Sheep Red Cells Research Articles

Immunomodulating Activity of Ginsenoside Rg1 from Panax Ginseng

The immunomodulatory activity of ginsenoside Rg1 from Panax ginseng was studied in mice using sheep red cells as the antigen. It was found that ginsenoside Rg1 at a dose of 10 mg/kg administered for three consecutive days before immunization increased the number of spleen plaque-forming cells, the titers of sera hemagglutinins as well as the number of antigen-reactive T-cells. Ginsenoside Rg1 also increased the number of T-helper cells with respect to the whole T-cell number and the splenocyte natural killer activity. Ginsenoside Rg1 induced an augmentation of the production of IL-1 by macrophages and exerted a direct mitogenic effect on microcultured thymus cells. Ginsenoside Rg1 also partly restored the impaired immune reactivity by cyclophosphamide treatment.

メン羊赤血球の遊離アミノ酸型と赤血球の生理的諸形質との関係

メン羊赤血球の遊離アミノ酸型と赤血球の生理的諸形質との関係

生薬・ウワウルシの薬理学的研究(第2報)免疫性炎症におけるArbutinとPrednisoloneあるいはDexamethazoneの併用効果

A study was carried out to examine the combined effect of arbutin isolated from the leaves of Arctostaphylos uva-ursi (L.) Spreng. (Bearberry leaf) and prednisolone or dexamethazone on Type IV allergic reaction-induced immuno-inflammation. Contact dermatitis caused by picryl chloride (PC-CD) and sheep red cell delayed type hypersensitivity (SRBC-DTH) in mice was not inhibited by the oral application of arbutin at 2 divided doses immediately before and 16 h after the application, but arbutin at doses of 10, 50 mg/kg 24 h after the application speedily decreased the swelling of PC-CD and SRBC-DTH. Arbutin plus prednisolone or dexamethazone showed the inhibitory effect on the swelling of PC-CD and SRBC-DTH stronger than that of prednisolone or dexamethazone alone. Prednisolone and dexamethazone decreased the weight of thymus and spleen in intact, PC-CD and SRBC-DTH mice, but arbutin did not show these effects. These results suggest that arbutin may increase the inhibitory action of prednisolone and dexamethazone on PC-CD and SRBC-DTH, but further investigations are required to understand the mechanism involved.

Antibacterial and antimalarial properties of peptides that are cecropin-melittin hybrids

Solid phase synthesis was used to produce 5 hybrid peptides containing sequences from the antibacterial peptide, cecropin A, and from the bee venom toxin, melittin. Four of these chimeric peptides showed good antibacterial activity against representative Gram-negative and Gram-positive bacterial species. The best hybrid, cecropin A(1–13)-melittin(1–13) was 100-fold more active than cecropin A against Staphylococcus aureus. It was also a 10-fold better antimalarial agent than cecropin B or magainin 2. Sheep red cells were lysed by melittin at low concentrations, but not by the hybrid molecules, even at 50 times higher concentrations.

Platelet-activating factor enhances complement-dependent phagocytosis of diamide-treated erythrocytes by human monocytes through activation of protein kinase C and phosphorylation of complement receptor type one (CR1)

Oligomerization of band 3 protein has been recently indicated as an early event in senescent or damaged red cell membrane followed by specific deposition of anti-band 3 antibodies and binding of complement C3 fragments. The band 3-anti-band 3-C3b complex is recognized by homologous monocytes, and phagocytosis ensues. This study shows that recognition of the anti-band 3-C3b complex by the monocyte C3b receptor type one (CR1) plays a crucial role in the process of removal of damaged red cells. Indeed, blocking of monocyte CR1 with an anti-CR1 monoclonal antibody abrogated phagocytosis of diamide-treated red cells. Platelet-activating factor (PAF) is a phospholipid mediator involved in inflammatory processes. Nanomolar (R)-PAF enhanced the CR1-dependent phagocytosis of diamide-treated human red cell and of sheep red cells coated with C3b, induced the fast translocation of protein kinase C to monocyte membrane compartment, and stimulated the phosphorylation of monocyte CR1. The biologically inert lyso-PAF and the enantiomer (S)-PAF were inactive. PAF receptor antagonists and inhibitors of protein kinase C blocked the enhancement of phagocytosis induced by PAF. Protein kinase C translocation, phosphorylation of CR1, and stimulation of this receptor to an active state capable of mediating phagocytosis represent a novel pathway by which PAF interferes with red cell homeostasis and possibly modulates inflammatory reactions and host mechanisms against infections.

