Blood Group-Active Surface Molecules of the Human Red Blood Cell

The surface of the human red blood cell is dominated by a small number of abundant blood group active proteins. The major proteins are the anion transport protein (band 3) which has AB(H) activity, and Glycophorin A which has MN activity. Band 3 and Glycophorin A are of equal abundance in the normal red cell membrane (approximately 10(6) copies of each) and the two proteins may associate together as a complex. The glucose transporter (band 4.5) had AB(H) activity and there are about 5 x 10(5) copies/red cell. Several polypeptides associate together to form the Rh complex. The major components of this complex (abundance 1-2 x 10(5) copies/red cell) are polypeptides of Mr 30,000, polypeptides of Mr 45,000-100,000 and Glycophorin B. The antigens of the Rh blood group system appear to be associated with the polypeptides of Mr 30,000 and those of Mr 45,000-100,000 (the latter also express AB(H) activity). Glycophorin B expresses the blood group 'N' antigen and the Ss antigens. Glycophorins C and D carry the Gerbich antigens and, together, these polypeptides comprise approximately 10(5) copies/red cell. The complete protein sequence of all the above-mentioned proteins is known, except for the Mr 30,000 and Mr 45,000-100,000 polypeptides of the Rh complex for which only partial sequences are available, and Glycophorin D, the sequence of which can be inferred from that of Glycophorin C. Several of the minor blood group active proteins at the red cell surface (abundance less than 1.2 x 10(4)/red cell) have been the subject of recent studies. The polypeptide expressing Cromer-related blood group antigens has been identified as decay-accelerating factor and that carrying the Ina/Inb antigens as CD44. The protein sequence of both of these proteins has been deduced form nucleotide sequencing. The polypeptides expressing Kell antigens, Lutheran antigens, Fy antigens, and LW antigens have also been identified and partially characterised.     

Diverse Effects of Cytochalasin B on Priming and Triggering the Respiratory Burst Activity in Human Neutrophils and Monocytes

Cytochalasin B, despite its potent enhancing effect on superoxide (O2-) release triggered by N-formyl-methionyl-leucyl-phenylalanine (FMLP) and many other agonists, significantly inhibited O2- release triggered by interleukin 8 (IL-8) and platelet-activating factor in human neutrophils. Cytochalasin B also enhanced changes in membrane potential stimulated by FMLP but inhibited those stimulated by IL-8. Using IL-8 as a triggering agonist, we found that the priming effect of tumor necrosis factor (TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on O2- release was slightly but significantly potentiated by cytochalasin B. O2- release triggered by TNF and GM-CSF was completely abolished by cytochalasin B. In contrast to these diverse effects of cytochalasin B on O2- release, changes in cytoplasmic pH stimulated by FMLP, IL-8, TNF, and GM-CSF were not or were only minimally affected by cytochalasin B. Unlike human neutrophils, human monocytes stimulated by FMLP showed inhibition of O2- release and changes in membrane potential in response to cytochalasin B, and the priming effect of TNF and GM-CSF on O2- release in human monocytes was completely abolished by cytochalasin B. These findings indicate the diverse effects of cytochalasin B on phagocytes and suggest distinct regulatory mechanisms according to the functions, agonists, and cell types.     

Inhibition of Apoptosis in Human Neutrophils by Helicobacter pylori Water-Soluble Surface Proteins

