Isolation and characterization of rhesus monkey milk lactoferrin

Rhesus monkey milk lactoferrin was isolated and its characteristics compared with those of human milk lactoferrin in order to assess the feasibility of using the rhesus monkey as an animal model for the study of iron absorption from milk. Monkey lactoferrin was isolated from pooled monkey milk by two chromatographic steps. Concentration of lactoferrin in milk, determined by rocket immunoelectrophoresis, demonstrated similar concentrations in both human and monkey milk, 1-2 mg/ml. Immunodiffusion of lactoferrins from several species using an antibody raised to money lactoferrin resulted in a cross-reaction only with monkey and human lactoferrin. Lactoferrins from cow, sheep, goat, dog, and rat milk were not recognized by the antibody. Amino acid analysis of monkey lactoferrin showed a composition very similar to human lactoferrin, as well as a similarity in the unusual amino acid sequence at the N-terminal of the protein. The carbohydrate moiety of monkey lactoferrin was investigated and shown to contain monosaccharides in similar proportions to those reported for human lactoferrin. In our opinion, the rhesus monkey is a promising model for the study of the role of lactoferrin in iron absorption in the infant, as well as of the other proposed actions of lactoferrin.     

人乳中乳铁蛋白含量的初步检测分析

目的 了解人乳乳铁蛋白水平.方法 利用酶联免疫吸附双抗体夹心法(ELISA),分别测定36名产妇产后2、3 d的初乳,6、7 d的过渡乳及42 d的成熟乳标本中乳铁蛋白水平.结果 初乳的乳铁蛋白含量最高,过渡乳组和成熟乳组依次降低,分别为初乳组(2.63 ± 1.11) g/L、过渡乳组(1.96 ± 1.05) g/L、成熟乳组(1.42 ± 1.01) g/L,三组差异有统计学意义(P ＜ 0.01).剖宫产与顺产产妇的乳铁蛋白差异无统计学意义(P ＞ 0.05).产妇年龄＜ 28岁与≥ 28岁的乳铁蛋白含量差异无统计学意义(P ＞ 0.05).结论 人乳乳铁蛋白在初乳、过渡乳、成熟乳中的下降趋势与国外报道相符,但三组测定值均低于国外水平,尤其是初乳乳铁蛋白明显低于国外报道.     

穿孔素和颗粒酶B在哮喘大鼠肺组织中的表达及重组人生长激素的干预作用

目的：探讨哮喘大鼠肺组织中穿孔素（PFP）、颗粒酶B（GzmB）的表达变化及重组人生长激素（rhGH）的干预作用。方法：30只雄性Sprague-Dawley大鼠随机分为正常对照组、哮喘模型组及rhGH干预组。通过苏木精-伊红染色观察卵蛋白激发作用及rhGH干预后哮喘大鼠气道的病理变化。TUNEL原位凋亡检测支气管上皮细胞凋亡,RT-PCR方法检测肺组织中PFP及GzmB mRNA的表达。结果：哮喘组支气管上皮细胞凋亡指数明显增高,而rhGH干预组的凋亡指数则明显低于哮喘组（P<0.05）;PFP和GzmB mRNA在对照组中呈低表达;在哮喘组的表达明显强于对照组（P<0.05）;rhGH进行干预后,PFP和GzmB mRNA表达明显低于哮喘组（0.48±0.08 vs 0.63±0.08;0.44±0.13 vs 0.71±0.15;均P<0.05）。PFP和GzmB mRNA表达与支气管上皮细胞凋亡率呈正相关（r分别为 0.800 和 0.806,P<0.05）。结论：PFP和GzmB参与了哮喘发病过程中的重要环节;rhGH可能通过降低PFP和 GzmB的表达抑制支气管上皮细胞的凋亡从而抑制气道重塑。     

