Antitumor vaccination with gene-transduced tumor cells expressing a fusion protein RM4/IFN-tau

Our previous studies showed that secretion of a fusion protein RM4/IFN-tau from a mouse myeloma cell line VKCK/RM4-IFN-tau curtailed its tumorigenicity. Inoculation of VKCK/RM4-IFN-tau tumor cells further induced a protective immunity against a secondary challenge of parental VKCK tumor cells, in which the predominant immune cellular components are CD8+ T cells. In this study, VKCK/RM4-IFN-tau cell line was again used to further characterize the protective immunity. We found that the reduced tumorigenicity of VKCK/RM4-IFN-tau was directly related to the amount of fusion protein secreted by tumor cells, and that CD8+ T cells derived from mice experienced with VKCK/RM4-IFN-tau tumor regression played an important role in the protective immunity in a chromium release assay in vitro and in an animal study in vivo by using T-cell subset depleted mice. Our animal studies also showed that not only the cytotoxic but also the memory T cells against the secondary challenge of parental VKCK cells could be adoptively transferred to normal BALB/c mice. In addition, our animal studies further showed that local vaccination of irradiated VKCK/RM4-IFN-tau cells was able to significantly inhibit established tumors in early stages in vivo. This study thus highlights the potential utility of this engineered VKCK/RM4-IFN-tau tumor cells secreting the fusion protein RM4/IFN-tau in cancer gene therapy.     

mB7-1-GPI融合蛋白对小鼠肺癌的治疗作用研究

目的 将mB7-1-GPI融合蛋白锚定于小鼠肺癌细胞膜上，制备肿瘤细胞疫苗，研究该瘤苗的抗肿瘤作用。方法将mB7-1-GPI融合蛋白锚定于小鼠肺癌细胞膜上，制备肿瘤细胞疫苗。采用流式细胞术检测该蛋白的细胞膜锚定作用。建立C57BL小鼠的肺癌模型，观察用mB7-1-GPI融合蛋白制备的肿瘤疫苗对荷瘤小鼠的免疫治疗作用。结果mB7-1-GPI融合蛋白能够锚定在小鼠肺癌细胞膜上，能有效刺激小鼠脾细胞增殖和分泌IL-2和IFN-γ。融合蛋白制备的肿瘤疫苗能够显著抑制荷瘤小鼠肿瘤的生长，并延长其生存期。结论mB7-1-GPI融合蛋白制备的肿瘤疫苗具有较强的抗肿瘤作用，有可能作为一种有效的新型疫苗用于肿瘤的治疗和预防。     

Antitumor effect of OK-432 (3)--mechanisms of tumor growth inhibition by OK-432 induced activated macrophages

Spleen cells and peritoneal exudate cells obtained from BALB/c mice which had received an i.p. injection of 0.1 mg of OK-432 4 days previous to sacrifice, were examined by Winn's neutralization assay for their antitumor activity against Meth-A sarcoma cells in BALB/c mice. Both of the cell preparations clearly inhibited the growth of admixed Meth-A cells, but when these same cell populations were treated on a Sephadex G-10 column, the effector activity seen in Winn's assay disappeared. The effector cells responsible for tumor inhibition were therefore considered to be cytotoxic macrophages. However, the inhibitory effect of these cytotoxic macrophages in Winn's assay was not evident in either X ray (300 rad)-irradiated BALB/c mice or in nu/nu BALB/c mice. These results indicate that the antitumor activity of cytotoxic macrophages is associated with a sequential immune mechanism in which T cells may play an important role.     

BCG及IL-2激活膀胱癌患者PBMC及TIL细胞的抗癌活性比较

 用Whitesides法从15例膀胱移行细胞癌患者外周血及癌手术标本中获得的外周血单个核细胞（PBMC）及肿瘤浸润淋巴细胞（TIL）,分别用活BCG、死BCG及IL－2刺激培养,计算细胞增殖倍数并测定其抗癌活性。结果,死BCG对细胞并无激活作用;IL－2对细胞的诱导增殖作用强于活BCG,而其激活这两种细胞的抗癌活性低于活BCG激活的抗癌活性。BCG对免疫受抑制的PBMC及YIL细胞的激活作用可能是BCG抗肿瘤重要作用机理之一。并为BCG的进一步临床免疫治疗奠定理论基础。     

