analysis with an additional 8-month follow-up. FUNDING: National Natural Science Foundation of China, Shanghai Municipal Science & Technology Commission Research Project, and Innovent Biologics. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

Accumulation of branched-chain amino acids reprograms glucose metabolism in CD8+ T cells with enhanced effector function and anti-tumor response.

Branched-chain amino acids (BCAAs) provide nutrient signals for cell survival and growth. How BCAAs affect CD8+ T cell functions remains unexplored. Herein, we report that accumulation of BCAAs in CD8+ T cells due to the impairment of BCAA degradation in 2C-type serine/threonine protein phosphatase (PP2Cm)-deficient mice leads to hyper-activity of CD8+ T cells and enhanced anti-tumor immunity. CD8+ T cells from PP2Cm-/- mice upregulate glucose transporter Glut1 expression in a FoxO1-dependent manner with more glucose uptake, as well as increased glycolysis and oxidative phosphorylation. Moreover, BCAA supplementation recapitulates CD8+ T cell hyper-functions and synergizes with anti-PD-1, in line with a better prognosis in NSCLC patients containing high BCAAs when receiving anti-PD-1 therapy. Our finding thus reveals that accumulation of BCAAs promotes effector function and anti-tumor immunity of CD8+ T cells through reprogramming glucose metabolism, making BCAAs alternative supplementary components to increase the clinical efficacy of anti-PD-1 immunotherapy against tumors.

Sintilimab plus bevacizumab biosimilar IBI305 and chemotherapy for patients with EGFR-mutated non-squamous non-small-cell lung cancer who progressed on EGFR tyrosine-kinase inhibitor therapy (ORIENT-31): first interim results from a randomised, double-blind, multicentre, phase 3 trial.

BACKGROUND: VEGF inhibitors can enhance the efficacy of immunotherapy. However, despite high initial response rates, almost all patients eventually develop treatment resistance to EGFR tyrosine-kinase inhibitors. We aimed to evaluate the efficacy and safety of sintilimab with or without IBI305 plus pemetrexed and cisplatin, compared with pemetrexed and cisplatin alone, for the treatment of patients with locally advanced or metastatic EGFR-mutated non-small-cell lung cancer (NSCLC) who had disease progression after receiving EGFR tyrosine-kinase inhibitor therapy. METHODS: This randomised, double-blind, multicentre, phase 3 trial was conducted at 52 hospitals in China. Eligible participants were adults aged 18-75 years with locally advanced or metastatic NSCLC and EGFRmut who progressed after receiving a EGFR tyrosine-kinase inhibitor, had an Eastern Cooperative Oncology Group performance status of 0 or 1 with at least one measurable lesion, and an estimated life expectancy of at least 3 months. Participants were randomly assigned (1:1:1) to receive sintilimab (200 mg) plus IBI305 (15 mg/kg) plus pemetrexed (500 mg/m2) and cisplatin (75 mg/m2), sintilimab plus pemetrexed and cisplatin, or pemetrexed and cisplatin (chemotherapy alone) using block randomisation with stratification according to sex and presence or absence of brain metastases. All study drugs were administered intravenously on day 1 of each cycle, once every 3 weeks. Except for cisplatin, which was only given in the first four cycles, treatment was given for 24 months or until disease progression, intolerable toxic effects, withdrawal of consent, death, or other protocol-specified conditions, whichever occurred first. The primary endpoint was progression-free survival in the intention-to-treat population. We herein report the first planned interim analysis, with progression-free survival results for the comparison between sintilimab plus IBI305 plus chemotherapy versus chemotherapy alone. The progression-free survival results for the sintilimab plus pemetrexed and cisplatin group are immature and not reported here. This study is registered with ClinicalTrials.gov, NCT03802240 (recruiting). FINDINGS: Between July 11, 2019, and July 31, 2021, 936 patients were screened and 444 were randomly assigned (148 to the sintilimab plus IBI305 plus chemotherapy group, 145 to the sintilimab plus chemotherapy group, and 151 to the chemotherapy alone group). Data cutoff for this interim analysis was July 31, 2021. After a median follow-up of 9·8 months (IQR 4·4-13·3), progression-free survival was significantly longer in the sintilimab plus IBI305 plus chemotherapy group versus the chemotherapy alone group (median 6·9 months [95% CI 6·0-9.3] vs 4·3 months [4·1-5·4]; hazard ratio 0·46 [0·34-0·64]; p<0·0001). The most common grade 3 or 4 treatment-related adverse events were decreased neutrophil count (30 [20%] in the sintilimab plus IBI305 plus chemotherapy group vs 26 [18%] in the sintilimab plus chemotherapy group vs 27 [18%] in the chemotherapy alone group), decreased white blood cell count (17 [11%] vs 12 [8%] vs 13 [9%]), and anaemia (18 [12%] vs ten [7%] vs 15 [10%]). Potentially treatment-related deaths occurred in six patients (intestinal obstruction, gastrointestinal haemorrhage, and myelosuppression in one patient each, and three deaths of unknown cause) in the sintilimab plus IBI305 plus chemotherapy group, and in one patient in the chemotherapy alone group (unknown cause). INTERPRETATION: In this interim analysis, sintilimab plus IBI305 plus cisplatin and pemetrexed was generally efficacious and well tolerated in patients with EGFR-mutated NSCLC who progressed after receiving EGFR tyrosine-kinase inhibitor therapy. FUNDING: Innovent Biologics and the National Natural Science Foundation of China. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

