三阴性乳腺癌干细胞的富集及CD44+CD24-/low、CXCR4表达测定

目的:探讨MDA-MB-231细胞经无血清培养富集三阴性乳腺癌干细胞,观察再成球、集落形成及CD44+CD24-/low、CXCR4表达。方法:将MDA-MB-231乳腺癌细胞进行微球体培养,取培养第7-9天的微球体,判断干细胞富集的程度;比较不同细胞浓度对癌球细胞成球率影响;流式细胞仪测定CD44+CD24-/low含量;Western blot法分析CXCR4蛋白表达;单个癌球细胞再成球能力;观察癌球与贴壁细胞集落形成。结果:1).在含20 ng/m L EGF,10 ng/m L b FGF,2%b27无血清培养基中可培养三阴性乳腺癌癌球,1×104/m L、2×104/m L、3×104/m L、4×104/m L、5×104/m L细胞浓度癌球细胞成球率分别为(5.61±0.02)%、(3.23±0.54)%、(2.28±0.48)%、(1.05±0.13)%、(0.91±0.01)%,组间比较差异有统计学意义P值均0.05。2).贴壁MDA-MB-231细胞与癌球细胞CD44+CD24-/low含量分别为(38.54±2.00)%VS(66.35±2.06)%,差异有统计学意义P=0.003。3).癌球细胞CXCR4蛋白表达高于贴壁MDA-MB-231细胞,灰度扫描分析差异有统计学意义,P=0.03。4).单个癌球细胞具有再成球能力。5).软琼脂糖集落形成能力癌球需200个细胞即可见集落形成,而贴壁细胞需1 000个MDA-MB-231细胞。结论:1.通过无血清培养可以富集三阴性乳腺癌干细胞,低细胞密度有利于癌球形成。2.癌球中CD44+CD24-/low含量高于贴壁MDA-MB-231细胞。3.CXCR4在癌球中表达高于贴壁MDA-MB-231细胞。     

Distribution of viruses in the water column of the ice-covered Rybinsk Reservoir and their contribution to heterotrophic bacteria mortality

Virioplankton and bacterioplankton abundance has been determined in the pelagic and littoral zones of the Rybinsk Reservoir during the ice-covered period. The role of viruses in heterotrophic bacterioplankton infection and mortality is assessed. At water temperatures between 0.3 and 0.9°C, the number of planktonic virus particles and planktonic bacteria varies from 37.1 × 10⁶ to 84.1 × 10⁶ particles/mL, (57.3 ± 2.1) × 10⁶ particles/mL on average and from 2.50 × 10⁶ to 6.11 × 10⁶ cells/mL, (3.66 ± 0.16) × 10⁶ cells/mL on average, respectively. The ratio of the virus number to the bacteria number varies from 8.8 to 27.9, being 16.5 ± 0.7 on average. Visually infected cells comprise 0.3–0.5% (1.5 ± 0.2% on average) of the total number of bacterioplankton. Infected bacterial cells contain from 5 to 107 (17 ± 4 on average) mature virus particles. The average virus-induced mortality of bacteria accounts for 13.0 ± 1.9% (variations range from 2 to 55%) of the daily bacterial production, indicating that viruses play an important role in the regulation of bacterioplankton production and abundance in the Rybinsk Reservoir during the ice-covered period.     

Manufacturing chimeric antigen receptor T cells from cryopreserved peripheral blood cells: time for a collect-and-freeze model?

