A protein fraction from aged garlic extract enhances cytotoxicity and proliferation of human lymphocytes mediated by interleukin-2 and concanavalin A

Fraction 4 (F4), a protein fraction isolated from aged garlic extract, enhanced cytotoxicity of human peripheral blood lymphocytes (PBL) against both naturalkiller (NK)-sensitive K562 and NK-resistant M14 cell lines. Although F4 treatment alone increased cytotoxicity, the effect was more remarkable when F4 was administered together with suboptimal doses of interleukin-2 (IL-2); combination treatment of 5 g/ml F4 plus 10 U/ml IL-2 for 72 h generated lymphokine-activated killer activity equivalent to that produced by 100 U/ml IL-2 alone against M14. F4 enhanced IL-2-induced proliferation and IL-2 receptor (Tac) expression of PBL without significant increase of IL-2 production. The enhancement of cytotoxicity both by F4 alone and by F4 plus IL-2 was abolished by anti-IL-2 antibody. F4 also enhanced concanavalin-A(ConA)-induced proliferation of PBL. Radiolabeled-ConA binding assays revealed that F4 treatment greatly augmented the affinity and slightly increased the number of ConA binding sites in PBL. F4 also enhanced ConA-induced IL-2 receptor (Tac) expression and IL-2 production of PBL. Anti-IL-2 antibody inhibited the effect of F4 on ConA-induced proliferation. These data suggest that IL-2 is involved in augmentative effects of F4. Our results indicate that F4 is a very efficient immunopotentiator and may be used for immunotherapy.     

Interleukin-5 induces tumor suppression by peritoneal exudate cells in mice

The antitumor activity of peritoneal exudate cells (PEC) induced by murine interleukin-5 (mIL-5) was examined using Meth-A sarcoma cells transplanted into the peritoneal cavity of mice. Although in vitro treatment of Meth-A sarcoma cells with mIL-5 did not result in inhibition of their growth, treatment of mice intraperitoneally with mIL-5 (1 g/day) from day –5 to +5 (tumor cells were inoculated on day 0) led to a significant increase in survival or even rejection of tumor cells. This antitumor effect depended on the dose of mIL-5. Interestingly, there was identical therapeutic activity when the protocol of days –10 to –1 was used as opposed to –5 to +5. In addition, post-treatment with mIL-5 from day +1 to +10 was ineffective. This suggests that the therapeutic activity of IL-5 is largely prophylactic. Under the former condition, the number of PEC was found to increase over 50-fold when compared to levels in control mice. Moreover, the antitumor effect of mIL-5 was completely abolished by subcutaneous injection of anti-mIL-5 monoclonal antibodies. The treatment of mice injected intraperitoneally with human IL-2 also resulted in an increase in survival. Winn assay experiments using PEC recovered from mIL-5-treated mice (1g/day, from day –10 to –1) revealed that these PEC could mediate antitumor activity against Meth-A sarcoma cells. Furthermore, when the cured mice were re-injected with Meth-A sarcoma cells or syngeneic MOPC 104E cells, they could reject Meth-A sarcoma cells but not MOPC 104E cells, indicating that immune memory had been generated. These results suggest that IL-5 augumented the PEC tumoricidal activity but we have no indication that the tumoricidal activity was mediated through a mIL-5-dependent mechanism.     

Prevention of lymph node metastases by adoptive transfer of CD4+ T lymphocytes admixed with irradiated tumor cells

CD4⁺8^– T lymphocytes with potent antitumor activity in vivo were obtained in peritoneal exudate cells by immunizing mice with irradiated MM48 tumor cells admixed with OK-432. These immune CD4⁺ T cells were used in adoptive immunotherapy for prevention of lymph node metastases after removal of the primary tumor. Complete cure of metastases was obtained by adoptive transfer of CD4⁺ T cells admixed with irradiated MM48 tumor cells, but not by CD4⁺ T cells alone. To analyze the curative effect of admixing tumor cells on the prevention of metastases, a model of 1-day tumor inoculated with macrophages was used. Administration of immune CD4⁺ T cells alone resulted in the regression of local tumor in more than half of the mice, although all of them eventually died of lymph node metastases. On the other hand, adoptive transfer of immune CD4⁺ T cells plus irradiated tumor cells resulted in the complete regression of local tumors in all the mice, which survived without any sign of metastasis. The curative effect of the immune CD4⁺ T cells obtained by admixing irradiated tumor cells was tumor-specific. Macrophages induced by OK-432 (tumoricidal), implanted together with tumor, assisted tumor regression more than did macrophages elicited by proteose peptone (nontumoricidal) in the same adoptive transfer system. Administration of recombinant interleukin-2 instead of stimulant tumor cells did not enhance, but rather eliminated the constitutive antitumor activity of CD4⁺ T cells. On the other hand, exogenous recombinant interleukin-1 was more effective in the enhancement of antitumor activity of the CD4⁺ T cells as compared with stimulant tumor cell administration. In this case, the activating states of macrophages at the implanted tumor site had no influence on the therapeutic efficacy. A possible role of macrophages for induction of tumor-specific cytotoxic T cells that were mediated by tumor-specific CD4⁺ T cells is discussed.     

