Xenogeneic bone marrow transplantation: II. Porcine-specific growth factors enhance porcine bone marrow engraftment in an in vitro primate microenvironment

Abstract: Establishment of mixed bone marrow chimerism in pig-to-primate transplantation, as a means of inducing specific immune tolerance, will require that both immune and nonimmune barriers be overcome. As a preliminary step in evaluating nonimmune barriers in this system, we have developed an in vitro model of engraftment in which long-term culture of porcine bone marrow-derived hematopoietic cells is supported on preformed primate bone marrow stromal layers. In the absence of cytokine supplementation, primate stromal cells were unable to support long-term porcine hematopoiesis in these cultures. Supplementation with porcine Steel Factor was required for long-term maintenance of hematopoietic progenitor cell content and total hematopoietic activity. Addition of porcine IL-3, in combination with porcine Steel Factor, increased long-term progenitor cell content and hematopoietic activity on primate stroma to levels comparable to that obtained in cultures on porcine stroma. The combination of porcine GM-CSF and Steel Factor increased progenitor cell content and hematopoietic activity early in the cultures, but had little effect in long-term cultures. The Steel Factor and IL-3 combination was species-specific in its action in these cultures, as the corresponding human cytokines were unable to effectively support long-term porcine hematopoiesis. Likewise, the combination of porcine cytokines had only minimal effects on long-term bone marrow culture of primate CD34+ cells I on primate stroma.     

肿瘤微环境对树突细胞功能状态的影响

目的：探讨肿瘤细胞分泌的可溶性细胞因子营造的微环境对树突细胞(DCs，dendritic cells)的分化发育的影响，以进一步揭示肿瘤的免疫逃逸机制。方法：用免疫磁珠从人外周血分离CD14^ 单核细胞，加入粒细胞巨噬细胞集落刺激因子(GM-CSF)、白细胞介素4(IL-4)和人白血病细胞Jurkat培养上清液体外培养DCs，以正常培养诱导的DCs作为对照，采用傅立叶变换红外光谱(FTIR)技术分析人白血病细胞培养上清液对DCs分化发育的影响。结果：正常培养DCs与肿瘤上清液培养的DCs相比，在细胞内蛋白质和核酸的相对含量和细胞内消耗葡萄糖重新合成磷脂的量方面差异无显著性，但是，在细胞的转录状态方面差异有显著性。结论：肿瘤细胞上清液培养液所营造的微环境细胞转录水平上对DCs的功能状态有明显的抑制作用。     

东部马脑炎病毒E2基因与GM-CSF共表达质粒的构建及其免疫原性初步研究

目的:利用粒细胞-巨噬细胞集落刺激因子(GM-CSF)作为基因佐剂,提高东部马脑炎病毒E2基因重组质粒的免疫效果.方法:分别扩增E2基因与GM-CSF基因,连接进入含有内部核糖体插入位点(IRES)的真核表达载体中,构建共表达质粒.Lipofectamine2000介导转染真核细胞BHK-21,反转录PCR检测E2和GM-CSF两个基因的转录,Western印迹法和间接免疫荧光法(IFA)检测细胞内E2基因表达产物的抗原性.通过肌肉注射免疫BALB/c小鼠,IFA法测定血清抗体效价,FACS测定免疫小鼠脾细胞中CD4 /CD8 淋巴细胞构成比,初步观察共表达质粒的免疫原性.结果:经酶切鉴定与序列测定证明重组共表达质粒中含有E2和GM-CSF两个基因且序列正确.转染细胞的反转录PCR可见E2和GM-CSF两个基因的转录.Western印迹结果可见转染细胞的裂解液在相对分子质量50 000位置有特异性条带.将转染细胞制成荧光抗原片,经IFA检测可见胞浆中出现特异荧光.免疫小鼠的最高血清抗体效价为1:160,但细胞免疫未观察到明显的提高.结论:构建的共表达质粒pE2-IRES-mGM-CSF具有良好的免疫原性,能有效刺激小鼠特异性体液免疫应答,提高了E2基因重组质粒诱导的血清抗体效价.     