PD-S15 融合蛋白体外特异性靶向PD-1 分子并快速扩增NK/T细胞

[摘要] 目的：探讨anti-PD-1（scFv）/IL-15/IL-15Rα-sushi （简称PD-S15）融合蛋白体外特异性结合PD-1 的能力及其对NK/T细胞增殖能力的影响。方法：化学合成人anti-PD-1（scFv）基因和人IL-15/IL-15Rα-sushi 融合基因,经过酶切连接构建重组表达质粒pUC57-PD-S15,用LipofectamineTM 2000 瞬时转染HEK293T细胞,收获细胞培养液上清,用Wb法检测细胞培养液上清中PDS15融合蛋白的表达;用不同配比的PD-S15/X-VIVOTM15 培养液对PBMC和TIL 分别诱导培养后,用流式细胞术检测PD-S15 融合蛋白体外特异性结合PD-1 的能力和对PBMC增殖及CD3+CD8+、CD3+CD4+、CD3-CD56+各细胞亚群比例的影响;用细胞计数法检测PD-S15 融合蛋白对TIL 增殖能力的影响。结果：pUC57-PD-S15 表达质粒经双酶切和测序验证构建成功并成功转染HEK293T细胞,目标蛋白相对分子质量大约为55 000,符合预期。PD-S15 融合蛋白体外具有PD-1 特异性结合能力（P<0.05）及NK/T细胞活化增殖能力（P<0.05）;与经典TIL 培养方案相比,PD-S15 培养方案体外活化扩增T细胞的能力更强（P＜0.01）。结论：PD-S15 融合蛋白体外能够特异性靶向PD-1 分子并快速扩增NK/T细胞,为后续从肿瘤组织内或外周血中选择性扩增CD8+PD-1+抗原特异性T细胞奠定了基础。     

树突状细胞与肾癌融合细胞疫苗诱导抗肿瘤免疫

目的探讨自体树突状细胞(DC)与肾癌融合细胞疫苗诱导特异性抗肿瘤免疫应答。方法利用肿瘤细胞纯化技术,从手术切除的肾癌组织中分离出高纯度的肾癌细胞,用10%胎牛血清(FCS)的RPMI-1640进行原代培养。分离外周血单核细胞(PBMCs),在含重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)和白细胞介素4(rhIL-4)的培养条件下,诱导分化为DC。用细胞融合技术将DC与肾癌细胞融合制备DC与肾癌融合细胞疫苗。用流式细胞仪检测肾癌细胞、DC和融合细胞的表型;用噻唑盐(MTT)比色法检测融合细胞刺激自体T细胞的增殖能力,乳酸脱氢酶(LDH)释放的细胞毒实验检测融合细胞刺激的细胞毒性T淋巴细胞(CTLs)的杀瘤活性。结果DC高表达主要组织相融性复合体(MHC)Ⅰ类分子、MHCⅡ类分子和共刺激分子(CD86,CD80);原代肾癌细胞高表达一种高分子量的糖蛋白(MUC-1);自体DC与肾癌融合细胞疫苗同时表达肾癌细胞和DC细胞的表面抗原,并能有效刺激自体T细胞增殖,诱导产生的CTLs对自体肾癌细胞具有显著的杀伤活性。结论自体DC与肾癌融合细胞疫苗能诱导特异性抗自体肾癌细胞的免疫应答,为自体DC与肾癌融合细胞疫苗在治疗肾癌的临床应用中提供了实验依据。     

Significant tumor regression induced by microencapsulation of recombinant tumor cells secreting fusion protein

Implantation of microencapsulated engineered cells secreting molecules with antineoplastic properties into tumors is a novel approach to cancer gene therapy. In this study, we constructed an engineered tumor cell line, VkCk/RM4-TNF-alpha, which secreted RM4/TNF-alpha fusion protein containing the chimeric antitumor antibody, F(ab')2 (RM4), recognizing the tumor antigen TAG72, as well as the TNF-alpha moiety. The engineered cells were encapsulated into microencapsules. The RM4/TNF-alpha fusion protein secreted by encapsulated VkCk/RM4-TNF-alpha cells could be diffused through the microencapsule membrane into the supernatant and exert a cytotoxic effect on L929 cells in vitro. The antigen-specific binding-reactivity of RM4/TNF-alpha for the TAG72 antigen was confirmed by immunohistochemical staining of rat LMCR tumor cells which expressed TAG72 antigen. Implantation of microencapsules containing VkCk/RM4-TNF-alpha cells into LMCR tumors in rats induced tumor regression as a result of tumor necrosis formation. Taken together, these data suggest that microencapsulation of recombinant tumor cells secreting antibody/cytokine fusion protein might be an alternative approach in the treatment of cancers.     