Neoadjuvant Nivolumab plus Chemotherapy in Resectable Lung Cancer.

BACKGROUND: Neoadjuvant or adjuvant chemotherapy confers a modest benefit over surgery alone for resectable non-small-cell lung cancer (NSCLC). In early-phase trials, nivolumab-based neoadjuvant regimens have shown promising clinical activity; however, data from phase 3 trials are needed to confirm these findings. METHODS: In this open-label, phase 3 trial, we randomly assigned patients with stage IB to IIIA resectable NSCLC to receive nivolumab plus platinum-based chemotherapy or platinum-based chemotherapy alone, followed by resection. The primary end points were event-free survival and pathological complete response (0% viable tumor in resected lung and lymph nodes), both evaluated by blinded independent review. Overall survival was a key secondary end point. Safety was assessed in all treated patients. RESULTS: The median event-free survival was 31.6 months (95% confidence interval [CI], 30.2 to not reached) with nivolumab plus chemotherapy and 20.8 months (95% CI, 14.0 to 26.7) with chemotherapy alone (hazard ratio for disease progression, disease recurrence, or death, 0.63; 97.38% CI, 0.43 to 0.91; P = 0.005). The percentage of patients with a pathological complete response was 24.0% (95% CI, 18.0 to 31.0) and 2.2% (95% CI, 0.6 to 5.6), respectively (odds ratio, 13.94; 99% CI, 3.49 to 55.75; P<0.001). Results for event-free survival and pathological complete response across most subgroups favored nivolumab plus chemotherapy over chemotherapy alone. At the first prespecified interim analysis, the hazard ratio for death was 0.57 (99.67% CI, 0.30 to 1.07) and did not meet the criterion for significance. Of the patients who underwent randomization, 83.2% of those in the nivolumab-plus-chemotherapy group and 75.4% of those in the chemotherapy-alone group underwent surgery. Grade 3 or 4 treatment-related adverse events occurred in 33.5% of the patients in the nivolumab-plus-chemotherapy group and in 36.9% of those in the chemotherapy-alone group. CONCLUSIONS: In patients with resectable NSCLC, neoadjuvant nivolumab plus chemotherapy resulted in significantly longer event-free survival and a higher percentage of patients with a pathological complete response than chemotherapy alone. The addition of nivolumab to neoadjuvant chemotherapy did not increase the incidence of adverse events or impede the feasibility of surgery. (Funded by Bristol Myers Squibb; CheckMate 816 ClinicalTrials.gov number, NCT02998528.).