Influence of carrageenan on peritoneal macrophages

BALB/c mice were given five intraperitoneal (i.p.) injections of lambda carrageenan (CGNA). Following this treatment, the viability, phagocytic capacity, bactericidal activity, interleukin 1 (IL-1)-producing ability and Ia + cell content of the peritoneal plastic-adherent macrophage population was analysed. The present study demonstrates the enhancement of bactericidal activity, inhibition of the augmented Ia + cell content after priming with sheep red bloo cells (SRBC), and impairment of IL-1 production by peritoneal macrophages, although their viability and phagocytic function remained unaltered.

Surface Properties ofAerococcus viridansin Relation to Virulence

Three strains of Aerococcus viridans isolated from cases of acute meningitis and two older culture-collection strains have been studied for properties which may be associated with bacterial virulence. None produced extracellular polysaccharide in sucrose-trypticase soy broth, nor were they adherent to human buccal epithelial, HeLa, MRC5 or HEP2 cells. Ultrastructural examination revealed a 'fuzzy layer' of indistinct morphology, 10-15 nm thick and 75-150 nm 'hairs' or fibrils; the distribution of both layers was uneven on the cell wall. The 'hairs' were not removed but became less distinct on treatment with 0.1 per cent trypsin; the 'fuzzy' layer was unaffected. The three meningitis strains, but not the older strains, showed haemagglutinating activity on red cells at 37°C with no action on sheep, ox, human (group O), fowl, guinea-pig or monkey red cells; rabbit and mouse cells gave inconclusive results. The haemagglutination of rat cells did not occur at 4°C or with heated (100°C, 10 min) or trypsinised (0.1 per cent) bacteria; it was inhibited by ant i-A. viridans serum or normal serum, 0.5 per cent formaldehyde and unheated or heated (100°C, 10 min) mucin. Haemagglutination was unaffected by the presence of glucose, fructose, galactose, mannose, rhamnose, arabinose, xylose or fucose. Neuraminidase treatment of red cells appeared to inhibit the haemagglutination but treatment of bacteria with N-acetylneuraminic acid gave inconsistent effects. The strains showed a wide range of reactions from hydrophobic to hydrophilic, low hydrophobicity being more marked with the older strains. Keywords: Aerococcus viridans ; Haemagglutination: Ultrastructure; Extracellular polysaccharide; Adherence; Hydrophobicity.

Inhibition of human serum complement activity by diisopropylfluorophosphate and selected anticholinesterase insecticides