A recombinant cell-permeable p53 fusion protein is selectively stabilized under hypoxia and inhibits tumor cell growth

More than 50% of human tumors contain a mutation in p53. Over 90% of tumors are solid tumors. Solid tumors have low oxygenated regions, called hypoxic regions where the tumor cells are more resistant to radio- and chemo-therapy than their well-oxygenated counterparts. In this study, we constructed a cell-permeable p53 fusion protein with selective stability in the hypoxic region. The fusion protein contained the TAT peptide for transduction across membranes, the oxygen-dependent degradation domain of hypoxia-inducible factor-1alpha and wild-type p53. This protein was effectively delivered into tumor cells where it exerted anticancer activity leading to the inhibition of cancer cell growth in vitro and the reduction of tumor weight in vivo. Hence, the fusion protein can be a novel protein drug for antitumor therapies, especially for hypoxic tumor cells.     

Antitumor efficacy of activated macrophages against murine glioma cells

A crucial manifestation of malignant gliomas is the regrowth of already-invaded neoplastic cells after surgical intervention. One possible approach for inhibiting such tumor growth is to utilize the tumoricidal potential of macrophages. In order to investigate the clinical application of this concept, peritoneal exudate cells (PEC) activated in vitro and in vivo by immunomodulating agents were tested for cytotoxic activity against murine glioma (203-glioma) cells. As immunomodulating agents, heat-killed Propionibacterium acnes (P. acnes), OK-432 and Concanavilin A supernatant (Con A sup) were used in these experiments. P. acnes was provided by Kowa Pharmaceutical Co., Tokyo, and OK-432 by Chugai Pharmaceutical Co., Ltd., Tokyo. Klinische Einheit (KE) units were used to express the strength of the preparation, with 1 KE equal to 0.1 mg of dried streptococci. Con A sup was produced by Con A pulsing of BALB/c splenocytes resuspended in complete medium. PEC harvested from mice to which 5% glycogen in saline had been inoculated intraperitoneally 6 d previously were activated in vitro by P. acnes (P. acnes-PEC), OK-432 (OK-432-PEC) and Con A sup (Con A-PEC). The cytotoxic activities of P. acnes-PEC, OK-432-PEC and Con A-PEC were approximately 25%, 65% and 60%, respectively. PEC were then collected from mice into which either 100 micrograms of P. acnes or 1 KE of OK-432 had been injected intraperitoneally several times. The antitumor effects of P. acnes-PEC and OK-432-PEC were about 35% and 50%, respectively. These activated PEC demonstrated cytotoxic activity against murine glioma in the tumor neutralization assay (Winn assay). Also, the antitumor efficacy of OK-432-PEC belonged mainly to adherent cells. Meningeal gliomatosis (MG) models were prepared for clinical studies. Viable 203-glioma cells (5 X 10(6) were injected percutaneously into the cisterna magna of C57BL/6 mice. The median survival time (MST) of the untreated group was 8.5 days. The MST of the groups treated by intraperitoneal and intracisternal administration of P. acnes were 26 and 33 days. This therapy significantly prolonged the survival time of these models, particularly by the intracisternal treatment. The differential cell count by Giemsa staining and latexphagocytic cell findings revealed that macrophages accounted for more than 90% of the P. acnes-PEC. These results may indicate that activated (PEC) macrophages were induced intracisternally by P. acnes and that activated macrophages induced intraperitoneally exerted antitumor effects in MG models.     