Amyloid Protein-Biofunctionalized Polydopamine Nanoparticles Demonstrate Minimal Plasma Protein Fouling and Efficient Photothermal Therapy.

Polydopamine (PDA) shows great application potential in photothermal therapy (PTT) of tumors due to its excellent photothermal performance. However, PDA rich in a large number of catechin structures, with strong adhesion, can readily attach to plasma proteins in blood to form protein corona, which greatly hinders the transfer efficiency to tumors and reduces the bioavailability. In this paper, a simple, rapid phase-transitioned albumin biomimetic nanocorona (TBSA) is used for the surface camouflage of PDA nanoparticles for minimal plasma protein fouling and efficient PTT. TBSA coating is formed by the BSA-derived amyloid through the hydrophobic aggregation near the isoelectric point and the rupture of disulfide bonds by tris(2-carboxyethyl) phosphine. The stable PDA@TBSA complexes are formed by camouflaging TBSA onto the surface of PDA through hydrophobic, electrostatic, and covalent binding between TBSA and PDA, which showed excellent anti-plasma protein adsorption properties profited from the surface charge of PDA@TBSA approaching equilibrium and the surface passivation of BSA. The plasma protein thickness of the PDA@TBSA surface is 6 times lower than that of PDA at adsorption saturation. In vitro and in vivo experiments have revealed that PDA@TBSA has an excellent photothermal antitumor effect compared to PDA. Both PDA and PDA@TBSA treatment plus 808 nm laser irradiation result in more than 70% inhibition on tumor cell proliferation. In addition, PDA@TBSA does not cause a significant inflammatory response and tissue damage. Taken together, the TBSA coating endows PDA with low-fouling functions in blood and improves the residence time of PDA in blood and enrichment in the tumor tissue. This work offers a novel and efficient strategy for the design of functional nanosystems exploiting the speciality of the biomolecular corona formation around nanomaterials.

[Research progress on chemical constituents and pharmacological activities of seabuckthorn and prediction of its Q-markers].

Seabuckthorn contains flavonoids, tannins, terpenoids, polysaccharides, and vitamins, which have anti-inflammation,anti-oxidation, liver protection, anti-cardiovascular disease, anti-aging, immune enhancing, anti-tumor, and anti-bacterial activities.We reviewed the papers focusing on the chemical constituents, pharmacological activities, and utilization of seabuckthorn. The quality markers(Q-markers) of seabuckthorn were predicted and analyzed based on original plant phylogeny, chemical composition correlation, traditional medicinal properties, pharmacodynamic correlation, traditional and extended efficacy, pharmacokinetics, metabolic processes, and measurable components. With this review, we aim to provide theoretical reference for the quality control and quality standard establishment of seabuckthorn, so as to promote the rational exploitation and utilization of seabuckthorn resources, and improve the healthy and sustainable development of seabuckthorn industry.

Oroxin B Attenuates Ovariectomy-Induced Bone Loss by Suppressing Osteoclast Formation and Activity.

BACKGROUND: Osteoclasts are the major players in bone resorption and have always been studied in the prevention and treatment of osteoporosis. Previous studies have confirmed that a variety of flavonoids inhibit osteoporosis and improve bone health mainly through inhibiting osteoclastogenesis. Oroxin B (OB) is a flavonoid compound extracted from traditional Chinese herbal medicine Oroxylum indicum (L.) Vent, exerts potent antitumor and anti-inflammation effect, but its effect on osteoclastogensis remains unknown. METHODS: We comprehensively evaluated the effect of OB on the formation and function of osteoclasts and the underling mechanism by bone marrow-derived macrophage in vitro. In vivo, we used mice ovariectomized model to verify the protective effect of OB. RESULTS: OB was found to inhibit osteoclast formation and bone resorption function in vitro, in a dose-dependent manner and the increased osteoclastic-related genes induced by RANKL (NFATc1, c-fos, cathepsin K, RANK, MMP9 and TRAP) were also attenuated following OB treatment. Mechanistical investigation showed OB abrogated the increased phosphorylation level of MAPK and NF-κB pathway, and diminished the expression of the vital transcription factors for osteoclastogenesis. OB also prevented ovariectomy (OVX)-induced bone loss by inhibiting osteoclast formation and activity in mice. CONCLUSION: Our study demonstrated that OB may act as an anti-osteoporosis agent by inhibiting osteoclast maturation and attenuating bone resorption.