The priming action of tumour necrosis factor-alpha (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on neutrophils activated by inflammatory microcrystals

We studied the effects of TNF-α or GM-CSF on the production of reactive oxygen species (as measured by chemiluminescence) and degranulation responses of neutrophils to opsonized inflammatory microcrystals. TNF-α in the 10–2000 pm or GM-CSF in the 2–200 pm concentration range caused the concentration-dependent amplification of neutrophil chemiluminescence responses to both calcium pyrophosphate dihydrate (CPPD) and monosodium urate monohydrate (MSUM) crystals. Degranulation responses, as measured by the extracellular release of the granule enzymes myeloperoxidase or lysozyme, were amplified by ≈ 50–100% for both MSUM or CPPD crystal-induced neutrophil activation when cells were pretreated with TNF-α at 2000 pm or GM-CSF at 75 pm.     

Granulocyte-macrophage colony stimulating factor does not improve neutrophil oxidative metabolism in a patient with variant X-linked chronic granulomatous disease

Variant X-linked chronic granulomatous disease (CGD) is characterised by a decreased but still measurable respiratory burst and cytochrome b content of phagocytes resulting in a clinically milder form of the disease. We examined the in vivo effect of recombinant human granulocyte-macrophage colony stimulating factor (rh-GM-CSF) on the neutrophil functions of a patient treated for liver abscess. The number of white blood cells was markedly increased at the highest dose of GM-CSF injected (30 g/kg per day). This was mainly due to a large increase in eosinophils and to a lesser extent in neutrophils. No change in the deficient neutrophil respiratory burst nitroblue tetrazolium (NBT)-reduction, superoxide (O ₂ ^– )-production and cytochrome b content was observed during 6 weeks of therapy with increasing doses of GM-CSF. No significant clinical improvement of the liver abscess was observed during treatment with GM-CSF.     

转粒细胞-巨噬细胞集落刺激因子基因真核表达质粒的构建及生物学活性鉴定

目的　构建小鼠转粒细胞 巨噬细胞集落刺激因子 (mGM CSF)真核表达质粒 pcDNA3 GM CSF ,转染红白血病细胞系FBL 3,并鉴定其活性。方法　采用分子克隆技术 ,构建mGM CSF真核表达质粒 pcDNA3 GM CSF ,电穿孔法将其导入红白血病细胞系FBL 3,G4 18筛选G4 18抗性细胞 ,限制性稀释法筛选G4 18抗性细胞克隆。PCR和RT PCR方法鉴定GM CSF基因整合和稳定表达。骨髓造血祖细胞增殖实验和骨髓造血祖细胞集落刺激实验鉴定其生物学活性。结果　采用PCR方法扩增mGM CSFcDNA序列 ,BamHⅠ和EcoRⅠ双酶切后装入 pcDNA3质粒 ,构建真核表达质粒 pcDNA3 GM CSF ,酶切和测序结果与预期相符 ,无插入、丢失、突变 ,方向正确。PCR和RT PCR结果显示 ,GM CSF基因整合到受体细胞染色体中并稳定表达。表达GM CSF的FBL 3 GM CSF细胞培养上清可明显刺激小鼠骨髓单个核细胞增殖 ,并能刺激干细胞形成克隆 ,形成克隆数为 (5 4 .6 7± 4 .83)个 ,形成率为 0 .5 4 7%。结论　构建mGM CSF真核表达质粒pcDNA3 GM CSF ,获得稳定表达该基因并具有生物学活性的细胞克隆 ,为制备转GM CSF基因瘤苗 ,探索白血病免疫治疗的可行性奠定基础     

Effects of GM-CSF, IL-3, and GM-CSG/IL-3 fusion protein on apoptosis of human myeloid leukemic cell line Tf-1 induced by irradiation

AIM: To observe the effects of three cytokines on the apoptosis of Tf-1 cells induced by y irradiation and investigate the relationship between apoptosis and caspase-3 activity. METHODS: Different cytokines GM-CSF, IL-3 and GM-CS/IL-3 fusion protein were added into the irradiated Tf-1 cells. MTT assay, morphology, flow cytometry, and DNA fragmentation assay were used to observe the effects of cytokines on apoptosis. The caspase-3 activity was determined with a fluorocytometer. RESULTS: Irradiated Tf-1 cells showed typical morphological characteristic of apoptosis demonstrated by transmission electron microscopy and were accumulated in G0/G1 phase. In the groups treated with growth factors after irradiation, three cytokines significantly increased the viability rate, distinctly decreased the apoptosis rate and the proportion of DNA fragmentation. When Tf-1 cells were irradiated by y     

