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.
Erythrocytes ; drug effects ; immunology ; physiology ; Humans ; Polyethylene Glycols ; pharmacology ; Rh-Hr Blood-Group System ; immunology

OBJECTIVETo study the changes of red cell immune function and T-lymphocyte subsets in children with bronchiolitis and their possible roles in the pathogenesis of bronchiolitis.

METHODSForty-five children with bronchiolitis and 30 healthy controls were enrolled. Red cell immune complex rosette (RBC-ICR) and red cell C3b receptor rosette (RBC-C3bRR) were detected. The percentages of CD3+, CD4+ and CD8+ cells were assayed by flow cytometry.

RESULTSRBC-C3bRR［(13.6 ± 6.2)% vs (18.0 ± 7.4)%] and the percentage of CD8+ cells [(21.6 ± 4.4)% vs (25.6 ± 5.2) %］ in the bronchiolitis group were lower than those in the control group (P<0.01). The percentage of CD3+ cells ［(59.9 ± 6.7)% vs (52.1 ± 8.3)%］ and CD4+ cells [(53.5 ± 6.2)% vs (46.8 ± 4.9)%] and RBC-ICR [(8.3 ± 3.5)% vs (6.1 ± 2.5)%] in the bronchiolitis group were higher than those in the control group (P<0.01). The percentage of CD4+ cells was positively correlated with RBC-ICR (r=0.63,P<0.05) and negatively correlated with RBC-C3bRR (r=-0.82,P<0.01).

CONCLUSIONSThere are dysfunctions of red cell immune and T-lymphocyte subsets in children with brochiolitis, which may play a role in the pathogenesis of brochiolitis.

Bronchiolitis ; etiology ; immunology ; Erythrocytes ; immunology ; Female ; Humans ; Infant ; Male ; Receptors, Complement 3b ; physiology ; Rosette Formation ; T-Lymphocyte Subsets ; immunology

OBJECTIVETo investigate whether miR-492 is involved in the post-transcriptional regulation of OK blood group antigen expression on red blood cells.

METHODSTwo 3'-UTR fragments of the BSG gene were synthesized with a chemical method, which respectively encompassed the BSG rs8259 TT or BSG rs8259 AA sites. The fragments were added with Xho I and Not I restriction enzyme cutting sites at both ends and cloned into a pUC57 vector, which in turn was constructed into a psiCHECK-2 vector and verified by sequencing. K562 cells were transfected with various combinations of miR-492 mimic and constructed psiCHECK2-BSG-T or psiCHECK2-BSG-A recombinant plasmid. A blank control group was set up. Each transfection experiment was repeated three times. The activity of Renilla reniformis luciferase was determined and normalized with that of firefly luciferase, and detected with a dual-luciferase reporter assay system. The data were subjected to statistical analysis.

RESULTSThe sequencing results confirmed that the recombinant psiCHECK2 plasmids containing the BSG rs8259 TT or rs8259 AA sites were constructed successfully. The results of dual-luciferase report gene detection showed that the miR-492 mimic could significantly inhibit psiCHECK2-BSG-T at a concentration over 100 nmol/L. However, it could not inhibit psiCHECK-BSG-A.

CONCLUSIONmiR-492 may be involved in the regulation of OK antigen expression on red blood cells with the BSG rs8259 TT genotype.

Basigin ; genetics ; Blood Group Antigens ; genetics ; Erythrocytes ; immunology ; Gene Expression Regulation ; Genotype ; Humans ; MicroRNAs ; physiology

Amphibian skin contains rich bioactive peptides. Especially, a large amount of antimicrobial peptides have been identified from amphibian skin secretions. Antimicrobial peptides display potent cytolytic activities against a range of pathogenic bacteria and fungi and play important defense roles. No antimicrobial peptides have been reported from toads belonging to the family of Pelobatidae. In this work, two novel antimicrobial peptides (Megin 1 and Megin 2) were purified and characterized from the skin venoms of spadefoot toad Megophrys minor (Pelobatidae, Anura, Amphibia). Megin 1 had an amino acid sequence of FLKGCWTKWYSLKPKCPF-NH2, which was composed of 18 amino acid residues and contained an intra-molecular disulfide bridge and an amidated C-terminus. Megin 2 had an amino acid sequence of FFVLKFLLKWAGKVGLEHLACKFKNWC, which was composed of 27 amino acid residues and contained an intra-molecular disulfide bridge. Both Megin 1 and Megin 2 showed potential antimicrobial abilities against bacteria and fungi. The MICs of Megin 1 against Escherichia coli, Bacillus dysenteriae, Staphylococcus aureus, Bacillus subtilis, and Candida albicans were 25, 3, 6.25, 3, and 50 μg·mL(-1), respectively. The corresponding MICs for Megin 2 were 6.25, 1.5, 12.5, 1.5, and 12.5 μg·mL(-1), respectively. They also exerted strong hemolytic activity against human and rabbit red cells. The results suggested that megin peptides in the toad skin of M. minor displayed toxic effects on both eukaryotes and prokaryotes. This was the first report of antimicrobial peptides from amphibians belonging to the family of Pelobatidae.
Amino Acid Sequence ; Amphibian Venoms ; chemistry ; immunology ; isolation & purification ; Animals ; Anura ; immunology ; Bacillus ; Candida albicans ; Erythrocytes ; physiology ; Escherichia coli ; Female ; Hemolysis ; Humans ; Male ; Peptides ; chemistry ; immunology ; isolation & purification ; Rabbits ; Sequence Alignment ; Skin ; chemistry ; immunology ; Staphylococcus aureus