Characterization of a quail suspension cell line for production of a fusogenic oncolytic virus

The development of efficient processes for the production of oncolytic viruses (OV) plays a crucial role regarding the clinical success of virotherapy. Although many different OV platforms are currently under investigation, manufacturing of such viruses still mainly relies on static adherent cell cultures, which bear many challenges, particularly for fusogenic OVs. Availability of GMP-compliant continuous cell lines is limited, further complicating the development of commercially viable products. BHK21, AGE1. CR and HEK293 cells were previously identified as possible cell substrates for the recombinant vesicular stomatitis virus (rVSV)-based fusogenic OV, rVSV-NDV. Now, another promising cell substrate was identified, the CCX.E10 cell line, developed by Nuvonis Technologies. This suspension cell line is considered non-GMO as no foreign genes or viral sequences were used for its development. The CCX.E10 cells were thus thoroughly investigated as a potential candidate for OV production. Cell growth in the chemically defined medium in suspension resulted in concentrations up to 8.9 × 10⁶ cells/mL with a doubling time of 26.6 h in batch mode. Cultivation and production of rVSV-NDV, was demonstrated successfully for various cultivation systems (ambr15, shake flask, stirred tank reactor, and orbitally shaken bioreactor) at vessel scales ranging from 15 mL to 10 L. High infectious virus titers of up to 4.2 × 10⁸ TCID₅₀/mL were reached in orbitally shaken bioreactors and stirred tank reactors in batch mode, respectively. Our results suggest that CCX.E10 cells are a very promising option for industrial production of OVs, particularly for fusogenic VSV-based constructs.     

Immunogenicity of recombinant human interleukin-2: Biological features and clinical relevance

The immunogenicity of recombinant interleukin-2 (rIL-2, EuroCetus, Amsterdam, Netherlands) was studied in seventy-six patients receiving different subcutaneous immunotherapy regimens. Patients presented with progressive metastatic renal cell carcinoma, malignant melanoma, colorectal cancer, B-cell lymphoma, and Hodgkin's disease. An enzyme immunoassay (EIA) was employed to screen patients for development of non-neutralizing antibodies against rIL-2, antibody specificity was confirmed by a standard Western blot. Neutralizing serum activity against rIL-2 was detected using a standard CTLL mouse proliferation assay. Additionally, serum levels of soluble interleukin-2 receptors and lymphocyte subsets expressing the CD56 natural killer (NK) associated antigen were measured.In a proportion of approximately 35% to 90% of the patients treated, non-neutralizing antibodies against rIL-2 could be detected after all treatment courses were evaluated. Antibodies were of the IgG, IgM, IgA and IgD subtypes. None of the 76 patients exhibited serum neutralizing activity after one treatment course. Five patients exhibited neutralizing anti-rIL-2 serum activity after two or more treatment courses of systemic rIL-2. In three of these patients, antibodies neutralized both recombinant and natural IL-2. Patients developing neutralizing anti-rIL-2 antibodies, exhibited significantly lower serum sIL-2 receptor levels upon the emergence of serum neutralizing activity than patients without antibody. Additionally, NK cell associated CD56 positivity was significantly lower in patients who exhibited neutralizing anti-rIL-2 serum activity than in patients who did not. A significant decrease in levels of soluble IL-2 receptors and CD56 NK cell positivity was observed, when comparing values prior to and after onset of serum neutralizing activity against rIL-2. However, while emergence of neutralizing antibodies to rIL-2 diminished rIL-2 induced biological activation, it did not coincide with abrogation of treatment response.Abbreviations rIL-2 recombinant interleukin-2 - EIA enzyme immuno assay - rIFN-2 recombinant interferon- 2     

Improved pulse sequences for measuring coupling constants in 13C, 15N-labeled proteins

Summary NMR pulse sequences for measuring coupling constants in ¹³C, ¹⁵N-labeled proteins are presented. These pulse sequences represent improvements over earlier experiments with respect to resolution and number of radiofrequency pulses. The experiments are useful for measuring J_NH , J_NCO, J_NC , J_H ^N _CO and J_H ^N _H . Applications to chymotrypsin inhibitor 2 (CI-2) are shown.     

3D 13C/1H NMR-based assignments for side-chain resonances of Lactobacillus casei dihydrofolate reductase. Evidence for similarities between the solution and crystal structures of the enzyme

Summary ¹³C-based three-dimensional ¹H–¹H correlation experiments have been used to determine essentially complete ¹³C and ¹H resonance assignments for the amino acid side chains of uniformly ¹³C/¹⁵N labelled L. casei dihydrofolate reductase in a complex with the drug methotrexate. Excellent agreement is observed between these assignments and an earlier set of partial assignments made on the basis of correlating nuclear Overhauser effect and crystal structure data, indicating that the tertiary structure of the enzyme is similar in solution and in the crystal state.To whom correspondence should be addressed.     