The lifetime of hypoxic human tumor cells

Purpose: For hypoxic and anoxic cells in solid tumors to be a therapeutic problem, they must live long enough to be therapeutically relevant, or else be rapidly recruited into the proliferating compartment during therapy. We have, therefore, estimated lifetime and recruitment rate of hypoxic human tumor cells in multicell spheroids in vitro, or in xenografted tumors in SCID mice.Materials and Methods: Cell turnover was followed by flow cytometry techniques, using antibodies directed at incorporated halogenated pyrimidines. The disappearance of labeled cells was quantified, and verified to be cell loss rather than label dilution. Repopulation was studied in SiHa tumor xenografts during twice-daily 2.5-Gy radiation exposures.Results: The longevity of hypoxic human tumor cells in spheroids or xenografts exceeded that of rodent cell lines, and cell turnover was slower in xenografts than under static growth as spheroids. Human tumor cells remained viable in the hypoxic regions of xenografts for 4–10 days, compared to 3–5 days in spheroids, and 1–3 days for most rodent cells in spheroids. Repopulation was observed within the first few radiation treatments for the SiHa xenografts and, with accumulated doses of more than 10 Gy, virtually all recovered cells had progressed through at least one S-phase.Conclusion: Our results suggest an important difference in the ability of human vs. rodent tumor cells to withstand hypoxia, and raise questions concerning the increased longevity seen in vivo relative to the steady-state spheroid system.     

Antitumor effect of tumor necrosis factor against various primarily cultured human cancer cells

The antitumor activity of tumor necrosis factor (TNF) against various primarily cultured human cancer cells (32 cases) was investigated by the 51Cr cytotoxic release assay and the tumor stem cell assay. Over 50% sensitivity (the ratio to the cytotoxicity in L929 cells) was noted in 4 of 14 cases of gastric cancer (28.6%), 7 of 9 cases of leukemic cells (77.8%), and 1 case each of pancreatic carcinoma and ovarian cancer. Scarcely any sensitivity, however, was observed in 1 case of acute promyelocytic leukemia or in some of the gastric cancer cases. No correlation was observed between the histological type of the cancer and TNF sensitivity. The above results seem to confirm that TNF has significant antitumor activity against human cancer cells.     

Antitumor effect of the tumor necrosis factor against various types of human cancer cells

The antitumor activity of tumor necrosis factor (TNA) against various human cancer cells (32 cases) was investigated by 51Cr cytotoxic release assay and tumor stem cell assay. Over 50% sensitivity (the ratio of cytotoxicity for L929 cells) was shown by 4 of 14 cases of gastric cancer (28.6%), 7 of 9 cases of leukemic cells (77.8%), and 1 case each of pancreatic carcinoma and ovarian cancer. However, scarcely any sensitivity was shown by APL, a portion of the gastric cancer cells, normal lymphocytes or colony-forming cells tested. No correspondence was observed between the histological type of the cancer and TNF sensitivity. The above results seem to confirm the significant antitumor activity of TNF against human cancer cells.     

scFv-sTRAIL融合蛋白靶向诱导肿瘤细胞凋亡的研究进展

随着分子生物学技术的进步和在分子水平上对恶性肿瘤发病机制认识的不断加深,以细胞受体、表面抗原、关键基因和细胞内调控分子为靶点的分子靶向治疗已成为辅助传统治疗的合理选择。抗体导向治疗是一种新颖的分子靶向治疗手段。目前已研发多种抗体导向类抗癌药物,这类药物由特异性识别肿瘤细胞的靶向部分和杀伤肿瘤的效应部分组成,因而具有较强的靶向杀伤肿瘤细胞作用,同时可有效降低药物对局部正常组织和全身的系统性毒性作用。利用基因工程技术将人类血清可溶性肿瘤坏死因子相关凋亡诱导配体(serum-soluble TNF-related apoptosis-inducing ligand,sTRAIL)与抗肿瘤单链抗体(single chain antibody fragment,scFv)融合,构建scFv-sTRAIL融合蛋白,是一种理想的抗体导向治疗策略。通过scFv与肿瘤细胞所特有的细胞表面抗原的特异结合,加强sTRAIL在肿瘤病灶的富集,靶向诱导肿瘤细胞凋亡,从而获得增强抗体的疗效和更高的药物安全性。     

