Correlation between DNA methylation of the ABO gene promoter CpG island and leukemia / 中国实验血液学杂志

Recent studies have found that ABO blood group antigen is also closely related to the onset and development of many diseases. More and more attention is being paid to the decrease of A/B blood group antigen caused by some tumors. This study was purpose to investigate the correlation between DNA methylation of the ABO gene promoter CpG island and leukemia. The relative contents of ABH antigen on the surface of RBC from kinds of blood disease patients and healthy individuals were detected by using flow cytometry and confocal laser scanning microscopy. The DNA sequences and CpG methylation of ABO gene promoter in patients with hematopathy and healthy individuals, as well as the -102 site methylation of ABO gene promoter in patients with hematopathy and healthy individuals were detected by PCR and MSP-PCR respectively. The results showed that RBC from leukemia patients displayed different degree of A/B antigen decrease. The sequences of ABO gene promotor of patients with hematopathy were not different from healthy individuals indicating high conservation of promoter sequences. Comparison of sequences between patients with hematopathy and healthy individual indicated that CpG islands on ABO gene promoter either from blood disease patients or from healthy individual had no methylated site in AA patients, but C residues at position -102, -101, -100, -99 and -97 on the promoter of ABO gene in AML, CML, ALL and some MDS patients were methylated. It is concluded that methylation of CpG islands in promoter of ABO gene may result in AB antigen decrease in patients with leukemia. The methylation sites -102, -101, -100, -99 and -97 may be specific for leukemia. The methylation of site -102 can be used as a molecular marker in differential diagnosis for leukemias.

B to O erythrocyte conversion by the recombinant alpha-galactosidase / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Human group O red blood cells have great benefit in specialized transfusion areas such as armed conflict and natural calamity. The group B antigen differs structurally from group O antigen only by the addition of one terminal alpha-linked galactose residue. In this study we aimed to remove the terminal galactose from group B red blood cell to get group O red blood cell.</p><p><b>METHODS</b>alpha-galactosidase cDNA was cloned by RT-PCR from Catimor coffee beans grown on Hainan Island of China. The vector for alpha-galactosidase cDNA expression was constructed and transferred into Pichia pastoris cells by electroporation. The transgenic cells were cloned by fermentation and the recombinant alpha-galactosidase was purified by ion exchange chromatography. After studying the biochemical characters of alpha-galactosidase, we have used it in converting human erythrocytes from group B to group O.</p><p><b>RESULTS</b>The purity of recombinant alpha-galactosidase was higher than 96%, which was thought to be suitable for the use of blood conversion. Enzymatically converted human group O red blood cells (ECHORBC) exhibited membrane integrity, metabolic integrity, normal cell deformation and morphology. There were no coagulation between ECHORBC and any group of human blood. The ECHORBC will keep normal structure and function for a period of 21 days at 4 degrees C in monoammoniumphosphate nutrient solution. Experiments with Rhesus monkeys and gibbons showed that transfusion of enzymatically converted erythrocytes was safe.</p><p><b>CONCLUSION</b>ECHORBC can be easily obtained from group B red blood cell by alpha-galactosidase digestion. This study suggests that ECHORBC could be transfused to patients safely and efficiently.</p>

Enhancing transfection efficiency of polyethylenimine by a hydrophobic peptide from bee venom / 中国实验血液学杂志

The study was aimed to investigate the possibility of enhancing transfection efficiency of branched polyethylenimine (BPEI) in HeLa cells by hydrophobic tail of bee venom peptide (melittin). Hydrophobic tail of melittin was synthesized and its membrane permeable activity was evaluated by hemolysis test. The peptide was mixed with BPEI and the transfection efficiency was determined in HeLa cells by using green fluorescent protein gene (GFP) as a reporter gene. The cytotoxicity of the mixture was analyzed by MTT assay at 24 hours after transfection. The results indicated that the synthesized peptide had permeable activity leading to hemolysis in both neutral and acidic solution. At optimal condition, the peptide could significantly improve the transfection efficiency of BPEI and the cytotoxicity of the mixture was lower than BPEI itself. It is concluded that hydrophobic tail of melittin may be a potential enhancer to improve transfection efficiency mediated by cationic polymers in difficult to transfect cells.