ADGRA1 negatively regulates energy expenditure and thermogenesis through both sympathetic nervous system and hypothalamus-pituitary-thyroid axis in male mice.

Adhesion G protein-coupled receptor A1 (ADGRA1, also known as GPR123) belongs to the G protein-coupled receptors (GPCRs) family and is well conserved in the vertebrate lineage. However, the structure of ADGRA1 is unique and its physiological function remains unknown. Previous studies have shown that Adgra1 is predominantly expressed in the central nervous system (CNS), indicating its important role in the transduction of neural signals. The aim of this study is to investigate the central function of Adgra1 in vivo and clarify its physiological significance by establishing an Adgra1-deficient mouse (Adgra1-/-) model. The results show that Adgra1-/- male mice exhibit decreased body weight with normal food intake and locomotion, shrinkage of body mass, increased lipolysis, and hypermetabolic activity. Meanwhile, mutant male mice present elevated core temperature coupled with resistance to hypothermia upon cold stimulus. Further studies show that tyrosine hydroxylase (TH) and ß3-adrenergic receptor (ß3-AR), indicators of sympathetic nerve excitability, are activated as well as their downstream molecules including uncoupling protein 1 (UCP1), coactivator 1 alpha (PGC1-α) in brown adipose tissue (BAT), and hormone-sensitive lipase (HSL) in white adipose tissue (WAT). In addition, mutant male mice have higher levels of serum T3, T4, accompanied by increased mRNAs of hypothalamus-pituitary-thyroid axis. Finally, Adgra1-/- male mice present abnormal activation of PI3K/AKT/GSK3ß and MEK/ERK pathways in hypothalamus. Overexpression of ADGRA1 in Neuro2A cell line appears to suppress these two signaling pathways. In contrast, Adgra1-/- female mice show comparable body weight along with normal metabolic process to their sex-matched controls. Collectively, ADGRA1 is a negative regulator of sympathetic nervous system (SNS) and hypothalamus-pituitary-thyroid axis by regulating PI3K/AKT/GSK3ß and MEK/ERK pathways in hypothalamus of male mice, suggesting an important role of ADGRA1 in maintaining metabolic homeostasis including energy expenditure and thermogenic balance.

[Study on protective effect of water extract from Sabia parviflora on liver injury in mice induced by acetaminophen].