Background aimsChimeric antigen receptor (CAR)-modified T-cell therapy has revolutionized outcomes for patients with relapsed/refractory B-cell malignancies. Despite the exciting results, several clinical and logistical challenges limit its wide applicability. First, the apheresis requirement restricts accessibility to institutions with the resources to collect and process peripheral blood mononuclear cells (PBMCs). Second, even when utilizing an apheresis product, failure to manufacture CAR T cells is a well-established problem in a significant subset. In heavily pre-treated patients, prior chemotherapy may impact T-cell quality and function, limiting the ability to manufacture a potent CAR T-cell product. Isolation and storage of T cells shortly after initial cancer diagnosis or earlier in life while an individual is still healthy are an alternative to using T cells from heavily pre-treated patients. The goal of this study was to determine if a CAR T-cell product could be manufactured from a small volume (50 mL) of healthy donor blood.MethodsCollaborators at Cell Vault collected 50 mL of whole peripheral venous blood from three healthy donors. PBMCs were isolated, cryopreserved and shipped to the Medical College of Wisconsin. PBMCs for each individual donor were thawed, and CAR T cells were manufactured using an 8-day process on the CliniMACS Prodigy device with a CD19 lentiviral vector.ResultsStarting doses of enriched T-cell numbers ranged from 4.0 × 10⁷ cells to 4.8 × 10⁷ cells, with a CD4/CD8 purity of 74–79% and an average CD4:CD8 ratio of 1.4. On the day of harvest, total CD3 cells in the culture expanded to 3.6–4.6 × 10⁹ cells, resulting in a 74- to 115-fold expansion, an average CD4:CD8 ratio of 2.9 and a CD3 frequency of greater than 99%. Resulting CD19 CAR expression varied from 19.2% to 48.1%, with corresponding final CD19+ CAR T-cell counts ranging from 7.82 × 10⁸ cells to 2.21 × 10⁹ cells. The final CAR T-cell products were phenotypically activated and non-exhausted and contained a differentiated population consisting of stem cell-like memory T cells.ConclusionsOverall, these data demonstrate the ability to successfully generate CAR T-cell products in just 8 days using cryopreserved healthy donor PBMCs isolated from only 50 mL of blood. Notably, numbers of CAR T cells were more than adequate for infusion of an 80-kg patient at dose levels used for products currently approved by the Food and Drug Administration. The authors offer proof of principle that cryopreservation of limited volumes of venous blood with an adequate starting T-cell count allows later successful manufacture of CAR T-cell therapy.     

Radioimmunoassays of plasma thymidine,uridine, deoxyuridine,and cytidine/deoxycytidine

Radioimmunoassay techniques have been developed for the assay of thymidine, uridine, deoxyuridine, and deoxycytidine. Plasma levels of the first three nucleosides have been measured, and an upper limit has been determined for the plasma concentration of deoxycytidine. The assays involve displacement of a [³H]pyrimidine nucleoside from the appropriate labeled rabbit immunoglobulin. By assaying a mixture of uridine and deoxyuridine in the presence and absence of borax, the concentrations of both nucleosides have been measured. In seven healthy adults, plasma levels of uridine were 21.1 ± 8.4 μm (mean ± SD) and of deoxyuridine were 0.62 ± 0.39 μm. In cancer patients, thymidine levels were 7.5 ± 2.7 × 10^?7m. The upper limit for plasma deoxycytidine levels in six healthy adults was 0.71 ± 0.1 μm.     

Heterotrophic microorganisms and viruses in the water of the Gorky reservoir during a period of anomalously high water temperature

During the anomalously hot summer of 2010, the water temperature in the Gorky reservoir reached 27–33°C. Pronounced cyanobacterial blooms occurred in the limnetic part of the reservoir. The average values for bacterioplankton abundance (11.58 ± 1.25 × 10⁶ cell/mL), biomass (886 ± 96 mg/m³), and production [169 ± 32 mg C/(m³ day)] were twice as high as in the year with temperatures comparable to long-term average values. These parameters were higher in the limnetic part than in the river one. The abundance (4.86 ± 0.75 × 10³ cell/mL) and biomass (138 ± 9 mg/m³) of heterotrophic nanoflagellates were 2.3 and 1.7 times higher, respectively, than in years with regular temperature regimes. The average number of plank-tonic viral particles (N _v) in 2010 was 48.89 ± 9.54 × 10⁶ particles/mL, while virus-induced bacterial mortality (VMB) accounted for 26.9 ± 4.6% of the bacterial production. The N _v and VMB values in the limnetic part of the reservoir were, respectively, 1.5 and 1.8 times higher than in the river one.     