Animal trials for mPEG-modified red blood cells / 中国实验血液学杂志

This study was aimed to investigate the survival rate and difference of transfused modified and unmodified RBC at 24 hours. The modified and unmodified RBC from mice, monkey, pig and human were labeled by using FITC, then these blood RBCs were transfused to homogeneous and heterogeneous animals. The result showed that 24 hour survival rate of unmodified mice RBC transfused to mice was 74%, while survival rate of 2.0 mmol/L mPEG-SPA modified mice RBC transfused to mice was 45%, difference between them was significant. The 24 hour survived rate of unmodified human RBC transfused to mice was 8%, while 24 hours survival rate of 2.0 mmol/L mPEG-SPA modified human RBC transfused to mice was 5% without statistical difference. The 24 hour survived rate of homogeneous transfusion of modified monkey RBC was 90%, while survival rate of modified human and pig RBC was zero on 24 hours after transfusion to monkey. It is concluded that RBC labeling methods and mice species are unrelated to 24 hours survival rate, but mPEG variety and concentration are related to mouse RBC life-span. It is incredible to use mouse RBC homogeneous transfusion result instead of human RBC to evaluate longevity and safety of modified human RBC. But modified human RBC transfused to mice can be a model to evaluate longevity of modified human RBC. It is very difficult to get the result about modified RBC life span by RBC transfusion among great heterogeneous mammal animals. So evaluation in large mammal animal models needs to be further studied.

Preliminary study on xenotransfusion from porcine red blood cell into Rhesus monkey / 中国实验血液学杂志

In order to study the possibility of xenotransfusion from porcine red blood cell (pRBC) to primate, the antigens on pRBC surface were modified to make it more compatible to primate sera. Porcine RBCs were subjected to both enzymatic removal of membrane alpha-Gal antigens with recombinant alpha-galactosidase (AGL) and covalent attachment of succinimid propionate-linked methoxypolyethyleneglycol (mPEG-SPA) to camouflage non-alphaGal antigens. The effects of double modifications were determinated by hemagglutination and clinical cross-match testing with rhesus sera. In vivo clearance rates and safety of modified pRBCs were measured after it was transfused into Rhesus monkey with or without immunosuppressant treatment. The validity of pRBC was detected in exsanguine Rhesus monkey model. The results showed that AGL could effectively remove alpha-Gal xenoantigens on pRBC membrane and reduce hemagglutination. The combination of mPEG modification with AGL treatment could significantly increased compatibility between pRBCs and Rhesus monkey sera. Modified pRBCs were detectable in Rhesus monkey blood at 12 hours after transfusion, and their survival time was 40 hours in the immunosuppressant-treated Rhesus monkey. In vivo survival rates of pRBCs were 38% in exsanguine Rhesus monkey at 8 hours after transfusion, and during that time, the hemoglobin and hematocrit of Rhesus monkey were maintained at the same level as before it lost blood. It is concluded that the modified pRBC can be safely transfused into Rhesus monkey and relieve the anemic symptom exsanguine Rhesus monkey. It suggested that pRBC can be hopefully used as a blood substitute for primate and human in the future.

Rh antigen stability of mPEG modified red blood cells / 中国实验血液学杂志

The objective of study was to investigate the Rh antigen stability of mPEG-modified RBC. RBC membrane protein SDS-PAGE technology was used to analyze the combination of the mPEG modified RBC membrane protein with mPEG molecules; the RBC ghost coagulation test and 4 degrees C CPD-preserved modified RBC mixed with matched blood were used to observe the stability of RBC Rh antigen camouflaged by mPEG. The results showed that the blood groups of stored mPEG-modified RBC were kept consistency before or after simulating transfusion, i.e. mixture of modified RBC with matched bloods, while the plasma hemoglobin after simulating transfusion was not only within the normal range during the storage, but also less than that before simulating transfusion even after incubation at 37 degrees C. The electrophoresis pattern stained with iodine and Coomassie blue displayed the bands of mPEG combined with RBC membrane protein and the slow mobility of membrane protein. The hemagglutination of PEGylation RBC ghosts did not take place and mPEG still covered the antigen. In conclusion, mPEG-SPA can bind the erythrocyte with its extracted membrane protein in both ghosts and living erythrocytes.