Background: Helicobacter pylori infection in humans causes persistent neutrophil infiltration into the gastric mucosa. It is believed that a prolongation of neutrophil life-span could contribute to the pathogenesis of H. pylori infection. We therefore examined whether the water-soluble surface proteins of H. pylori can influence the apoptosis of neutrophils. Methods: After neutrophils were incubated with H. pylori water extract (HPWE), neutrophil apoptosis was evaluated by TUNEL assay, Hoechst 33342 staining, electron microscopy and ELISA for cytosolic oligonucleosome-bound DNA for up to 48 h. To investigate the regulatory mechanisms of neutrophil apoptosis associated with HPWE, mRNA expression and protein production of Fas, Fas ligand (FasL) and tumor necrosis factor receptor 1 (TNF-R1) were analyzed by RT-PCR, ribonuclease protection assay, Northern blot and Western blotting. Cell surface expression of these death factors was also measured by flow cytometry. Results: HPWE inhibited neutrophil apoptosis and cytotoxicity for up to 48 h. The mRNA and protein expression of FasL and the cell surface expression of Fas, FasL and TNF-R1 in HPWE-treated neutrophils were suppressed compared with the controls. Conclusion: The water-soluble surface proteins of H. pylori could suppress neutrophil apoptosis. This may be caused by the suppression of FasL expression in neutrophils and Fas, FasL and TNF-R1 expression on the surface of neutrophils.     

Heterogeneity of Human Monocytes: An Optimized Four-Color Flow Cytometry Protocol for Analysis of Monocyte Subsets

Monocytes are central mediators in the development of atherosclerotic plaques. They circulate in blood and eventually migrate into tissue including the vessel wall where they give rise to macrophages and dendritic cells. The existence of monocyte subsets with distinct roles in homeostasis and inflammation suggests specialization of function. These subsets are identified based on expression of the CD14 and CD16 markers. Routinely applicable protocols remain elusive, however. Here, we present an optimized four-color flow cytometry protocol for analysis of human blood monocyte subsets using a specific PE-Cy5–conjugated monoclonal antibody (mAb) to HLA-DR, a PE-Cy7-conjugated mAb to CD14, a FITC-conjugated mAb to CD16, and PE-conjugated mAbs to additional markers relevant to monocyte function. Classical CD14⁺CD16⁻ monocytes (here termed “Mo1” subset) expressed high CCR2, CD36, CD64, and CD62L, but low CX₃CR1, whereas “nonclassical” CD14^loCD16⁺ monocytes (Mo3) essentially showed the inverse expression pattern. CD14⁺CD16⁺ monocytes (Mo2) expressed high HLA-DR, CD36, and CD64. In patients with stable coronary artery disease (n = 13), classical monocytes were decreased, whereas “nonclassical” monocytes were increased 90% compared with healthy subjects with angiographically normal coronary arteries (n = 14). Classical monocytes from CAD patients expressed higher CX₃CR1 and CCR2 than controls. Thus, stable CAD is associated with expansion of the nonclassical monocyte subset and increased expression of inflammatory markers on monocytes. Flow cytometric analysis of monocyte subsets and marker expression may provide valuable information on vascular inflammation. This may translate into the identification of monocyte subsets as selective therapeutic targets, thus avoiding adverse events associated with indiscriminate monocyte inhibition.     

Cell Surface and Enzyme Markers of Cord Blood Lymphocytes

Summary. Human cord blood (CB) lymphocytes were studied with several markers for T- and B-cells and the results compared with those of adult peripheral blood (PB) samples. The proportion of E-rosettes was significantly lower in CB (mean 24.7±13.5 SD) than in PB (67.5 ± 7.3 SD). Treatment with neuramidase produced a marked increase in the proportion of E-rosettes in CB (mean 47±13.9 SD), still below the PB values. The proportion of CB lymphocytes showing block positivity with α-naphthyl-acetate-esterase correlated closely with the percentage of E-rosettes in neuraminidase treated cells. The percentage of H-rosettes (human RBC) was significantly higher in CB (7.2±6.0) than in PB (3.2±1.6 SD). Re-rosetting experiments showed that in CB about 30% of the E-positive cells formed H-rosettes, in contrast to 5% in PB. These findings indicate that in CB the real number of T-lymphocytes is higher than shown by conventional E-rosette formation.
The proportion of B-lymphocytes, tested by surface immunoglobulins and by rosette formation with mouse RBC (M-rosettes), was similar in CB and in adult PB. A slight increase in cells with IgM on the surface was found in CB. The overall proportion of lymphocytes with negative B and T markers in CB is three times greater than in adult PB. Levels of the enzyme terminal deoxynucleotidyl transferase were marginally increased in CB; in two out of 41 samples the levels were above those found in normal bone marrow. CB may be a suitable model for the study of lymphocyte subsets with negative B and T markers in man.     