血清GDF-15、hs-cTnT对冠状动脉旁路移植术后新发房颤及近期主要心血管事件的预测效能研究

摘要 目的：分析血清生长分化因子15（GDF-15）、高敏心肌肌钙蛋白T（hs-cTnT）对冠状动脉旁路移植术后新发心房颤动（房颤）及近期主要心血管事件的预测效能。方法：选择自2020年1月至2022年1月在我院行冠状动脉旁路移植术的140例冠心病患者作为研究对象,根据术后是否新发房颤,分为房颤组（46例）和非房颤组（94例）。检测两组术前血清GDF-15、hs-cTnT水平,使用多因素Logistic回归分析血清GDF-15、hs-cTnT与术后新发房颤的关系;随访6个月,观察主要心血管事件发生情况,通过受试者工作特征曲线下面积（AUC）评价血清GDF-15、hs-cTnT对术后新发房颤及近期主要心血管事件的预测效能。结果：两组患者年龄、性别、体重指数等一般资料比较无差异（P＞0.05）;房颤组SYNTAX积分高于非房颤组,差异有统计学意义(P＜0.05);房颤组血清GDF-15、hs-cTnT水平均高于非房颤组（P＜0.05）;经多因素Logistic回归分析,SYNTAX积分、血清GDF-15、hs-cTnT均是冠状动脉旁路移植术后新发房颤的独立预测因素（P＜0.05）;经ROC曲线分析,血清GDF-15联合hs-cTnT预测冠状动脉旁路移植术后新发房颤的AUC为0.933,大于SYNTAX积分的0.790,预测近期主要心血管事件的AUC为0.925,大于SYNTAX积分的0.750（P＜0.05）。结论：血清GDF-15联合hs-cTnT对冠状动脉旁路移植术后新发房颤及近期主要心血管事件均具有良好的预测效能,值得临床予以重视。     

燕麦-绿豆间作效应及氮素转移特性

为探究燕麦(Avena sativa)-绿豆(Phaseolus radiatus)间作效应及氮素转移特性, 在不施氮肥的大田试验条件下, 设置3种种植模式(燕麦单作、绿豆单作和燕麦-绿豆间作), 采用传统挖根法和¹⁵N同位素标记法进行研究。结果表明, 间作系统中燕麦侵袭力强于绿豆, 绿豆生长受到抑制。整个生育期, 间作燕麦地上部干物质积累量比单作增加14.9%-33.1%, 2年成熟期间作燕麦的氮素积累量比单作分别提高53.1%和44.8%; 间作减少了开花结荚期绿豆氮素积累量和根瘤重量, 降低了绿豆的固氮效率, 绿豆的固氮效率2年平均降低23.7%, 生物固氮量平均减少11.66%。间作绿豆向燕麦的氮素转移率2年平均值达31.7%, 氮素转移量为212.16 kg∙hm^-2。燕麦-绿豆间作降低了开花结荚期绿豆的根瘤固氮酶活性和固氮效率, 但绿豆体内氮素转移增加了燕麦对氮素的吸收利用, 实现了地上部与地下部生长的相互调节和促进, 优化了农田生态系统的氮素管理。     

Soil 13C–15N dynamics in an N2-fixing clover system under long-term exposure to elevated atmospheric CO2

Reduced soil N availability under elevated CO₂ may limit the plant's capacity to increase photosynthesis and thus the potential for increased soil C input. Plant productivity and soil C input should be less constrained by available soil N in an N₂‐fixing system. We studied the effects of Trifolium repens (an N₂‐fixing legume) and Lolium perenne on soil N and C sequestration in response to 9 years of elevated CO₂ under FACE conditions. ¹⁵N‐labeled fertilizer was applied at a rate of 140 and 560 kg N ha^?1 yr^?1 and the CO₂ concentration was increased to 60 Pa pCO₂ using ¹³C‐depleted CO₂. The total soil C content was unaffected by elevated CO₂, species and rate of ¹⁵N fertilization. However, under elevated CO₂, the total amount of newly sequestered soil C was significantly higher under T. repens than under L. perenne. The fraction of fertilizer‐N (f_N) of the total soil N pool was significantly lower under T. repens than under L. perenne. The rate of N fertilization, but not elevated CO₂, had a significant effect on f_N values of the total soil N pool. The fractions of newly sequestered C (f_C) differed strongly among intra‐aggregate soil organic matter fractions, but were unaffected by plant species and the rate of N fertilization. Under elevated CO₂, the ratio of fertilizer‐N per unit of new C decreased under T. repens compared with L. perenne. The L. perenne system sequestered more ¹⁵N fertilizer than T. repens: 179 vs. 101 kg N ha^?1 for the low rate of N fertilization and 393 vs. 319 kg N ha^?1 for the high N‐fertilization rate. As the loss of fertilizer‐¹⁵N contributed to the ¹⁵N‐isotope dilution under T. repens, the input of fixed N into the soil could not be estimated. Although N₂ fixation was an important source of N in the T. repens system, there was no significant increase in total soil C compared with a non‐N₂‐fixing L. perenne system. This suggests that N₂ fixation and the availability of N are not the main factors controlling soil C sequestration in a T. repens system.