Molecular forms of lactoferrin in stool and urine from infants fed human milk

The molecular forms of lactoferrin (LF) were examined in stools and urine collected at 2.5 or 5 wk of age from very low birth wt infants fed either a cow's milk formula or a fortified human milk preparation. LF was not found by Western blotting in excreta from infants fed cow's milk. In contrast, intact and fragmented forms of LF were detected in stools and concentrated urine of each infant who received human milk. Only intact LF was detected in the fortified human milk preparation, whereas many types of LF fragments were present in the stools and urine. The approximate molecular wt of the most prominent fragments were 44, 38, 34, and 32 kD. However, the stools also displayed lower molecular wt fragments that were not found in urines of those infants. The LF fragments in those excreta were similar in size to those produced in vitro by limited digestion of apo-LF with trypsin. Furthermore, fragments produced by in vitro proteolysis were immunoreactive in an ELISA for LF. Thus, the fragments of LF in stools of very low birth wt infants fed human milk appeared to be produced by in vivo proteolysis, and the close resemblance between the LF fragments in the stools and urine suggests that the urinary LF fragments originated in the gastrointestinal tract. It remains unclear, however, whether the whole LF molecules that were fragmented were derived solely from ingested LF in human milk or in part from LF produced by the infant in response to human milk feedings.     

Expression and activity of the Ca(2+)-atpase enzyme in human neonatal erythrocytes

The plasma membrane Ca(2+)-ATPase (PMCA) is one of the main regulators of Ca(2+) homeostasis. We studied the perinatal alteration of the abundance and the activity of PMCA molecules in human erythrocytes in pre-term and full-term neonates and children at the age of 1-4 years. The lower abundance of the 4b isoform was associated with lower enzyme activity in full-term neonates compared to children. Although the number of PMCA molecules was higher in pre-term neonates, their total PMCA activities were identical to those of full-term neonates. Our findings suggest that the abundance of PMCA molecules changes during the perinatal development. The same activity at higher enzyme molecule numbers might indicate a potential immaturity of the enzyme in the pre-term infant.     

Expression, activity, and function of phosphodiesterases in the mature and immature ductus arteriosus

A patent ductus arteriosus is due in large part to increased sensitivity of the premature ductus to PGE2. After PGE2 stimulation, cAMP concentrations are higher in the immature than in the mature ductus. cAMP concentrations depend on the rates of adenyl cyclase production and phosphodiesterase (PDE)-mediated degradation. We used ductus from immature (n = 25) and mature (n = 21) fetal sheep to investigate whether a developmental increase in PDE activity could explain the diminished cAMP accumulation that follows PGE2 stimulation in the mature ductus. With advancing gestation, mRNA expression of the smooth muscle PDE isoforms (PDE1A, 1B, 1C, 3A, 3B, 4D, and 5A) increased in the ductus as did their hydrolytic activities. Selective inhibitors of PDE1, PDE3, and PDE4 relaxed the mature and immature ductus in the presence of inhibitors of prostaglandin and nitric oxide production. The mature ductus required higher concentrations of each of the PDE inhibitors to inhibit its tension to the same extent as in the immature ductus. There were no developmental changes in PDE expression in the fetal aorta. In conclusion, we observed a developmental increase in cAMP and cGMP PDE activity that contributes to the decreased sensitivity of the late-gestation ductus arteriosus to vasodilators like PGE2.     

Effect of 1human milk prostaglandins and lactoferrin on respiratory syncytial virus and rotavirus

The effect of lactoferrin and prostaglandins E and F_2α on the growth of rotavirus and respiratory syncytial virus in cell culture was investigated. Lactoferrin inhibited the growth of respiratory syncytial virus at a concentration tenfold lower than that normally present in human milk. The prostaglandins had no effect on either virus growth, even at a concentration of 100-fold more than that found in human milk. Lactoferrin may have some antiviral properties in human milk in addition to its known antibacterial functions.     

Effects of iron-unsaturated human lactoferrin on hydrogen peroxide-induced oxidative damage in intestinal epithelial cells

Human milk (HM) contains various bioactive antioxidants. Lactoferrin (Lf) has been assumed to be one of the major antioxidants in HM. We examined the antioxidative properties of iron-unsaturated human Lf (apo-hLf, the major form of Lf in HM) in two intestinal epithelial cell lines: (1) An intestinal epithelial cell line (IEC-6) were preincubated for 24 h with either 50 microg/mL of apo-hLf, iron-saturated human Lf (holo-hLf), iron-unsaturated bovine transferrin (apo-bTf), or 800 ng/mL of the iron-chelating compound deferoxamine (DFX), followed by hydrogen peroxide (H2O2) challenge to induce oxidative stress. Survival rates were significantly higher in the cells preincubated with apo-hLf and DFX than those preincubated with holo-hLf. (2) Caco-2 cells were preincubated with or without apo-hLf for 24 h, followed by an H2O2 challenge. Intracellular oxidative stress was assessed by a fluorescent probe, 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA). Fluorescent intensity of cell images and cell homogenates was significantly lower in the cells preincubated with apo-hLF than those preincubated without apo-hLF. Our study indicates that apo-hLf alleviates H2O2-induced oxidative damage in intestinal cells due to the iron-chelating capacity. Therefore, Lf in HM may act as an antioxidant in the gastrointestinal tract (GIT).     