Supplementation of antioxidant micronutrients reduces stress and improves immune function/response in periparturient dairy cows and their calves

BackgroundPeriparturient period induces stress in cows which fluctuates hormonal and metabolic function and causes immune suppression. Apart from impairing the health, production, and reproduction of cows, it also influences the well-being of newborn calves by decreasing the colostrum quality. Micronutrients are known for optimal health and production and their effects on parturition stress, immune response in both cow and its calf need to be explored.AimThe aim of this study was to see the effect of oral supplementation of micronutrients during the prepartum period on the health status of crossbred dairy cows and subsequently on their newborn calves.MethodsA total of 42 healthy multiparous cows were selected and randomly divided into five groups with seven cows in each group, i.e. control (Basal Diet, BD), VA group (BD + vitamin A, 10⁵ IU), Zn group (BD + zinc sulphate, 60 ppm), VE group (BD + vitamin E, 2500 IU), and combined supplementation (CS) group (BD + combination of VA, Zn, and VE). The supplements were offered in compounded concentrate DM (100 g) to individual cows once daily before the morning feeding and the remaining portion was incorporated in the TMR. Feeding was started one month before the expected days of calving till calving. Blood samples were collected from cows at days -15, -7, -3, 0, +3, +7, and +15 relative to the day of calving. Blood samples from newborn calves and milk samples of cows were collected at days 0, +3, +7, and +15. Milk somatic cell counts (SCC) were estimated using a cell counter. Cortisol was estimated by ELISA kit in blood and milk plasma of cows and in the blood plasma of their calves. Total immunoglobulins (Ig) were estimated in milk of cows and serum of calves using zinc sulphate turbidity method. Blood neutrophils from cows and calves were studied for phagocytic activity (PA) using nitro blue tetrazolium (NBT) assay.Data were analysed by repeated-measures two-way ANOVA using the mixed procedure of SAS, and the pairwise comparison was performed using a multiple comparison test (Tukey).ResultsCombined supplementation of micronutrients decreased (P < 0.05) maternal blood plasma (control vs. CS group, 5.98 ± 0.20 vs. 3.86 ± 0.23 ng/mL) and milk plasma (3.96 ± 0.13 vs. 2.71 ± 0.10 ng/mL) cortisol, milk SCC (3.05 ± 0.11 vs. 2.12 ± 0.10 × 10⁵ cells/mL) and increased (P < 0.05) total milk Ig concentration (18.80 ± 0.11 vs. 23.04 ± 0.57 mg/mL) and the PA of blood neutrophils (0.84 ± 0.03 vs. 1.07 ± 0.03). Similarly, lower blood cortisol concentration (9.69 ± 0.35 vs. 6.02 ± 0.18 ng/mL) and higher (P < 0.05) total Ig (23.26 ± 0.11 vs. 30.34 ± 0.70 mg/mL) and PA of blood neutrophils (0.37 ± 0.02 vs. 0.52 ± 0.02) were observed in the calves born to CS group of cows as compared to the control. Highest (P < 0.05) positive effects (lower stress levels and higher immune response) of treatment were noticed in CS group followed by VE group and then Zn group. However, VA group didn’t differ from the control group.ConclusionOur results indicate that micronutrient interventions during the prepartum period can improve the health status of dairy calves and subsequently the well-being of their calves.     

Soluble CD23 in Human Immunodeficiency Virus Type 1 Infected Patients

The level of sCD23 produced in the course of human immunodeficiency virus (HIV) infection was measured in patients grouped according to the Centers for Disease Control by using an immunoradiometric assay. Soluble CD23 was evaluated in supernatants of peripheral blood mononuclear cell (PBMC) (10⁶ cells/ml) stimulated by phytohemagglutinin (PHA). Compared with healthy controls (m±S.D. = 1.0 ±0.34 U/ml, n = 7), higher values were observed in some of the patients of group II (asymptomatic) (m±S.D. = 2±1.33, n = 9) and some of the patients of group IV (AIDS) (m±S.D. = 1.3 ±1.40, n = 8). Those results prompted us to compare the plasma levels of sCD23 in group II and group IV HIV-infected patients and in healthy individuals. Soluble CD23 plasma levels in healthy patients (n = 42) ranged from 0 to 1.5 U/ml (m±S.D. = 0.9±0.33), in group II patients (n = 17) from 0 to 3 U/ml (m±S.D. = 0.92±0.83) and in group IV patients (n =73) from 0 to 2.9 U/ml (m±S.D. = 1.15±0.71). The differences between the patients and the healthy individuals were not statistically significant but individual sCD23 values higher than 2 U/ml were obtained in 6% of the group II patients and 16.7% of the group IV patients. Increased values of sCD23 were obtained in plasma from patients with secondary infectious diseases (groups IV-C1 and IV-C2) and from patients without secondary infectious diseases (group II, group IV-A and group IV-B). Elevated values of sCD23 were detected even in patients with low counts of CD4⁺ T cells and CD8⁺ T cells in their peripheral blood. sCD23 has numerous activities including control of IgE synthesis and cytokine-like properties. Our results show a disarray of sCD23 in HIV-infected patients which could be involved in drug reactions, allergic manifestations and the IgE-level increase. Further investigations should attempt to define the role of sCD23 in clinical manifestations of HIV infection.     