Activation of the human complement (C′) system, a major line of defense against infections, requires the participation of serine esterases. Since the widely used anticholinesterase insecticides inhibit serine esterases, the present study evaluated potencies of carbaryl, carbofuran, dichlorvos, and paraoxon to inhibit C′ activities of a panel of normal human sera. C′-mediated lysis of sheep red cells was measured with a modified assay (1) incorporating suboptimal concentrations of sensitizing antibody and (2) exhibiting increased sensitivity to serine esterase inhibitors. Test chemicals were added to diluted sera 2 hr prior to incorporation into C′ reaction mixtures. Potencies to inhibit C′ and serum cholinesterase (CHE) were compared to potencies of diisopropylfluorophosphate (DFP), a potent serine esterase inhibitor and a standard probe for C′ esterases. At 0.5 to 3.0 m m, carbaryl, carbofuran, dichlorvos, and DFP produced a dose-dependent inhibition of lysis, whereas paraoxon was not inhibitory. On a molar basis, carbaryl was three times more potent than DFP, and inhibited lysis 15–25 and 26–45% at 1.0 and 3.0 m m, respectively. Carbofuran, dichlorvos, and DFP were equipotent. Mean IC50's for inhibition of CHE (a marker for occupational exposure to organophosphates and carbamates) by DFP, paraoxon, dichlorvos, carbofuran, and carbaryl were 1.0 × 10 −8, 4.1 × 10 −8, 1.0 × 10 −7, 3.3 × 10 −6, and 1.8 × 10 −5 m, respectively. Potencies of the insecticides to inhibit CHE did not predict absolute or relative potencies to inhibit serum C′ activity.

Decreased frequency of bone marrow NK progenitors in aplastic anaemia.

Twelve patients with aplastic anaemia were studied with regard to the frequency of NK progenitors in the bone marrow (BM) to investigate the mechanism of depressed NK activity and low NK cell count in the peripheral blood. NK cell (CD16+ cell) count and NK (K562) activity were significantly decreased (P less than 0.02 and P less than 0.001, respectively) in the patients as compared to eight healthy control subjects. Nylon wool non-adherent (NW-NA) BM mononuclear cells (MNC) of each patient and control were prepared. Mature T and NK cells were extensively depleted by sheep red cell rosette formation followed by a centrifugation on Ficoll-Hypaque and monoclonal antibody mediated complement dependent cytolysis. Those selected BM cells were cultured in the presence of recombinant interleukin 2 (rIL2). Generation of NK activity was significantly decreased (P less than 0.01) in the patients with aplastic anaemia. Frequency of NK progenitors in the selected BM cells assayed by the limiting dilution method was significantly decreased in those patients (P less than 0.05). The frequency of BM NK progenitors relative to NW-NA BM cells were related to NK cell count (P less than 0.01). Those results indicate that depressed NK activity in aplastic anaemia is closely related to decreased NK cell count which is probably due to decreased production of NK cells in the BM.

A reverse passive haemagglutination test for detection of Japanese encephalitis virus antigens in cerebrospinal fluid

A rapid sensitive and specific reverse passive haemagglutination test (RPHA) was developed for the detection of Japanese encephalitis virus (JEV) antigens in human cerebrospinal fluid (CSF). Sheep red cells were sensitized with five monoclonal antibodies (109, 112, 203, 204 and 301) reactive with envelope glycoprotein of JEV. Viral antigens were detected in CSF from 35 of 58 (60%) clinical cases of JE when the five MAb coated cells were used in combination. An IgM capture ELISA detected JEV specific antibodies in CSF among 52 of these 58 cases (90%). While 29 specimens contained both antigen and IgM antibodies, 23 had only IgM antibodies and 6 had only antigen. RPHA proved valuable for detection of viral antigens in CSF samples obtained within 10 days after onset of clinical symptoms. Amongst the five MAbs used, the individual antigen detection rates were 44, 12, 43, 12 and 36%, for MAbs 109, 112. 203. 204 and 301. respectively.

Inhibition of Human Serum Complement Activity by Diisopropylfluorophosphate and Selected Anticholinesterase Insecticides