Destruction of IgG-Sensitized Erythrocytes by Human Blood Monocytes: Modulation of Inhibition by IgG

The in vitro interaction between monocytes and erythrocytes sensitized with non-complement binding IgG antibodies (i.e. the Rh antibody anti-D:EAIgG anti-D) is completely inhibited by low concentrations of IgG (e.g. 30–100 μg/ml). However, the interaction between monocytes and erythrocytes sensitized with IgG anti-A (EAIgG anti-A) is not inhibited by IgG. The findings presented in this paper indicate that this difference is probably due to the difference in the number of IgG antibody molecules per EAIgG. Thus, the higher the number of IgG antibody molecules per EAIgG, the less the interaction between EAIgG and monocytes is inhibited by IgG.
A second factor which proved to have a strong influence on inhibition by IgG was the number of EAIgG per monocyte. When the number of EAIgG per monocyte was increased from 1 to 32, the percentage of inhibition by a fixed amount of IgG (50 μg/ml) decreased significantly. This in vitro effect is only evident when relatively weakly sensitized erythrocytes are used and, in vivo , destruction of these weakly sensitized red cells (e.g. EAIgG anti-D) is confined to the spleen. Since a considerable haemoconcentration occurs in this organ, it is conceivable that a high EAIgG:macrophage ratio is accomplished. The latter data are an indication that this high ratio may allow interaction between weakly sensitized erythrocytes and splenic macrophages despite the presence, in vivo , of a high concentration of IgG, and that, in this way, in the spleen, the inhibitory effect of IgG is overcome.     

The Effect of Adrenaline,Insulin and Hydrocortisone on Human Peripheral Blood Lymphocytes Studied by Cell Surface Markers

Changes in numbers of peripheral blood lymphocytes from healthy individuals were calculated from samples collected before and after parenteral administration of adrenaline, insulin and hydrocortisone, respectively. A marked increase in circulating lymphocytes was noted in response to adrenaline and insulin. However, subpopulation analysis showed a decrease in the proportion of T-lymphocytes, estimated as cells forming rosettes with sheep red blood cells after incubation in the cold and a corresponding increase in proportion of lymphocytes having receptors for C3 (non-T lymphocytes). In contrast, lymphocyte numbers were unaffected by hydrocortisone. The results indicate that a decreased proportion of circulating T-lymphocytes and an increase of non-T lymphocytes may be the result of adaptive changes in response to various forms of stress and hence is to be expected in several clinical conditions.     

Effect of the Mucoactive Drug Nacystelyn on the Respiratory Burst of Human Blood Polymorphonuclear Neutrophils

In lung diseases such as chronic obstructive pulmonary disease (COPD) or cystic fibrosis, the activation of phagocytic cells produces high amounts of cytotoxic reactive oxygen species (ROS) that are partly implicated in the pathogenic process. In this study, the ex vivo antioxidant activity of nacystelyn (NAL), a recently developed mucoactive thiol-containing agent, was investigated using the respiratory burst of human blood polymorphonuclear neutrophils (PMNs). The ROS generation was induced by serum-opsonized zymosan and assessed with luminol- and lucigenin-enhanced chemiluminescence (ECL). The activity of NAL was compared with N-acetylcysteine (ACC) and captopril, other thiol-containing pharmacological agents having documented antioxidant properties. The three drugs significantly inhibited the ECL response of activated PMNs in the presence of luminol, a luminogenic agent which mostly reflects the production of hydroxyl and hypohalite radicals. NAL was more efficient than the other two drugs: the concentrations producing a 50% inhibition (IC₅₀) of total luminol-ECL were 290 μM, 1580 μM and 760 μM for NAL, ACC and captopril, respectively. The inhibition of the lucigenin-ECL response of activated PMNs was less marked for all compounds suggesting a poorer reactivity with superoxide radicals. These findings demonstrate that NAL, at concentrations obtainable in vivo by inhalation, impairs the PMNs chemiluminescence response related to hydroxyl and hypohalite radicals production. As those radicals are highly cytotoxic, NAL appears as a promising agent in the prevention of oxidative lung damage caused by an active inflammatory response.     