Prostate Tumor-Derived Exosomes Down-Regulate NKG2D Expression on Natural Killer Cells and CD8+ T Cells: Mechanism of Immune Evasion

Tumor-derived exosomes, which are nanometer-sized extracellular vesicles of endosomal origin, have emerged as promoters of tumor immune evasion but their role in prostate cancer (PC) progression is poorly understood. In this study, we investigated the ability of prostate tumor-derived exosomes to downregulate NKG2D expression on natural killer (NK) and CD8⁺ T cells. NKG2D is an activating cytotoxicity receptor whose aberrant loss in cancer plays an important role in immune suppression. Using flow cytometry, we found that exosomes produced by human PC cells express ligands for NKG2D on their surface. The NKG2D ligand-expressing prostate tumor-derived exosomes selectively induced downregulation of NKG2D on NK and CD8⁺ T cells in a dose-dependent manner, leading to impaired cytotoxic function in vitro. Consistent with these findings, patients with castration-resistant PC (CRPC) showed a significant decrease in surface NKG2D expression on circulating NK and CD8⁺ T cells compared to healthy individuals. Tumor-derived exosomes are likely involved in this NKG2D downregulation, since incubation of healthy lymphocytes with exosomes isolated from serum or plasma of CRPC patients triggered downregulation of NKG2D expression in effector lymphocytes. These data suggest prostate tumor-derived exosomes as down-regulators of the NKG2D-mediated cytotoxic response in PC patients, thus promoting immune suppression and tumor escape.     

Mobilization and engraftment of peripheral blood stem cells in healthy related donors >55 years old

Background aimsThe increasing scarcity of young related donors has led to the use of older donors for related allogeneic hematopoietic stem cell transplantation (HSCT). This study analyzed the influence of age on the results of mobilization of peripheral blood stem cells (PBSCs) in healthy donors as well as on the engraftment and outcome of HSCT.MethodsA retrospective analysis from a single center was performed comparing the results of PBSC mobilization from related healthy donors according to their age.ResultsThe study included 133 consecutive related donors. The median age was 50 years (range, 4–77 years); 70 (53%) donors were males, and 44 (33%) were >55 years old. All donors were mobilized with granulocyte colony-stimulating factor for 5 days. The peak CD34⁺ cell count in peripheral blood was higher in younger than in older donors (median, 90.5 CD34⁺ cells/μL [range, 18–240 CD34⁺ cells/μL] versus 72 CD34⁺ cells/μL [range, 20–172.5 CD34⁺ cells/μL], P = 0.008). The volume processed was lower in younger than in older donors (16,131 mL [range, 4424–36,906 mL] versus 18,653 mL [range, 10,003–26,261 mL], P = 0.002) with similar CD34⁺ cells collected (579.3 × 10⁶ cells [range, 135.14 × 10⁶–1557.24 × 10⁶ cells] versus 513.69 × 10⁶ cells [range, 149.81 × 10⁶–1290 × 10⁶ cells], P = 0.844). There were no differences in time to recovery of neutrophils and platelets or in the incidences of acute and chronic graft-versus-host disease, overall survival, non-relapse mortality and relapse incidence.ConclusionsDonors >55 years old mobilized fewer CD34⁺ cells and required a greater volume to collect a similar number of CD34⁺ cells. The outcome of HSCT was not influenced by donor age. Donor age should not be a limitation for related allogeneic HSCT.     