Inhibition of Human Serum Complement Activity by Diisopropylfluorophosphate and Selected Anticholinesterase Insecticides. CASALE, G. P., BAVARI, S., AND CONNOLLY, J. J. (1989). Fundam. Appl. Toxicol. 12, 460–468. Activation of the human complement (C') system, a major line of defense against infections, requires the participation of serine esterases. Since the widely used anticholinesterase insecticides inhibit serine esterases, the present study evaluated potencies of carbaryl, carbofuran, dichlorvos, and paraoxon to inhibit C' activities of a panel of normal human sera. C'-mediated lysis of sheep red cells was measured with a modified assay (1) incorporating suboptimal concentrations of sensitizing antibody and (2) exhibiting increased sensitivity to serine esterase inhibitors. Test chemicals were added to diluted sera 2 hr prior to incorporation into C' reaction mixtures. Potencies to inhibit C' and serum cholinesterase (CHE) were compared to potencies of diisopropylfluorophosphate (DFP), a potent serine esterase inhibitor and a standard probe for C' esterases. At 0.5 to 3.0 mM carbaryl, carbofuran, dichlorvos, and DFP produced a dose-dependent inhibition of lysis, whereas paraoxon was not inhibitory. On a molar basis, carbalyl was three times more potent than DFP, and inhibited lysis 15–25 and 26–45% at 1.0 and 3.0 mM respectively. Carbofuran, dichlorvos, and DFP were equipotent. Mean IC50's for inhibition of CHE (a marker for occupational exposure to organophosphates and carbamates)by DFP, paraoxon, dichlorvos, carbofuran, and carbaryl were 1.0×l0−8 4.1×l0−8, 1.0×l0−7 3.3×l0−6and 1.8×l0−5M, respectively. Potencies of the insecticides to inhibit CHE did not predict absolute or relative potencies to inhibit serum C' activity.

Thiol-dependent K:Cl transport in sheep red cells: VIII. Activation through metabolically and chemically reversible oxidation by diamide.

The sulfhydryl (SH) oxidant diamide activated in a concentration-dependent manner ouabain-resistant (OR), Cl-dependent K flux in both low potassium (LK) and high potassium (HK) sheep red cells as determined from the rate of zero-trans K efflux into media with Cl or Cl replaced by NO3 or methane sulfonate (CH3SO3). Diamide did not alter the OR Na efflux into choline Cl. The diamide effect on K efflux appeared after 80% of cellular glutathione (GSH) was oxidized to GSSG, its disulfide. The stimulation of K efflux was completely reversed during metabolic restitution of GSH, a process that depended on the length of exposure to and the concentration of diamide. The action of diamide on both the K:Cl transporter and GSH was also fully reversed by the reducing agent dithiothreitol (DTT). Diamide apparently oxidized the same SH groups alkylated by N-ethylmaleimide (NEM) (Lauf, P.K. 1983. J. Membrane Biol. 73:237-246). Like NEM, diamide activated K:Cl transport several-fold more in LK cells than in HK cells, and the effect on LK cells was partially inhibited by anti-L1, the allo-antibody known to inhibit OR K fluxes.

Na+-K+ pump activities of high- and low-potassium sheep red cells with internal magnesium and calcium altered by A23187.

1. Sheep erythrocytes were treated with the divalent metal ionophore A23187 to alter the cellular magnesium (Mgi) and calcium (Cai) composition. Ouabain-sensitive Na+-K+ pump fluxes were measured using rubidium as a potassium congener in media where Cl- was replaced by NO3-. 2. A23187, per se, had no effect on ouabain-sensitive rubidium influx. However, lowering the concentration of cellular magnesium [( Mg]i) and increasing that of calcium [( Ca]i) decreased Na+-K+ pump flux. 3. Ouabain-sensitive rubidium influx was found to be a saturating function of [Mg]i in high-potassium (HK) red cells with a Hill coefficient of about 1.8 and an apparent half-activation constant (K0.5) of 0.46 mmol/(l original cells). In low-potassium (LK) cells, in the absence and presence of the Na+-K+ pump stimulatory L-antibody, ouabain-sensitive rubidium influx was also saturated with Mgi yielding Hill coefficients of close to 1.8 and K0.5 values of 0.20 and 0.30 mmol/(l original cells), respectively. 4. When [Ca]i was raised at constant [Mg]i ouabain-sensitive rubidium influx was inhibited at about 700 mumol/(l cells) in both HK, and in anti-L-treated LK red cells. 5. These data exclude the possibility that the Na+-K+ pump turnover, known to be different in HK red cells, and in LK red cells in the absence and presence of anti-L (Joiner & Lauf, 1978b), is based on differences in the activation by MgATP, and that Cai interacts with the Na+-K+ pump cycle differently in the two red cell cation types.