Processing of Human Cord Blood by Three Different Procedures for Red Blood Cell Depletion and Mononuclear Cell Recovery

BACKGROUND AND OBJECTIVES: Human cord blood (CB) is an important source of stem cells which may be used for hematopoietic reconstitution as an alternative to bone marrow transplantation. Banking of CB would be accomplished by removing red blood cells (RBC) and plasma from CB collections. Our aim was to compare three different procedures for CB processing. MATERIALS AND METHODS: Poligeline, hydroxyethyl starch gel (HES) and gelatin were used as separation media in processing 79 CB units for RBC depletion and mononuclear cell (MNC) recovery. RESULTS: The best MNC recoveries were obtained performing the HES- and the gelatin-based procedures (80.9 and 84.7%, respectively), but the gelatin procedure allowed us to obtain the highest RBC depletion (96.4%); CD34+ cell recovery was higher using HES or gelatin as separation media (85.6 and 85.9%, respectively). CONCLUSION: The best results, as far as RBC removal and MNC recovery are concerned, were obtained by using gelatin as RBC sedimentation medium. Gelatin is a low-cost, animal-derived reagent, which has been successfully used for CB transplantation; the procedure is simple to perform and appears to be suitable for large-scale banking in view of CB transplantation.     

A Human Leukemia Cell with Both B and T Cell Surface Receptors

Bone-marrow-derived (B) and thymus-derived (T) lymphocytes can be distinguished by the presence of a number of receptors and differentiation antigens. The presence of these markers has facilitated the identification and characterization of the mononuclear cells in a number of animal and human lymphoid malignancies. We describe here the immunological properties of human leukemia cells that are highly unusual, since they simultaneously bear the receptor for sheep erythrocytes characteristic of human T lymphocytes and the receptor for antigen-antibody-complement complexes characteristic of human B lymphocytes. A small number (about 2%) of normal human lymphocytes bearing both of these receptors was also identified.     

Heterogeneity of Red Blood Cell Velocity in Skeletal Muscle Decreases with Increased Flow

Objective: Effective material exchange between blood and tissue depends on the heterogeneity of microvascular flow. The objective was to address inconsistencies between intravital studies regarding this dependency. We tested the hypothesis that heterogeneity of red blood cell velocity (V_RBC) in capillary beds varies with the strength of metabolic stimulus and with capillary bed geometry. Methods: We used videomicroscopy to measure V_RBC in a bed of 10–24 capillaries at the surface of extensor digitorum longus (EDL) muscle in anesthetized rats. The coefficient of variation (CV = standard deviation/mean; an index of spatial heterogeneity) was computed in the same bed before and after (i) 1, 2, 4, or 8 Hz supramaximal muscle contraction or (ii) adenosine superfusion (10^?7–10^?3 M). Beds with or without arteriolar—venular capillary shunts were used. Results: Although control V_RBC differed between beds (shunt: 232 μm/s; no shunt: 130 μm/s), the percentage increases in postcontraction V_RBC did not (range: 111–326%). In both beds, control CV varied greatly (overall range: 28–117%) and 2–8 Hz muscle contractions reduced CV significantly by 25%. Similar results were obtained for adenosine. In confirmatory experiments using the rat cremaster muscle, contractions (4 Hz) and adenosine (10^?4 M) also reduced CV. Based on all data, CV = 63–0.022 V_RBC (r = 0.82, P < 0.001). Conclusions: The heterogeneity of V_RBC decreased with metabolic stress, regardless of capillary bed geometry. We propose that both the large variability in control CV and the relatively shallow dependence of CV on velocity could be responsible for the present inconsistencies between intravital studies.     