A pilot study on establishment of human/pig hematopoietic chimera model in fetal and neonatal pigs / 中国实验血液学杂志

This study was aimed to explore the feasibility of transplanting human cord blood stem cells (HSC) into pre-immune fetal and neonatal pigs, and to investigate the self-renewal of HSC in the recipient pigs. The fetus and neonate were manipulated in sterile separated room and human donor cells were injected into fetus via fetus muscle or umbilical vein (dissectted womb) or into neonate via umbilical vein before cutting it. Human CD45(+) cells s were detected by labeling with human anti-CD45 antibody and analyzed by fluorescence activated cell sorting (FACS). The results showed that tested pigs developed as well as control and a definite proportion of human cells existed in peripheral blood of chimeric pig on day 60 after transplantation. In conclusion, the fetus and neonate pigs can tolerate a definite proportion of human antigens, and to establish the human/pig model of hematopoietic chimerism is possible.

Effect of human plasma on preservation of red blood cells / 中国实验血液学杂志

In order to study whether plasma can affect the structure and function of red blood cells during their storage period, the differences of pH value, concentration of K(+), Na(+), osmotic fragility, plasma hemoglobin, AchE, ATP, 2.3-DPG, P50 in suspended RBC, washed RBC, and RBC with various plasma volume at different storage times were compared. The results showed that plasma helped the blood to keep the RBC at high pH value, low K(+), high Na(+) and maintain RBC-ATP, oxygen carry capacity and deformability, but no effect on maintenance of osmotic fragility, and levels of plasma hemoglobin, AchE, ATP and 2.3-DPG was found in preservated blood. In conclusion, human plasma may be in favour of the preservation of red blood cells.

Comparison of modification of surface xenoantigens on bovine and porcine erythrocytes / 中国实验血液学杂志

This study was aimed to explore impact of removal of cell membrane G alalpha1-3Gal beta1-4Glc NAc epitopes (called alpha-Gal) and chemical modification of other xenoantigen on bovine red blood cell (bRBC) and porcine red blood cell (pRBC) antigenicity and to compare their modified erythrocytes, in order to provide basis for development of human blood substitute with rich source, high safety and efficacy. bRBC and pRBC were subjected to both enzymatic removal of membrane alpha-Gal with recombinant coffee bean alpha-galactosidase (rC alpha-GalE) and covalent attachment of benzotriazole carbonate-linked methoxypolyethylene glycol (mPEG-BTC, MW = 20 kD). The effects of treatment were measured by hemagglutination, flow cytometric assay of IgG binding and clinical cross-match testing to human sera. The results showed that although alpha-galactosidase treatment reduced hemagglutination titers to levels similar to negative control, the combination of the treatments was most effective. Clinically used cross-match tests between bRBC, pRBC and human sera demonstrated increased compatibility. Bovine RBC were more robust than pRBC, and had less xenoantigens, and had longer half life than pRBC in vivo. These characteristics suggested that bRBCs were more suitable to investigation as an alternatives to hRBC in clinical transfusion than pRBC. These data suggested that strategies to remove or mask xenoantigens on bRBC reduce antigenicity sufficiently to allow in vitro cross-match compatibility to human sera, and therefore bRBC following modification may be considered as human blood substitute.

Preparation of transfusable human universal red blood cell with recombinant alpha-galactosidase / 中国实验血液学杂志

In order to meet the demand for safe transfusion in special conditions and to utilize the donated blood supply efficiently, technology has been developed to convert erythrocytes from type A, B, or AB to "universal donor" blood. Conversion of blood type B to O was performed by means of recombinant alpha-galactosidase digestion. The results showed that blood type B to O was converted successfully, 1 transfusion unit of red cells of group B (100 ml totally) could converted to universal blood cells in the optimal conditions including pH 5.6, 26 degrees C, 2 hours, obturation and sterilization. It is concluded that the universal red blood cells converted from group B to group O are conformed to demand of identification rules of biological products, no harmful effects of alpha-galactosidase on cell structure and function are observed. The converted red cells can stored in 4 degrees C for 21 days.