Ex vivo activation and expansion of natural killer cells from patients with advanced cancer with feeder cells from healthy volunteers

Background aimsCulturing natural killer (NK) cells from patients with advanced cancer is difficult and has restricted the generation of sufficient cell numbers for autologous adoptive NK-cell therapy. The aim of this study was to establish a novel method for ex vivo NK-cell expansion from patients with cancer.MethodsNK cells (CD3^?CD56⁺) were isolated from peripheral blood mononuclear cells from healthy volunteers and cancer patients, and NK^? fractions were used as feeder cells. Purified NK cells were co-cultured with feeder cells in AIM-V medium (Invitrogen, Carlsbad, CA, USA) supplemented with 5% human serum and 1000 units/mL human interleukin-2.ResultsNK cells co-cultured with feeder cells from healthy volunteers (feeder-HV) expanded more than NK cells co-cultured with feeder cells from cancer patients (feeder-CP). During the 14-day culture period, NK cells from patients with advanced cancer co-cultivated with feeder-HV expanded on average 300-fold. NK cells co-cultivated with feeder-CP expanded on average 169.4-fold. Cultures grown in the presence of feeder-HV contained 93.8 ± 7.0% (mean ± standard deviation; n = 6) CD3^?CD56⁺ NK cells, and cultures grown in the presence of feeder-CP contained 83.6 ± 15.9% CD3^?CD56⁺ NK cells. Feeder-HV caused a relative increase in CD3⁺CD4⁺ T cells, whereas feeder-CP did not induce changes. Interleukin-15, a cytokine that induces NK-cell proliferation, was detected in the culture supernatants of feeder-HV but not in those of feeder-CP.ConclusionsFeeder cells obtained from healthy volunteers have the potential to expand and activate NK cells from patients with advanced cancer. The novel NK-cell expansion method described here provides a technique for acquiring the large numbers of highly active NK cells from patients with cancer for autologous adoptive immunotherapy.     

Combined treatment of Pseudomonas aeruginosa biofilms with bacteriophages and chlorine

Bacterial biofilms are a growing concern in a broad range of areas. In this study, a mixture of RNA bacteriophages isolated from municipal wastewater was used to control and remove biofilms. At the concentrations of 400 and 4 × 10⁷ PFU/mL, the phages inhibited Pseudomonas aeruginosa biofilm formation by 45 ± 15% and 73 ± 8%, respectively. At the concentrations of 6,000 and 6 × 10⁷ PFU/mL, the phages removed 45 ± 9% and 75 ± 5% of pre‐existing P. aeruginosa biofilms, respectively. Chlorine reduced biofilm growth by 86 ± 3% at the concentration of 210 mg/L, but it did not remove pre‐existing biofilms. However, a combination of phages (3 × 10⁷ PFU/mL) and chlorine at this concentration reduced biofilm growth by 94 ± 2% and removed 88 ± 6% of existing biofilms. In a continuous flow system with continued biofilm growth, a combination of phages (a one‐time treatment at the concentration of 1.9 × 10⁸ PFU/mL for 1 h first) with chlorine removed 97 ± 1% of biofilms after Day 5 while phage and chlorine treatment alone removed 89 ± 1% and 40 ± 5%, respectively. For existing biofilms, a combined use of a lower phage concentration (3.8 × 10⁵ PFU/mL) and chlorination with a shorter time duration (12 h) followed by continuous water flushing removed 96 ± 1% of biofilms in less than 2 days. Laser scanning confocal microscopy supplemented with electron microscopy indicated that the combination treatment resulted in biofilms with lowest cell density and viability. These results suggest that the combination treatment of phages and chlorine is a promising method to control and remove bacterial biofilms from various surfaces. Biotechnol. Bioeng. 2013; 110: 286–295. © 2012 Wiley Periodicals, Inc.     

Isolation and characterization of exosomes from blood plasma of breast cancer and colorectal cancer patients

A simple approach for isolation of exosomes from blood plasma samples has been proposed. Using this approach it is possible to obtain highly purified preparations of microvesicles no larger than 100 nm. The presence of different subpopulations of exosomes isolated by this method has been recognized in the blood plasma of healthy donors and cancer patients. Universal markers CD9, CD24, and CD81 are applicable for routine typing of exosomes isolated from blood plasma samples.     