Interaction of Human Tumor Viruses with Host Cell Surface Receptors and Cell Entry

Currently, seven viruses, namely Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpes virus (KSHV), high-risk human papillomaviruses (HPVs), Merkel cell polyomavirus (MCPyV), hepatitis B virus (HBV), hepatitis C virus (HCV) and human T cell lymphotropic virus type 1 (HTLV-1), have been described to be consistently associated with different types of human cancer. These oncogenic viruses belong to distinct viral families, display diverse cell tropism and cause different malignancies. A key to their pathogenicity is attachment to the host cell and entry in order to replicate and complete their life cycle. Interaction with the host cell during viral entry is characterized by a sequence of events, involving viral envelope and/or capsid molecules as well as cellular entry factors that are critical in target cell recognition, thereby determining cell tropism. Most oncogenic viruses initially attach to cell surface heparan sulfate proteoglycans, followed by conformational change and transfer of the viral particle to secondary high-affinity cell- and virus-specific receptors. This review summarizes the current knowledge of the host cell surface factors and molecular mechanisms underlying oncogenic virus binding and uptake by their cognate host cell(s) with the aim to provide a concise overview of potential target molecules for prevention and/or treatment of oncogenic virus infection.     

The Surface Morphology of Mitogen-Stimulated Human Peripheral Blood Lymphocytes

The changes occurring in surface morphology during the transformation of normal peripheral blood lymphocytes by phytohaemagglutinin (PHA) are described and the surface ultrastructure of the PHA-induced blasts is compared with that of pokeweed mitogen (PWM)-and rabbit anti-beta2-microglobulin antiserum-induced blasts. Both mitogen-specific and non-specific changes were observed and their possible relationship to the activation of lymphocyte subpopulations is discussed. Similar surface characteristics found in various tissue lymphoid cells are also described.     

C3b Receptor-Negative Peripheral Blood Neutrophils

Expression of peripheral blood neutrophil (PBN) C3b receptors, as assessed by rosette formation with C3b-coated ox erythrocytes, was examined and compared with neutrophil alkaline phosphatase (NAP) activities in both normal and haematologically abnormal conditions. The results indicate that a small percentage of normal PBN are apparently C3b receptor-negative and that these neutrophils do not appear to differ with respect to age from those with detectable C3b receptors. Examination of PBN C3b receptors from 154 cases of various haematological disorders revealed a significant proportion of cases with increased numbers of C3b receptor-negative neutrophils. These abnormalities did not appear to be related to peripheral leucocyte counts, NAP activities or serum lysozyme concentrations and it is suggested that the increased numbers of C3b receptor-negative PBN may be related to intravascular factors such as immune complexes.     

人脐血造血细胞体外扩增与表面标志的关系

目的：对脐血造血细胞体外扩增及移植的最佳时机选择进行探讨。方法：使用SCF，rIL－3，rIL－1β和rlL－6长期培养人脐血造血细胞，观察其体外扩增与表面标志的变化。结果：培养14天细胞增殖达高峰，28天下降，但仍为第7天的29．88倍。CD细胞第14天达高峰，28天后仍维持一定水平。HLA－DR+细胞培养14～21天达高峰，第35天仍较高。CD细胞培养第7天显著增多，14～21天达高峰，28天后明显下降。结论：此培养体系能够促进脐血造血细胞增殖、扩增，并维持其存活。14天造血细胞数量最多，状态最佳，适于移植。     

The Endotoxin-induced Coagulant Activity of Human Monocytes

Leucocyte suspensions, exposed to endotoxin in vitro, develop coagulant activity which has been identified as tissue factor. Pure suspensions of polymorphonuclear (PMN) neutrophils, lymphocytes and monocytes were exposed to endotoxin and tested for tissue factor activity after 4 h incubation at 37 degrees C. The results indicate that the monocyte is the cell primarily responsible for the endotoxin-induced coagulant activity of mixed leucocyte suspensions, the small amount of activity demonstrated in PMN neutrophil and in lymphocyte suspensions at high cell concentrations being accounted for by monocyte contamination of less than 1%.