Preliminary in vitro study on modification of RBC with mPEG-BTC for resolving the problem of clinical difficulty in blood matching / 中国实验血液学杂志

The aim of this study was to find an effective solution for difficulty of blood matching. Twenty nine cases with clinical difficult in blood matching were collected, classified by their etiological factors, and analyzed with all the antibodies in serum. RBC from health donor were incubated with mPEG-BTC at 25 degrees C for 1 hour. The coagulation of patient serum and donor RBC before and after mPEG-BTC camouflage was detected and compared by polybrene and antihuman globulin reagents. The result showed that 29 cases with difficult blood matching mainly suffered form blood diseases and tumors. The main antibody were Rh and autoantibody. Donor RBC modified by mPEG showed no coagulation with the blood serum in the patients with problems of blood matching. In conclusion, the modification of RBC with mPEG-BTC provides a useful strategy for resolving problem of clinical difficulty in blood matching.

Selective myelo-protection by MDR1 and MnSOD genes regulated by a specific promoter / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the specific protection of myeloid cells from chemotherapeutic agents and radiation.</p><p><b>METHODS</b>The recombinant retroviral vectors containing MDR1 gene and MnSOD gene regulated by APN myeloid promoter were constructed and introduced into myeloblastic cell line KG1a and hepatoma cell line BEL7402. The resistance of the cells to antitumor drugs and radiation were analyzed by cell survival assay. In vivo, the murine bone marrow cells were isolated and infected by the retroviral particles, which were transplanted into recipient mouse treated with paclitaxel or X-ray. The murine white blood cell (WBC) was counted in order to assay the effects of MDR1 or MnSOD gene on hematopoiesis in the course of chemotherapy and radiotherapy.</p><p><b>RESULTS</b>The resistance to chemotherapeutic agents such as cochicine, Vp-16, vincristine, doxorubcin and paclitaxel were elevated markedly by 10.6, 10.4, 11.2, 4.2 and 14.2 folds in KG1a cell line transduced with MDR1 gene. The resistance to radiation increased 3.7 folds at the dose of 10 Gy compared with parental cells in KGla cell line transduced with MnSOD gene derived by APN promoter. In contrast, the chemosensitivity and the radiosensitivity showed no significant change in BEL 7402 cell line transduced with MDR1 gene and MnSOD gene. In vivo, the WBC counts in the mouse introduced with MDR1 gene or MnSOD gene were higher than those in the control mouse (P < 0.01).</p><p><b>CONCLUSION</b>The expression of MDR1 gene and MnSOD gene regulated by APN myeloid promoter is effective on myelo-specific protection without enhancing the resistance of tumor cells in vitro. The hematopoiesis can be reconstituted in vivo during anticancer drug or radiation treatment. This study may provide experimental evidence and new clues for myeloprotection of cancer patients being treated with chemotherapy and/or radiotherapy.</p>

Fermentation and purification of recombinant alpha-galactosidase from Pichia pastoris / 生物工程学报