Genetic control of red-cell nucleoside transport and its association with the B blood-group locus and nucleoside phosphorylase activity in sheep.

Nucleoside transport in sheep red cells is controlled by two allelomorphic genes, the gene for nucleoside transport deficiency (NuI) being dominant to that for the functional presence of carrier-mediated nucleoside transport activity (Nui). Sheep are also polymorphic with respect to their red-cell nucleoside phosphorylase (NP) activity, some having high activities and others low activities of this enzyme. The gene for high activity (NPH) is incompletely dominant to that for low activity (NPL). Inheritance data indicate that the Nu locus is genetically linked to that for the B blood-group system and, in addition, exerts a pleiotropic effect on NP activity, Nu permeability stabilizing the heat-labile NPL gene product. Nu-permeable cells have a higher ATP content than Nu-impermeable red cells, and within the Nu-impermeable subgroup, NP deficiency causes a further reduction in red cell ATP concentration. It is concluded that the nucleoside inosine supplements glucose as a physiological energy substrate in sheep red cells.

Binding and activation of C4 and C3 on the red cell surface by non-complement enzymes

We investigated the binding of C4 and C3 to red cell surfaces by non-complement enzymes. Cell bound C components were quantitated by a radioimmunoassay, the chain structure of bound components was analyzed by Western blotting and the hemolytic activity of bound components was determined. Trypsin, chymotrypsin, plasmin, elastase, thrombin, kallikrein and enzymes from Bacillus subtilis, Staphytococcus aureus and Streptomyces griseus all were found capable of binding C4b and C3b to sheep red cells. C4b bound by any of these enzymes was hemolytically active; both classical and alternate pathway activity of C3 could be demonstrated for most enzymes except plasmin and thrombin. In addition, trypsin and the bacterial enzymes were also able to generate the classical pathway C3-convertase from C4b + C2. The hemolytic efficiency of enzyme bound C4b and C3b was about the same as for these molecules bound by complement enzymes. In contrast, the process of binding by the non-complement enzymes was several hundred-fold less efficient than by cell bound complement enzymes. The results demonstrate that several enzymes can replace the Cl and C42 enzymes in the classical pathway and are able to initiate the alternative pathway by activating C3 and binding C3b to the cell surface.

A two-stage culture system for the induction of antibody-forming cell clones in cultures of normal human blood lymphocytes

A two-stage culture method is described for the induction of a specific antibody response to sheep red cells (SRC) in microcultures at limiting dilutions of human peripheral blood lymphocytes (PBL). PBL from normal donors were cultured for 4 days with antigen and EBV using well defined conditions. The cells were then distributed in 10 μl microcultures at different cell densities in order to estimate the frequency of responding units. The culture well were tested for the presence of anti-SRC antibody by the spot test. The results show that the expression of antibody-forming cell clones in the second stage microcultures is strictly dependent on the presence of both antigen and EBV during the first stage cultures. The efficiency of the system was improved by the addition of 4% polyethylene glycol (PEG, MW 6000) in the first stage and its removal in the second stage and by the use of human serum (instead of fetal calf) in both stages. This approach permits the separation of different cellular events, occurring when human B cells are stimulated by antigen and represents a useful approach for studying the mechanisms of the specific immune response in man.

In human monocytes a strong correlation exists between expression of the M3 antigen, Fc-mediated phagocytic activity and failure to participate in extracellular antibody-dependent cytotoxicity.