The aim of this study was to observe the protective effect of water extract from Sabia parviflora on mice with acute liver injury induced by acetaminophen, and investigate its possible mechanism. Fifty-eight Kunming mice were divided into 6 groups, 8 in the normal group, 10 in the model group, 10 in the biphenyl diester group, and 10 each in the low, medium and high dose groups. After adaptive feeding for one week, the mice in normal group were intragastrically administered with an equal volume of 0.5% sodium carboxymethylcellulose sodium(CMC-Na), and the mice in other groups were intragastrically administered with corresponding drugs at 20 mL·kg~(-1) once a day. Then acetaminophen(200 mg·kg~(-1)) was administered after the above drug administration except the normal group. The behavior and signs of the experimental animals were observed every day and the samples were taken for experiments on the next day of the final administration. The liver mass and mass index were calculated. The blood was collected from the abdominal aorta and centrifuged to obtain the serum for detecting aspartate aminotransferase(AST) activity and alanine aminotransferase(ALT) activity. The liver tissue homogenate was used to detect superoxide dismutase(SOD) activity, glutathione(glutathione, r-glutamyl cysteingl+glycine, GSH) activity and malondialdehyde(MDA) content. Liver tissue was analyzed for histological analysis. The results showed that S. parviflora could alleviate the lipid peroxidation damage in the liver caused by acetaminophen, reduce the ALT and AST activities in serum, increase the levels of SOD and GSH in liver tissue, decrease the content of MDA in liver tissue, and inhibit the apoptosis. S. parviflora could also improve the live histopathological profile, protect liver cells and restore liver function. Among them, the high dose had the most significant effect and showed dose-effect relationship. This study indicated that S. parviflora had a significant protective effect on acetaminophen-induced liver injury in mice, and its mechanism may be related to its anti-oxidation effect and inhi-bitory effect on apoptosis.

Effect of Boric Acid Supplementation on the Expression of BDNF in African Ostrich Chick Brain.

The degree of brain development can be expressed by the levels of brain brain-derived neurotrophic factor (BDNF). BDNF plays an irreplaceable role in the process of neuronal development, protection, and restoration. The aim of the present study was to evaluate the effects of boric acid supplementation in water on the ostrich chick neuronal development. One-day-old healthy animals were supplemented with boron in drinking water at various concentrations, and the potential effects of boric acid on brain development were tested by a series of experiments. The histological changes in brain were observed by hematoxylin and eosin (HE) staining and Nissl staining. Expression of BDNF was analyzed by immunohistochemistry, quantitative real-time PCR (QRT-PCR), and enzyme linked immunosorbent assay (ELISA). Apoptosis was evaluated with Dutp-biotin nick end labeling (TUNEL) reaction, and caspase-3 was detected with QRT-PCR. The results were as follows: (1) under the light microscope, the neuron structure was well developed with abundance of neurites and intact cell morphology when animals were fed with less than 160 mg/L of boric acid (groups II, III, IV). Adversely, when boric acid doses were higher than 320 mg/L(groups V, VI), the high-dose boric acid neuron structure was damaged with less neurites, particularly at 640 mg/L; (2) the quantity of BDNF expression in groups II, III, and IV was increased while it was decreased in groups V and VI when compared with that in group I; (3) TUNEL reaction and the caspase-3 mRNA level showed that the amount of cell apoptosis in group II, group III, and group IV were decreased, but increased in group V and group VI significantly. These results indicated that appropriate supplementation of boric acid, especially at 160 mg/L, could promote ostrich chicks' brain development by promoting the BDNF expression and reducing cell apoptosis. Conversely, high dose of boric acid particularly in 640 mg/L would damage the neuron structure of ostrich chick brain by inhibiting the BDNF expression and increasing cell apoptosis. Taken together, the 160 mg/L boric acid supplementation may be the optimal dose for the brain development of ostrich chicks.

[The effects of Shadu Cao Mixture on immune functions of immunosuppression mice].

OBJECTIVE: To investigate the effects of Shadu Cao Mixture (SDCM, traditional Chinese medicine) on immune functions of immunosuppression mice. METHODS: Fifty BALB/C mice were randomly divided into blank control group, model group, SDCM low-dose, middle-dose and high-dose group. Except the blank control group, other groups were intraperitoneal injected with cyclophosphamide (40 mg/kg) to establish immunosuppression mice model. The blank control group and model group received gavage administration with nonnal saline, while the other groups received gavage administration with different doses of SDCM (10, 20, 40 m/kg for 15 days) respectively. The number of leukocytes and serum levels of interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in peripheral blood, spleen index, and the function of NK cells were measured. RESULTS: Compared with the model group , SDCM increased the number of leukocytes and serum concentrations of IL-2, TNF-α and IFN-γ in peripheral blood and improved the spleen index and the function of NK cells significantly (P < 0.05-0.01). CONCLUSION: SDCM could remarkably enhance the immune functions of immunosuppression mice induced by cyclophosphamide.