In order to obtain an adequate supply of alpha-galactosidase for research and practical use, the fermentation, purification and identification of the recombinant coffee bean a-galactosidase were carried out. Baffled flasks containing 100mL BMGY were inoculated with the pPIC9K-Gal/GS115 strain and allowed to grow at 30 degrees C, 250- 300r/min until a maximum optical density at 600nm (OD600) between 2.0 to 6.0 was attained. Entire 400 mL seed culture was transferred aseptically to the 5-liter fermenter, which contained 4 liter sterilized basal salts medium and 4% glycerol. The batch culture grew at 30 degrees C, pH 5.0 until the glycerol was completely consumed, and a glycerol feed was initiated to increase the cell biomass prior to induction with methanol. The culture was centrifuged at 8000 x g and the supernatant was collected. Following ultrafiltration, the retentate was balanced in 20 mmol/L sodium formicate buffer, pH 3.8 and loaded onto a cation-exchange column, HiTrap SP. The column was washed with the same buffer and bound proteins were eluted with 1 mol/L NaCl. The fractions containing recombinant a-galactosidase were pooled and concentrated with PEG20 000. Subsequently, the biochemical properties of the enzyme were determined with typical methods. At last, the fresh human blood A and B erythrocytes were incubated with the purified alpha-galactosidase at 26 degrees C for 2 4 hours. Hemagglutinins were assayed by the standard method. After an elapsed fermentation times (EFT) of 18h, the fed-batch phase was initiated to increase the cell biomass. A cellular yield of nearly 200 g/liter wet cells was achieved when induction was initiated. 72h later, the alpha-galactosidase activity against artificial substrate PNPG (PNP-alpha-galactopyranoside) achieved 36 000u per liter culture. The crude fementation supernatant contained few impurities as detected by SDS-PAGE. The supernatant was purified by cation-exchange chromatography, the target alpha-galactosidase was eluted with 40% 1mol/L NaCl and showed a 41kD band on SDS-PAGE. After concentration, the final recovery was about 41%. The Michaelis constant of the recombinant alpha-galactosidase was determined as 0.275 mmol/L, which slightly lower than the nature enzyme and suggested a higher affinity with specific substrate. When human blood type B erythrocytes pretreated with 100u/mL recombinant alpha-galactosidase reacted with bood type B antiserum, no hemagglutination occurred. This suggested that the B antigens had been removed by the enzyme successfully. These results demonstrated that the recombinant alpha-galactosidase could be produced in largescale and made it possible to explore the application of alpha-galactosidase in more fields.

Camouflage of HLA-I antigen in lymphocyte surface / 中国实验血液学杂志

The objective of this study was to investigate the method and effect of blocking the specific reaction between lymphocyte HLA-I antigen and its antibody. The lymphocytes were disposed with 12 mmol/L methoxypolyethelene glycol benzotriazol carbonate (mPEG-BTC) in concentration gradient in PBS (pH 7.4) at 22 degrees C. The effect of the modified lymphocytes was detected by microlymphocytotoxicity assay. The results showed that lymphocytes modified by mPEG-BTC did not react with related HLA-I antibodies in microcytotoxicity test. It is suggested that the specific reaction between HLA-I antigen of lymphocyte and HLA-I antibodies can be completely camouflaged by mPEG-BTC in PBS (pH 7.4) under 22 degrees C room temperature.

Preliminary study on conversion of RhD positive red blood cells to RhD negative by modification with methoxy polyethylene glycol / 中国实验血液学杂志

Rh is a very important blood group like ABO blood system in transfusion medicine. It causes severe transfusion reaction and hemolytic disease of the newborn (HDN) if RhD blood group does not match between the donor and the recipient. The population of RhD negative is only about 0.2% - 0.5% in Chinese. Conversion of RhD positive RBCs to RhD negative is very important in clinical transfusion. This study was to try to modify RhD antigen located on the surface of A, B, O and AB red blood cells in order to convert RhD positive to RhD negative by the modification of four kinds of methoxypolyethylene glycol (mPEG) derivatives and to observe the effect of mPEG modification on cell morphology, structure and function. The result demonstrated that modification efficiency of mPEG-BTC (mPEG-benzotriazole carbonate) was better than other three kinds of mPEG derivatives. It could camouflage RhD antigen efficiently when the concentration reached to 1 mmol/L. The result also showed that there were no harmful effects of mPEG modification on cell morphology, osmotic fragility, hemolysis, AchE, cholesterol, ATP, 2,3-DPG and deformability. It is suggested that success in converting RhD positive RBCs to RhD negative was preliminarily achieved.

Recent advance on blood group antigen modification of porcine erythrocytes / 中国实验血液学杂志

Advances in the field of xenotransplantation raise the intriguing possibility of using porcine red blood cells (pRBCs) as an alternative source for blood transfusion. Serologically, pRBCs share a number of characteristics with human red blood cells (RBCs), so pRBCs are considered the most likely donor for xenotransfusion. However, xenoantigens on porcine erythrocytes play major roles in antibody-mediated RBC destruction. Although the alphaGal epitope (Galalpha1, 3Galbeta1, 4GalNAc-R) is the major xenoantigen on porcine erythrocytes and is responsible for the binding of the majority of human natural antibodies, other non-alphaGal xenoantigens have been identified. The importance of these non-alphaGal xenoantigens in binding human natural antibodies and subsequently triggering immunological responses cannot be underestimated.