静注人免疫球蛋白联合万古霉素治疗小儿败血症的疗效及外周血NLR、PCT变化观察

摘要 目的：观察静注人免疫球蛋白联合万古霉素治疗小儿败血症的疗效及外周血中性粒细胞/淋巴细胞比值（NLR）、降钙素原(PCT)变化。方法：选取2011年1月~2020年1月我院收治的败血症患儿80例为研究对象,按数字随机表法分为对照组和观察组各40例,对照组给予万古霉素治疗,观察组在对照组基础上给予静注人免疫球蛋白治疗,比较两组临床疗效、症状改善时间和住院时间、NLR、PCT、超敏C反应蛋白(hs-CRP)、白细胞计数（WBC）、免疫功能及不良反应发生率。结果：观察组治疗有效率高于对照组（87.50%vs65.00%）（P<0.05）。观察组神经系统症状改善时间、体温改善时间、拒奶改善时间和住院时间为（6.22±1.05）d、（3.88±0.25）d、（5.10±0.86）d、（8.71±2.05）d,均短于对照组的（8.76±1.53）d、（6.22±0.64）d、（7.53±1.46）d和（11.24±3.36）d,比较差异有统计学意义（P<0.05）。治疗后观察组外周血NLR、PCT、hs-CRP、WBC水平为（1.35±0.20）、（0.80±0.34）mg/mL、（3.56±0.62）g/L、（9.12±1.80）×10⁹/L,均显著低于对照组的（1.83±0.32）、（2.19±0.73）mg/mL、（9.78±2.64）g/L和（12.26±2.59）×10⁹/L,比较差异有统计学意义（P<0.05）。治疗后观察组CD4⁺、CD3⁺、CD4/CD8为（42.77±11.36）％、（41.27±11.26）％、（1.70±0.33）,均显著高于对照组的（35.80±9.32）％、（35.66±9.40）％和（1.29±0.25）,比较差异有统计学意义（P<0.05）。两组不良反应发生率比较无差异（10.00%vs7.50%）（P>0.05）。结论：静注人免疫球蛋白联合万古霉素治疗小儿败血症的疗效显著,可降低炎症因子,提高免疫功能,且安全性较高。     

微创旋切术用于老年乳腺良性肿块患者对手术指标、应激反应和免疫功能的影响

摘要 目的：对比分析微创旋切术和传统开放手术对老年乳腺良性肿块患者手术指标、应激反应、免疫功能的影响及安全性。方法：收集我院2015年4月~2019年2月因乳腺良性肿块需手术治疗的老年患者148例,按不同手术方式分为观察组和对照组,每组74例。观察组予以微创旋切术,对照组予以传统开放手术,比较两组手术指标,手术前后应激反应、免疫功能,术后乳房美观性和并发症的发生情况。结果：观察组切口长度、手术时间、出血量、住院时间和术后疼痛评分分别为(2.35±1.45)cm、(18.27±4.51)min、(5.07±1.02)mL、(4.98±1.20)d和(2.88±1.13)分,均低于或短于对照组(P<0.05);术后血清去甲肾上腺素(NE)、肾上腺素(E)和皮质醇(Cor)水平分别为(71.03±3.02)ng/mL、(68.22±7.23)ng/mL和(101.82±13.29)mmol/L,均明显低于对照组(P<0.05);手术后免疫功能中CD4⁺、CD3⁺和CD4⁺/CD8⁺分别为(27.27±3.70)%、(44.87±6.13)%和(1.22±0.07),均显著高于对照组(P<0.05);术后乳房美观性优良率为98.6%,明显高于对照组(P<0.05),总并发症发生率为2.7%,显著低于对照组(P<0.05)。结论：与传统开放手术相比,微创旋切术用于老年乳腺良性肿块患者的效果较好,手术时间短,出血少,患者术后疼痛较轻,住院时间短,可有效保护患者的免疫功能,降低应激反应,安全性高。     

Determination of ferulic acid by flow injection chemiluminescence analysis based on enhancement of the N‐bromobutanimide–eosin–CrCl3 system in alkaline solution