BACKGROUNDThe influence of inflammatory processes has been one of the hot topics in discussions of the etiology of chronic venous insufficiency (CVI). Erythrocytes are very important in controlling inflammatory immunity and innate immune reactions. The purpose of this study was to analyze the correlation between the development of CVI and the change of CD35, Fy6 on erythrocytes, and interleukin-8 (IL-8) levels.

METHODSA group of 43 patients with CVI were studied in parallel with 8 healthy individuals serving as control subjects. Control subjects were those with normal findings on lower extremity duplex examinations. We used an erythrocyte flow cytometer to examine the expression of both CD35 and Fy6 on red blood cells, and an enzyme-linked immunosorbent assay analysis method to measure plasma IL-8 levels. We also analyzed the change of IL-8 levels under the influence of erythrocytes using a modified method of the hemaimmune reaction.

RESULTSCompared with normal control subjects, CD35 expression increased significantly among patients with CVI classified as C4 without lipodermatosclerosis, but tended to decrease and reach the lowest level among patients classified as C5-C6. Fy6 expression increased significantly among patients in the early stages of CVI, but tended to decrease remarkably among patients classified as C5-C6. The inflammatory response intensified at the C5-C6 classification, where high levels of IL-8 coexisted with a low expression of Fy6. The increase in IL-8 in the CVI group was higher than in the control group in association with the complete blood cells, regardless of the presence of erythrocytes, when inactive tumour cells were added, whereas the level of IL-8 in the CVI group was significantly lower than in the control group.

CONCLUSIONSAbnormalities of erythrocyte innate immunity represents a fundamental derangement in CVI. These inadequate inflammatory responses may lead to local tissue and microvascular damage of the lower extremity.

Chronic Disease ; Duffy Blood-Group System ; blood ; Erythrocytes ; immunology ; physiology ; Humans ; Interleukin-8 ; blood ; physiology ; Leg ; blood supply ; Receptors, Cell Surface ; blood ; Receptors, Complement 3b ; blood ; Venous Insufficiency ; immunology

BACKGROUND: The red blood cell (RBC) deformability test is a useful method for measuring the ability of RBCs to adapt their shape to the flow conditions. Using this test, several investigators have shown the relationship between RBC deformability and numerous clinical conditions. For the quality control (QC) of RBC deformability test, we evaluated whether frozen-thawed-deglycerolized RBCs can be used as QC materials. METHODS: Packed RBCs were frozen with 40% (wt/vol) glycerol and stored at -80degrees C for 3 months. For 10 different frozen RBC panels, RBCs were thawed, deglycerolized and stored at 4degrees C for 4 weeks. Using microfluidic ektacytometer, we measured RBC deformability of the thawed RBCs. The stability of thawed RBCs was tested once a day for 28 days of storage time and was analyzed by simple regression analysis. The precision of the test using thawed RBCs was analyzed for 7 days of storage time by calculation of CV values of intra-assay (10 measurements/assay) and between-day measurements. RESULTS: Frozen-thawed-deglycerolized RBCs were stable for 1 week. Within-run and between-day precisions of the RBC deformability test during 7 days of storage of thawed RBCs were 1.4-2.9%, and 1.9-2.8%, respectively. CONCLUSIONS: Frozen-thawed-deglycerolized RBCs used in RBC deformability test showed satisfactory within-run and between-run precisions and stability for one week after thawing, and may be used as QC materials for this test.
Blood Preservation ; Cryopreservation ; Cryoprotective Agents/chemistry/isolation & purification ; *Erythrocyte Deformability ; Erythrocytes/immunology/physiology ; Glycerol/chemistry/isolation & purification ; Hematologic Tests/standards ; Humans ; Quality Control

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.
ABO Blood-Group System ; immunology ; Chromatography, Ion Exchange ; Electrophoresis, Polyacrylamide Gel ; Erythrocytes ; drug effects ; Fermentation ; physiology ; Hemagglutination ; drug effects ; Humans ; Pichia ; genetics ; metabolism ; alpha-Galactosidase ; genetics ; metabolism ; pharmacology