B cell and B cell-related pathways for novel cancer treatments

B cells are recognized as the main effector cells of humoral immunity which suppress tumor progression by secreting immunoglobulins, promoting T cell response, and killing cancer cells directly. Given these properties, their anti-tumor immune response in the tumor micro-environment (TME) is of great interest. Although T cell-related immune responses have become a therapeutic target with the introduction of immune checkpoint inhibitors, not all patients benefit from these treatments. B cell and B cell-related pathways (CCL19, −21/CCR7 axis and CXCL13/CXCR5 axis) play key roles in activating immune response through humoral immunity and local immune activation via tertiary lymphoid structure (TLS) formation. However they have some protumorigenic works in the TME. Thus, a better understanding of B cell and B cell-related pathways is necessary to develop effective cancer control. In this review, we summarize recent evidences regarding the roles of B cell and B cell-related pathways in the TME and immune response and discuss their potential roles for novel cancer treatment strategies.     

The in vitro effects of stem cell factor, interleukin 3 and granulocyte-macrophage colony stimulating factor on haemopoietic progenitor cells from premature infants

Summary. Circulating haemopoietic progenitor cells from premature infants were assessed for their ability to respond to interleukin 3, granulocyte-macrophage colony stimulating factor and stem cell factor (SCF) in vitro. All three cytokines increased the number of colonies derived from burst forming units erythroid (BFU-E), colony forming units granulocyte-macrophage (CFU-GM) and multi-lineage progenitors (CFU-Mix) grown in the presence of erythropoietin (Epo). The size and haemoglobin content of BFU-E derived colonies also increased in the presence of the cytokines. Of those tested, SCF was found to be the most potent additive to Epo for the enhanced growth of BFU-E and CFU-Mix. In short-term liquid cultures without Epo, SCF alone induced globin synthesizing cells. Progenitors from premature infants were at least as responsive to all three cytokines as those from healthy adults. The use of SCF in combination with Epo in the prevention or treatment of anaemia in premature infants warrants further investigation.     

重组人粒细胞巨噬细胞刺激因子联合重组表皮生长因子治疗儿童急性白血病化疗性口腔黏膜炎的临床疗效分析

目的　回顾性分析重组人粒细胞巨噬细胞刺激因子（rhGM-CSF）含漱联合重组表皮生长因子喷涂治疗儿童急性白血病化疗引起的口腔黏膜炎的临床疗效。方法　回顾性分析2021年9月至2022年4月就诊于中国医科大学附属盛京医院第二小儿血液内科107例次急性白血病化疗后并发口腔黏膜炎的患儿资料,按rhGM-CSF与重组人酸性成纤维细胞生长因子（rh-aFGF）联合应用与否分为联合治疗组和单独治疗组,联合治疗组共51例次,单独治疗组共56例次。联合治疗组将rhGM-CSF 100ug加入氯化钠注射液250 mL,冷藏保存,每日含漱6次（分别于三餐前后各1次）,再予外用rh-aFGF喷涂口腔黏膜破溃处,每日3次。单独治疗组的56例患儿仅用rh-aFGF喷涂口腔黏膜破溃处,每日3次。从患儿发生口腔黏膜炎即开始应用治疗药物至口腔黏膜炎愈合,比较两组患儿治疗口腔黏膜炎的临床疗效、口腔黏膜炎愈合时间,以及药物不良反应的发生情况。结果　联合治疗组患儿有效率明显高于单独治疗组（84% vs 68%,P=0.047）,联合治疗组患儿口腔溃疡愈合时间明显缩短,联合治疗组（4.80±2.73）d,单独治疗组（9.04±2.81）d,（P<0.01）,治疗期间无不良反应发生。结论　对于儿童急性白血病并发化疗性口腔黏膜炎,应用rhGM-CSF含漱联合rh-aFGF喷涂的方式治疗疗效显著,有效缓解疼痛,缩短治愈时间,有推广价值。