Increased Thymic Cell Turnover under Boron Stress May Bypass TLR3/4 Pathway in African Ostrich.

Previous studies revealed that thymus is a targeted immune organ in malnutrition, and high-boron stress is harmful for immune organs. African ostrich is the living fossil of ancient birds and the food animals in modern life. There is no report about the effect of boron intake on thymus of ostrich. The purpose of present study was to evaluate the effect of excessive boron stress on ostrich thymus and the potential role of TLR3/4 signals in this process. Histological analysis demonstrated that long-term boron stress (640 mg/L for 90 days) did not disrupt ostrich thymic structure during postnatal development. However, the numbers of apoptotic cells showed an increased tendency, and the expression of autophagy and proliferation markers increased significantly in ostrich thymus after boron treatment. Next, we examined the expression of TLR3 and TLR4 with their downstream molecular in thymus under boron stress. Since ostrich genome was not available when we started the research, we first cloned ostrich TLR3 TLR4 cDNA from thymus. Ostrich TLR4 was close to white-throated Tinamou. Whole avian TLR4 codons were under purify selection during evolution, whereas 80 codons were under positive selection. TLR3 and TLR4 were expressed in ostrich thymus and bursa of fabricius as was revealed by quantitative real-time PCR (qRT-PCR). TLR4 expression increased with age but significantly decreased after boron treatment, whereas TLR3 expression showed the similar tendency. Their downstream molecular factors (IRF1, JNK, ERK, p38, IL-6 and IFN) did not change significantly in thymus, except that p100 was significantly increased under boron stress when analyzed by qRT-PCR or western blot. Taken together, these results suggest that ostrich thymus developed resistance against long-term excessive boron stress, possibly by accelerating intrathymic cell death and proliferation, which may bypass the TLR3/4 pathway. In addition, attenuated TLRs activity may explain the reduced inflammatory response to pathogens under boron stress.

Formononetin suppresses the proliferation of human non-small cell lung cancer through induction of cell cycle arrest and apoptosis.

Formononetin is a novel herbal isoflavonoid isolated from Astragalus membranaceus and possesses antitumorigenic properties. In the present study, we investigated the anti-proliferative effects of formononetin on human non-small cell lung cancer (NSCLC), and further elucidated the molecular mechanism underlying the anti-tumor property. MTT assay showed that formononetin treatment significantly inhibited the proliferation of two NSCLC cell lines including A549 and NCI-H23 in a time- and dose-dependent manner. Flow cytometric analysis demonstrated that formononetin induced G1-phase cell cycle arrest and promoted cell apoptosis in NSCLC cells. On the molecular level, we observed that exposure to formononetin altered the expression levels of cell cycle arrest-associated proteins p21, cyclin A and cyclin D1. Meanwhile, the apoptosis-related proteins cleaved caspase-3, bax and bcl-2 were also changed following treatment with formononetin. In addition, the expression level of p53 was dose-dependently upregulated after administration with formononetin. We also found that formononetin treatment increased the phosphorylation of p53 at Ser15 and Ser20 and enhances its transcriptional activity in a dose-dependent manner. Collectively, these results demonstrated that formononetin might be a potential chemopreventive drug for lung cancer therapy through induction of cell cycle arrest and apoptosis in NSCLC cells.

Structural and functional diversity of CLAVATA3/ESR (CLE)-like genes from the potato cyst nematode Globodera rostochiensis.