A simple and sensitive flow injection chemiluminescence method has been developed for the determination of ferulic acid (FA) based on the significant enhancement effect of FA on the CL signal of the N‐bromobutanimide (NBS)–eosin–CrCl₃ system in alkaline solution. Under optimum conditions, the enhanced CL intensity is linearly related to the concentration of FA in its pharmaceutical preparations and human plasma samples. The corresponding linear regression equations were established over the 4.0 × 10^–10–1.0 × 10^–7 g/mL for FA tablets and 2.0 × 10^–10–1.0 × 10^–7 g/mL for plasma samples. The limit of detection for FA tablets and limit of quantification for plasma samples were 2.8 × 10^–10 g/mL (3 σ) and 3.04 × 10^–10 g/mL (10 σ), respectively. A complete analysis could be performed within 40 s, including washing and sampling, giving a throughput of ≈90/h. The proposed method was successfully applied to the determination of FA in pharmaceutical preparations and human plasma samples with satisfactory results. The recoveries of pharmaceutical preparations and human plasma samples at three different concentrations were 97.8–102.6% and 96.7–104.0%, respectively. Furthermore, the possible mechanism of CL reactions was also discussed briefly. Copyright © 2013 John Wiley & Sons, Ltd.     

Effect of host plant on susceptibility of whitefly Bemisia tabaci (Homoptera: Aleyrodidae) to the entomopathogenic fungus Beauveria bassiana (Ascomycota: Hypocreales)

We determined host plant effect on susceptibility of whitefly Bemisia tabaci to the entomopathogenic fungus Beauveria bassiana under controlled conditions. Insects were reared on cucumber, eggplant, tomato or cabbage. Fungal suspensions of 1×10⁴, 10⁵, 10⁶, 10⁷ and 10⁸ conidia/mL were applied on second-instar nymphs. Nymphal survival significantly differed among different host plant species on which the nymphs were reared. Ten days after inoculation with 1×10⁸ conidia/mL, percent survival was 4.2±0.7, 9.6±0.4, 13.4±0.8, and 24.3±0.9% on cucumber, eggplant, tomato and cabbage, respectively. Average survival times of nymphs were also significantly influenced by host plant species. After inoculation with 1×10⁸ conidia/mL, survival times were 4.8±0.15, 6.0±0.11, 5.7±0.13, and 6.2±0.08 days for nymphs reared on cucumber, eggplant, tomato, and cabbage, respectively. Virulence also differed depending on host plant species; 10 days after inoculation, LC₅₀ values were 4.6×10⁴, 1.6×10⁵, 4.2×10⁵ and 2.1×10⁶ conidia/mL on cucumber, eggplant, tomato and cabbage, respectively. Nymphs on cucumber showed highest susceptibility.     

The influence of viruses on bacterioplankton of the offshore and coastal parts of the Barents Sea

The viral and bacterioplankton communities of the Barents Sea were investigated using a combination of methods of electron and epifluorescence microscopy for the first time. The quantitative composition of the communities and the nature of their interactions were also determined. Our study showed that during the summer the abundance and biomass of bacterioplankton reached 0.4–4.0 × 10⁶ cells/mL and 25.09–84.21 mg/m³ in offshore waters and 0.4–1.8 × 10⁶ cells/mL and 19.63–100.19 mg/m³ in coastal waters, respectively. In both regions, the number of viruses (1.7–35.8 × 10⁶ and 14.5–32.4 × 10⁶ particles/mL) exceeded the number of bacteria by 2–31 and 13–60 times, respectively; the average viral production was 0.7510⁶ and 1.74 × 10⁶ particles/mL/day, respectively. The proportion of infected cells in the total bacterioplankton (7% on average) and virus-induced mortality of bacteria (8%) were much lower in offshore than in coastal waters (14 and 20%, respectively).     