Effects of anthracyclines on oxygenated and hypoxic tumor cells

The cytotoxic effects of anthracyclines and other chemotherapeutic agents were examined in normally aerated and hypoxic Sarcoma 180 and EMT6 tumor cells in vitro. Adriamycin, daunomycin, and mitomycin C were selectively toxic to hypoxic Sarcoma 180 cells. The augmented sensitivity was not the result of an increase in susceptibility of oxygen-deprived cells toward antitumor agents in general. 1,3-Bis(2-chloroethyl)-1-nitrosourea, for example, exhibited equal cytotoxicity toward normally aerated and hypoxic cells, while streptonigrin was selectively toxic toward normally aerated cells. The cellular levels of [3H]daunomycin in both Sarcoma 180 and EMT6 cells were not different under the two conditions of oxygenation, and no greater production of either the alcohol or aglycone metabolites of daunomycin occurred in hypoxic cells, compared with their normally aerated counterparts. In addition, analysis of cellular pellets for residual drug remaining after exhaustive extraction showed no significant difference between normally aerated and hypoxic cells. The effects of reoxygenation of hypoxic cells on their sensitivity to mitomycin C and to Adriamycin were studied in both Sarcoma 180 and EMT6 cells. The enhanced efficacy of mitomycin C as a cytotoxic agent observed under hypoxia was reversed after a 2-hr reoxygenation. In contrast, the augmented toxicity of Adriamycin toward hypoxic cells was not reversible in either cell line after 2 or 4 hr of reoxygenation. The results suggest that neither the formation of a reactive oxygen species nor direct involvement of an alkylating agent generated by drug metabolism is an obligatory step in the cytotoxic action of these anthracyclines.     

Antitumor effect of carboplatin on in vitro three-dimensional tumor]

We studied the antitumor effect of Carboplatin on the three-dimensional tumor in vitro. First, we observed the three-dimensional growth of cultured tumor cell lines (Hep 2, KB and HT 29 cells), which were developed in the combined culture system of fibrin matrix and agar culture. The tumor cells developed a 3 x 3 mm tumor in diameter in the in vitro 10 day-culture system. This size was large enough to allow the histologic study. When we applied Carboplatin to the three-dimensional tumors, histologic change of the three-dimensional tumor developed from Hep 2 and KB cells were observed in a dose dependent manner. However, no remarkable histologic change was observed in the three-dimensional tumor developed from HT 29 cells. These results suggest the experimental system of three-dimensional tumor can predict histologically the antitumor effect of Carboplatin on various tumors at the solid tumor level.     

Antitumor effect of Cepharanthin in the double grafted tumor system

The antitumor effect of Cepharanthin (CR) in a new experimental mouse model was studied. Intratumoral administration of CR strongly inhibited the growth of Meth-A solid tumors in male BALB/c mice and led to a complete regression of tumors and resistance to reinoculated tumor. Subsequently, the antimetastatic effect of CR was examined in the double grafted tumor system, in which mice first received simultaneous intradermal inoculations of Meth-A in both right (10(6) cells) and left (2 x 10(5) cells) flanks and were then injected with 0.5 mg of CR in the right tumor on days 3, 4 and 5. CR inhibited the growth of not only the right but also the left, nontreated tumor. Immunized spleen cells were taken from mice which had been cured with the intratumoral administration of CR. Adoptive transfer of CR immunized spleen cells caused the complete regression of Meth-A tumors. The effector cell activity was lost only after treatment with anti-Lyt-1 antibody. These results suggest that intratumoral administration of CR might induce Lyt-1 positive cytotoxic cells in the spleen and the left, non-treated tumor. In BALB/c nude mice, CR inhibited the growth of the right tumor but did not the left tumor. Therefore, the antitumor activity of CR on the left tumor in the double grafted tumor system is associated with a sequential immune mechanism in which T cells may play an important role. The antitumor effect of CR on the right tumor is direct cytotoxic effect. TILs (tumor infiltrating lymphocytes) obtained from left and right sides tumors treated with CR were examined by Winn assay for their antitumor activity against Meth-A sarcoma in BALB/c mice. TILs from both sides clearly inhibited the growth of admixed Meth-A cells and seems to play an important role on antitumor effect in both tumors.     