Plant CLAVATA3/ESR-related (CLE) peptides have diverse roles in plant growth and development. Here, we report the isolation and functional characterization of five new CLE genes from the potato cyst nematode Globodera rostochiensis. Unlike typical plant CLE peptides that contain a single CLE motif, four of the five Gr-CLE genes encode CLE proteins with multiple CLE motifs. These Gr-CLE genes were found to be specifically expressed within the dorsal esophageal gland cell of nematode parasitic stages, suggesting a role for their encoded proteins in plant parasitism. Overexpression phenotypes of Gr-CLE genes in Arabidopsis mimicked those of plant CLE genes, and Gr-CLE proteins could rescue the Arabidopsis clv3-2 mutant phenotype when expressed within meristems. A short root phenotype was observed when synthetic GrCLE peptides were exogenously applied to roots of Arabidopsis or potato similar to the overexpression of Gr-CLE genes in Arabidopsis and potato hairy roots. These results reveal that G. rostochiensis CLE proteins with either single or multiple CLE motifs function similarly to plant CLE proteins and that CLE signaling components are conserved in both Arabidopsis and potato roots. Furthermore, our results provide evidence to suggest that the evolution of multiple CLE motifs may be an important mechanism for generating functional diversity in nematode CLE proteins to facilitate parasitism.

Alternative splicing: a novel mechanism of regulation identified in the chorismate mutase gene of the potato cyst nematode Globodera rostochiensis.

Chorismate mutase (CM) secreted from the stylet of plant-parasitic nematodes plays an important role in plant parasitism. We isolated and characterized a new nematode CM gene (Gr-cm-1) from the potato cyst nematode, Globodera rostochiensis. The Gr-cm-1 gene was found to exist in the nematode genome as a single-copy gene that has two different alleles, Gr-cm-1A and Gr-cm-1B, both of which could give rise to two different mRNA transcripts of Gr-cm-1 and Gr-cm-1-IRII. In situ mRNA hybridization showed that the Gr-cm-1 gene was exclusively expressed within the subventral oesophageal gland cells of the nematode. Gr-cm-1 was demonstrated to encode a functional CM (GR-CM-1) potentially having a dimeric structure as the secreted bacterial *AroQ CMs. Gr-cm-1-IRII, generated by retention of intron 2 of the Gr-cm-1 pre-mRNA through alternative splicing (AS), would encode a truncated protein (GR-CM-1t) lacking the CM domain with no CM activity. The quantitative real-time reverse transcription-PCR assay revealed that splicing of the Gr-cm-1 gene was developmentally regulated; Gr-cm-1 was up-regulated whereas Gr-cm-1-IRII was down-regulated in early nematode parasitic stages compared to the preparasitic juvenile stage. Low-temperature SDS-PAGE analysis revealed that GR-CM-1 could form homodimers when expressed in Escherichia coli and the dimerization domain was retained in the truncated GR-CM-1t protein. The specific interaction between the two proteins was demonstrated in yeast. Our data suggested that the novel splice variant might function as a dominant negative isoform through heterodimerization with the full-length GR-CM-1 protein and that AS may represent an important mechanism for regulating CM activity during nematode parasitism.

[Effect of jiawei yupingfeng powder on T-lymphocyte subsets in patients with senile chronic bronchitis in acute onset stage].

OBJECTIVE: To explore the effect of Jiawei Yupingfeng Powder (JYP) on T-lymphocyte subsets in patients with senile chronic bronchitis in acute onset stage (SCB-AOS). METHODS: Patients were divided, according to the randomized controlled principle, into two groups, the 44 patients in the treated group and the 40 in the control group. The conventional western medicinal therapy was given to both groups, but to the treated group, JYP was administered additionally. The therapeutic course to them was 14 days. Changes of CD3, CD4, CD8 and the ratio of them were observed. RESULTS: Increase of CD3, CD4 and CD4/CD8 (P < 0.05) and decrease of CD8 (P < 0.01) were significantly shown in the treated group after treatment, but no change in the control group. Besides, comparison of the total effective rate in the two groups also showed significant difference (P < 0.05). CONCLUSION: Cellular immune function disturbance exists in patients with SCB-AOS, JYP could enhance the efficacy by way of modulating cellular immune function.