Plasma proteomic signature of age in healthy humans

To characterize the proteomic signature of chronological age, 1,301 proteins were measured in plasma using the SOMAscan assay (SomaLogic, Boulder, CO, USA) in a population of 240 healthy men and women, 22–93 years old, who were disease‐ and treatment‐free and had no physical and cognitive impairment. Using a p ≤ 3.83 × 10^?5 significance threshold, 197 proteins were positively associated, and 20 proteins were negatively associated with age. Growth differentiation factor 15 (GDF15) had the strongest, positive association with age (GDF15; 0.018 ± 0.001, p = 7.49 × 10^?56). In our sample, GDF15 was not associated with other cardiovascular risk factors such as cholesterol or inflammatory markers. The functional pathways enriched in the 217 age‐associated proteins included blood coagulation, chemokine and inflammatory pathways, axon guidance, peptidase activity, and apoptosis. Using elastic net regression models, we created a proteomic signature of age based on relative concentrations of 76 proteins that highly correlated with chronological age (r = 0.94). The generalizability of our findings needs replication in an independent cohort.     

Feasibility and cost analysis of day 4 granulocyte colony-stimulating factor mobilized peripheral blood progenitor cell collection from HLA-matched sibling donors

BackgroundGuidelines recommend treatment with 4–5 days of granulocyte colony-stimulating factor (G-CSF) for optimal donor peripheral blood progenitor cell (PBPC) mobilization followed by day 5 collection. Given that some autologous transplant recipients achieve adequate collection by day 4 and the possibility that some allogeneic donors may maximally mobilize PBPC before day 5, a feasibility study was performed evaluating day 4 allogeneic PBPC collection.MethodsHLA-matched sibling donors underwent collection on day 4 of G-CSF for peripheral blood (PB) CD34⁺ counts ≥0.04 × 10⁶/mL, otherwise they underwent collection on day 5. Those with inadequate collected CD34⁺ cells/kg recipient weight underwent repeat collection over 2 days. Transplant and PBPC characteristics and cost analysis were compared with a historical cohort collected on day 5 per our prior institutional algorithm.ResultsOf the 101 patient/donor pairs, 50 (49.5%) had adequate PBPC collection on day 4, with a median PB CD34⁺ cell count of 0.06 × 10⁶/mL. Day 4 donors were more likely to develop bone pain and require analgesics. Median collected CD34⁺ count was significantly greater, whereas total nucleated, mononuclear and CD3⁺ cell counts were significantly lower, at time of transplant infusion for day 4 versus other collection cohorts. There were no significant differences in engraftment or graft-versus-host disease. Cost analysis revealed 6.7% direct cost savings for day 4 versus historical day 5 collection.DiscussionDay 4 PB CD34⁺ threshold of ≥0.04 × 10⁶/mL identified donors with high likelihood of adequate PBPC collection. Day 4 may be the optimal day of collection for healthy donors, without adverse effect on recipient transplant outcomes and with expected cost savings.     

Zoledronate-activated Vγ9γδ T cell-based immunotherapy is feasible and restores the impairment of γδ T cells in patients with solid tumors

Gamma/delta (γδ) T cells play a role in innate immunity and exhibit cytotoxicity toward a large range of tumor types. Recent studies have shown that aminobisphosphonates may be applied to a culture in which a large number of γδ T cells are proliferated ex vivo. We carried out a clinical study of 25 patients with various solid tumors to determine further the safety, immunologic effect and feasibility of zoledronate-activated Vγ9γδ T cell-based immunotherapy. No severe toxicity was observed. In the cells used for the first treatment, the total cell number, frequency and number of CD3⁺ Vγ9⁺ γδ T cells were 409 ± 284 × 10⁷ cells, 56 ± 33% and 255 ± 242 × 10⁷ cells, respectively. Aminobisphosphonate therapy or chemotherapy resulted in the suppression of CD3⁺ Vγ9⁺ γδ T-cell proliferation. The numbers of CD3⁺ T cells, CD3⁺ Vγ9⁺ γδ T cells and CD27^? CD45RA^? Vγ9⁺ subsets in peripheral blood were significantly lower in patients than in healthy subjects (P <y 0.05). From such an impaired immunologic condition, the numbers and frequencies of CD3⁺ Vγ9⁺ γδ T cells and CD27^? CD45RA^? subsets significantly increased in patients treated with this immunotherapy. Zoledronate-activated Vγ9γδ T cell-based immunotherapy that restores the number of Vγ9γδ T cells in cancer patients may provide another mode of adoptive immunotherapy.