Selective removal, recovery, and characterization of immunoglobulins from human colostrum

Investigations into the mechanisms by which Ig in human colostrum influence the development and maturation of both the gastrointestinal and the immune systems of human milk-fed term and preterm infants have been restricted by the paucity of purified human milk Ig. We have developed a simple adsorption procedure for the selective removal and quantitative recovery (95-100%) of intact Ig (secretory IgA, IgG, and IgM) present in human colostral whey. The procedure exploits the rapid, ionic-strength dependent, thiophilic adsorption of Ig during a single pass of colostral whey through a column of beaded agarose with immobilized thioether-sulfone ligands (Anal Biochem 1986;159:217-226). The purity and composition of the adsorbed Ig were verified by SDS-PAGE and sensitive silver-staining protein detection procedures; proteins of approximately 78-80 kD (secretory component), 50-60 kD (heavy chain), and 25 kD (light chain) were observed. The identity, structural integrity, and relative concentrations of the recovered Ig were confirmed by high-performance size-exclusion chromatography, Ouchterlony immunodiffusion, rocket immunoelectrophoresis and ELISA. These results were analyzed and compared with reported values for the concentration of human milk Ig. Thus, the use of thiophilic adsorption appears to facilitate 1) selective removal of Ig from colostrum, enabling the evaluation of remaining components for growth- and immune-potentiating properties, and 2) selective immobilization and recovery of Ig from colostrum under conditions consistent with preserved biologic activity.     

Comparison of recombinant granulocyte colony-stimulating factor,recombinant human granulocyte-macrophage colony-stimulating factor and placebo for treatment of septic preterm infants

BACKGROUND: To reduce morbidity and mortality adjuvant cytokine therapy was administered to septic neonates with variable results. The objective of this case series was to compare the effectiveness of recombinant human granulocyte-macrophage colony-stimulating factor (rhuGM-CSF) and recombinant granulocyte colony-stimulating factor (rG-CSF) with that of placebo in correcting neutropenia induced by sepsis. METHODS: Symptomatic, septic premature neonates with or without a positive blood culture were eligible. Twenty-eight patients were randomized: 10 received rG-CSF (5 microg/kg/dose i.v. twice a day); 10 received rhuGM-CSF (4 microg/kg/dose i.v. twice a day) and 8 received placebo for a maximum of 7 days, or until an absolute neutrophil count (ANC) of 10,000 cells/mm was reached. RESULTS: A significant increase in the ANC above the baseline was present on Day 2 in the rG-CSF group (P = 0.015) and on Day 5 in the rhuGM-CSF (P = 0.002) and placebo (P = 0.027) groups. The ANC of the rG-CSF group was significantly above that in the rhuGM-CSF and placebo groups on Day 7 (P = 0.03). Mortality and neonatal intensive care unit morbidity was not significantly different between the groups. CONCLUSION: The neutrophil count in the rG-CSF-treated group increased significantly faster than that in the placebo or rhuGM-CSF group.     

In vivo effect of recombinant human granulocyte colony-stimulating factor on phagocytic function and oxidative burst activity in septic neutropenic neonates

Neutrophil dysfunction may contribute to an increased risk of sepsis in very-low-birth-weight (VLBW) neonates. The current study was designed to determine whether recombinant human granulocyte colony-stimulating factor (rhG-CSF) affects absolute neutrophil count (ANC), phagocytic function, and oxidative burst in neutropenic VLBW neonates. Fourteen ventilated VLBW neonates were treated with rhG-CSF (10 microg/kg/day x 3 days i.v.). Phagocytic activity and oxidative burst were assessed before and after treatment with rhG-CSF using flow cytometry and fluorescence labeled opsonized Staphylococcus aureus. Control (nonseptic, nonneutropenic, n = 4), preeclamptic neutropenic (PET; nonseptic, n = 5), and septic neutropenic (n = 5) neonates with a gestational age ranging from 24 to 30 weeks were studied. In both PET and septic neonates, posttreatment phagocytosis more than doubled, but did not achieve matching control levels, whereas rhG-CSF treatment maintained the level of the phagocytic activity in the control group. The oxidative burst increased in all groups, but, again, PET and septic groups did not achieve matching control values. These effects occurred independent of a 2- to 12-fold increase in ANC. These results suggest that other disease-specific factors delay full functional recovery even after rhG-CSF treatment. We speculate that PET and septic neonates may remain susceptible to infection due to deficient neutrophil-killing capacity, even though their ANC returns to normal ranges. Augmenting immune function beyond the immediate period of ANC recovery suggests that prophylaxis with rhG-CSF may be an important risk reduction strategy for susceptible VLBW neonates.     