Human fresh blood monocytes can phagocytize and lyse antibody-coated target cells by contact with membrane Fc receptors. Recently, the monocyte differentiation antigen Leu M3 has been described to be associated with monocyte/macrophage maturation pathway and to be linked to functionally distinct monocyte subsets. In the present study peripheral blood monocytes were separated into M3+ and M3- subsets, and evaluated for their ability to mediate antibody-driven effector functions. A clear cut functional difference could be demonstrated. M3+ monocytes phagocytize antibody-coated sheep red cells but do not carry out contact-mediated extracellular lysis. In contrast, M3- monocytes have exactly the opposite functional features and they mediate cytolysis without exhibiting any phagocytic activity. By using phenotypically defined clones of the U937 histiocytic cell line, the segregation of different lytic abilities and their linkage of the M3 phenotype have been confirmed.

A novel method for the purification of sheep red cell rosetting lymphocytes

A rapid, direct method for the purification of sheep red cell rosetting lymphocytes (ERFC) was developed. The whole procedure, including rosette formation, density separation and hemolysis could be completed within 10 min. A mixture of human peripheral blood mononuclear cells (PBMC) and 2-aminoethylisothiouronium bromide hydrobromide-treated sheep erythrocytes (E AET) was layered onto Ficoll-Paque without any pretreatment and centrifuged at 600 × g for 2.5 min. The pellet was then immediately treated with an NH 4Cl solution containing 10% FCS and hemolysis was completed within 1 min. The purity of ERFC separated in one cycle of the procedure was 98%, the viability 99% and the yield 56% of the initial lymphocyte count. The re-rosetting ability of the prepared cells, after hemolysis, was 95%. The lymphocytes in the fraction prepared by the same method contained 94.3% CD2(OKT11) + cells, 90% of which were CD3(OKT3) + cells (T cells) and 9% were CD16(Leulla) + cells (NK cells).

Phagocytic cell function in aged subjects

In order to study the activity of phagocytic cells in normal and pathological aging, we compared normal young and aged subjects and patients with Alzheimer's (AD) or Parkinson's (PD) disease. Blood granulocytes and monocytes were separately assayed for ingestion of three different particle species (opsonized zymosan, immunoglobulin-coated sheep red cells (IgG-SRC) and glutaraldehyde-treated sheep red cells (G-SRC]. The superoxide anion production induced by these particles was also measured. All granulocyte responses to zymosan and IgG-SRC were depressed in the three aged groups as compared to young controls. Hence, only functions involving a specific receptor (Fc or C3b receptor) seemed affected. Monocyte activity was slightly decreased in the same groups. No difference was found between AD or PD patients and normal aged subjects. Hence the phagocytic and oxidative defects we found were a consequence of aging.

Base-line O2 extraction influences cerebral blood flow response to hematocrit

We have shown that the fall in cerebral blood flow (CBF) as hematocrit (Hct) rises is due to the independent effects of increasing red blood cell (RBC) concentration and arterial O2 content (CaO2). In the present study, we tested the hypothesis that the magnitude of the effect of RBC concentration depends on the base-line cerebral fractional oxygen extraction (E). E is the ratio of O2 demand (cerebral O2 consumption, CMRO2) to supply (cerebral O2 transport: OT = CBF x CaO2) and is assumed to be inversely related to tissue O2 availability. Pentobarbital-anesthetized 1- to 7-day-old sheep were first exchange transfused with plasma to lower Hct to 20%. Base-line E was set to either high or low levels by induction of hypocarbia [arterial CO2 partial pressure (PaCO2) = 15.3 +/- 0.7 mmHg, means +/- SE; n = 7] or hypercarbia (PaCO2 = 62.7 +/- 1.1 mmHg; n = 5), respectively. A second isovolemic exchange transfusion with pure methemoglobin-containing adult sheep red cells then raised Hct (to 38.5 +/- 0.5%) with no significant increase in CaO2. PaCO2 was maintained and other variables (oxyhemoglobin affinity, pH, mean arterial blood pressure) with potential effect on CBF did not change.(ABSTRACT TRUNCATED AT 250 WORDS)