Endostatin-cytosine deaminase fusion protein suppresses tumor growth by targeting neovascular endothelial cells

Endostatin, an angiogenesis inhibitor tested in multiple clinical trials, selectively targets neovascular endothelial cells, suppressing tumor growth. To enhance the therapeutic efficacy of endostatin, we fused endostatin with cytosine deaminase, which converts a prodrug 5-flucytosine into a cytotoxic 5-fluorouracil. This therapeutic strategy was developed based on the observation that the endostatin-green fluorescence protein gene and endostatin-luciferase gene selectively target to endothelial cells in vitro and to the tumor site in vivo, respectively. When we used the endostatin-cytosine deaminase fusion protein to treat s.c. grafted tumors or experimental metastasis tumors, our results showed that endostatin-cytosine deaminase treatment provided stronger tumor growth suppression and increased mean survival time of the mice compared with the treatments of endostatin alone, cytosine deaminase alone, or endostatin plus cytosine deaminase. The endostatin-cytosine deaminase protein significantly inhibited the growth of endothelial cells and preferentially induced tumor cell apoptosis. This endostatin-cytosine deaminase fusion approach opens an avenue for cancer-targeting therapy.     

树突细胞-食管癌融合细胞体外诱导特异性抗食管癌免疫反应

背景与目的:树突细胞(dendritic cells,DC)是人体专职的抗原呈递细胞,以DC为基础的DC/肿瘤细胞融合疫苗可以有效地诱导特异性抗肿瘤免疫应答.本研究旨在探讨成熟的DC与人食管癌细胞株ECl09细胞融合疫苗体外诱导特异性抗食管癌细胞的免疫反应.方法:从健康志愿者外周血中分离出单个核细胞(peripheral blood mononuclear cells,PBMC),在重组人粒/巨噬细胞集落刺激因子(recombinant human granulocyte-macrophage-colony-stimulating factor,rhGM-CSF)和白介素(interleukin-4,IL-4)作用下体外诱导DC,采用聚乙二醇(polyethylene glyco1,PEG)融合法使DC与EC109细胞融合制备DC/EC109细胞融合疫苗,四甲基偶氮唑盐(MTT)实验检测融合疫苗刺激T淋巴细胞增殖的活性,乳酸脱氢酶(LDH)实验检测融合疫苗活化的细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)在体外特异性杀伤EC109细胞的活性,并与对人胃癌细胞株SGC7901及人乳腺癌细胞株MCF7的杀伤作用进行比较.结果:DC与EC109细胞的融合效率最高达到22.25%.DC/EC109细胞融合疫苗能有效地刺激T淋巴细胞的增殖反应,其刺激T淋巴细胞增殖的能力显著高于DC或EC109细胞(P＜0.05).DC/EC109细胞融合疫苗活化的CTL对EC109细胞具有特异性的杀伤作用,对EC109细胞的杀伤作用显著强于对SGC7901细胞及MCF7细胞的杀伤作用(P＜0.05).结论:DC/EC109细胞融合疫苗能有效诱导抗EC109细胞的特异性免疫应答.     

Lipoproteins in hypoxic tumor cells as traps of free radicals

We examined the role of main cell protective mechanisms in retaining the high resistance of ascitic cells (EAC, ZAH) to lipid peroxidation with respect to different stages of tumor-organism metabolic interactions. The following mechanisms were studied: (1) the activity of main EAC enzymatic antioxidants (GSH-Px, SOD); (2) changes in lipid metabolism, especially the content of the main PUFA (llnoleic and arachidonic fatty acids) in EAC cells; (3) comparison of intracellnlar resistance between EAC/ZAH cytoplasmic sections (containing LP-granules or not) to lipid peroxidation (initialized directly by UV-light). We found that the high resistance to lipid peroxidation was typical for cytoplasma sections (without LP-grannles) on all stages of tumor development in vivo. The intracellular LP-granules become the main sensitive targets for FR-action, but only in the chronic hypoxia state of EAC/ZAH tumor cells. The latter effect developed in close correlation with the following metabolic interactions: (1) increasing the proportion of PUFA (especially, arachidonic and linoleic acids) transported to EAC tumor cells from host organs and accumulated mainly in ttunor LPgranules, and (2) decreasing the cα-tocopherol content of these hypoxic EAC cells while no activation of the main cell antioxidative enzymes (GSH-Px, SOD) took place. The vitality and high resistance of EAC stationary cells were accompanied by the ‘paradoxical’ state with great differences between the resistance of the intracellnlar PUFA-rich granules and other cytoplasmic sections. A similar state was found in stationary ZAH cells. The cell state is in good agreement with the Dormandy’s suggestion that PUFA-rich granules can trap reactive radical species preventing their interaction with ‘critical’ PUFA-membranes.