Haematological improvement by long-term administration of recombinant human granulocyte-colony stimulating factor and recombinant human erythropoietin in a patient with severe aplastic anaemia

A 6-year-old girl with post-hepatitic severe aplastic anaemia was referred to our hospital. Haematological examination showed a haemoglobin level of 5.2 g/dl, platelet count of 8,000/l, and white blood cell count of 130/l with 17% neutrophils. She was treated with recombinant human granulocytecolony stimulating factor (15 g/kg/day i.v.) and cyclosporin A (6 mg/kg/day p.o.). The absolute neutrophil count gradually increased, but Hb and platelets were not improved. The intravenous administration of recombinant human erythropoietin (100 U/kg three times a week) was started, and the reticulocyte count reached 20000/l on day 12. The platelets increased to 81000/l after 16 months of combined administration of recombinant human granulocyte-colony stimulating factor, recombinant human erythropoietin and cyclosporin A. After 20 months of combined administration, the haematological results were: Hb, 13.1 g/dl; platelets 80000/l: WBC, 9500/l with 40% neutrophils. After recombinant human granulocyte-colony stimulating factor treatment, the myeloid elements of the bone marrow and the number of granulocyte-macrophage colony forming units increased. Bone marrow erythropoiesis and erythroid colonies also increased after recombinant human erythropoietin administration. The clinical course suggested a beneficial effect of haemopoietic growth factors and cyclosporin A in post-hepatitic aplastic anaemia.     

重组人类肥胖抑素在大肠杆菌中的大规模纯化

目的 探讨产率较高,高度纯化大肠杆菌表达重组肥胖抑素的方法,深入研究肥胖抑素的分子结构,生物活性及临床应用,对在大肠杆菌中表达的重组人类肥胖抑素进行纯化。方法 温度诱导表达携有人为肥胖基因（ｏｂ）片段的大肠杆菌重组子,采用酸沉淀结合凝胶过滤层析的方法从包涵体中纯化目的蛋白。结果 １周内从３Ｌ培养液（１０ｇ）湿菌体中可获得７０ｍｇ高度纯化的重组人类肥胖抑素。结论在短期内成功获得了高产高纯化的重组人类     

Superoxide dismutase activity in colostrum, transitional and mature human milk

Colostrum and mature human milk are rich sources of nutrients and contain biologically active molecules that are essential for specific antioxidant functions. The aim of the present study was to determine the activity of copper, zinc superoxide dismutase (CuZnSOD) and manganese superoxide dismutase (MnSOD) activity in different phases of lactation. Specific enzyme activity was determined in colostral milk (3rd-5th days after delivery), and in mature milk in the third week (15-20 days), and the fourth and seventh months of lactation. In the third week of lactation, the activity of CuZnSOD and MnSOD was significantly higher in comparison to the colostral phase. In the fourth month of lactation, the activity of both enzymes was suppressed, while in the seventh month of lactation the MnSOD activity was increased, and the CuZnSOD activity was not significantly changed. These findings show that the activities of superoxide dismutases significantly change during different phases of lactation.     

重组人生长激素的临床应用和研究进展

生长激素是由脑垂体前叶嗜酸性细胞分泌的一种蛋白质,直接或间接通过胰岛素样生长因子对生长和代谢发挥作用.生长激素可作用于人体多种组织的靶细胞,具有广泛的生理作用.随着临床研究不断深入,重组人生长激素的应用范围不再局限于治疗儿童矮小症,其在成人生长激素缺乏症、神经系统疾病如缺血缺氧性脑病、脑外伤、脑性瘫痪、阿尔茨海默病等疾病以及烧伤领域、辅助生殖等方面均有一定的临床意义.该文就国内外重组人生长激素临床应用和研究最新进展作一综述.