The A, M, N, and P1 blood group substances in butanol extracts of human erythrocytes

Aqueous phase and butanol phase extracts of group A1, O, M, N, P1 and P2 human erythrocytes perpared by extraction of red cell stromata with n-butanol were examined for the presence of blood group substances in inhibition of agglutination experiments using antisera and test cells of human origin. Group A substance was recovered in both aqueous phase and butanol phase extracts, M and N substance in aqueous phase extracts only, and P1 substance only in butanol phase extracts. Use of high concentrations of extract resulted in detection of P1 activity in aqueous phase extracts and of N specific inhibition in the aqueous phase extracts of group M erythrocytes. Distilled water appeared to be superior to digitonin and hypotonic phosphate buffer solutions for preparation of stromata for the recovery of group A and M substances in the butanol extracts but not for group N and P1 substances.     

The A, M, N, and P1 Blood Group Substances in Butanol Extracts of Human Erythrocytes

Aqueous phase and butanol phase extracts of group A₁, O, M. N, P₁ and P₂ human erythrocytes prepared by extraction of red cell stromata with n-butanol were examined for the presence of blood group substances in inhibition of agglutination experiments using antisera and test cells of human origin. Group A substance was recovered in both aqueous phase and butanol phase extracts, M and N substances in aqueous phase extracts only, and P₁ substance only in butanol phase extracts. Use of high concentrations of extract resulted in detection of P₁ activity in aqueous phase extracts and of N specific inhibition in the aqueous phase extracts of group M erythrocytes. Distilled water appeared to be superior to digitonin and hypotonic phosphate buffer solutions for preparation of stromata for the reccvery of group A and M substances in the butanol extracts but not for group N and P₁ substances.     

The I, i, and HI blood group antigens in extracts of human erythrocytes

Extracts of stromata of human adult and newborn (cord) erythrocytes were prepared with n-butanol. The aqueous phase and butanol phase extracts were examined for the presence of blood group I, i, HI and H substances in inhibition of agglutination experiments. The recovery of HI activity in both the aqueous and butanol phase extracts of adult red cells but only in the aqueous phase of cord cells is reported heare for the first time. The i specificity was present in the aqueous phase but not in the butanol phase of cord erythrocytes, also not previously reported. Although the recovery of I but not i substance in aqueous phase extracts of adult cells had been shown by other workers, examination of the butanol phase extracts for I and i substances had not been documented. In the present study, Ii substances were not demonstrable in any of the butanol phase extracts of adult and cord erythrocytes.     

The I, i, and HI Blood Group Antigens in Extracts of Human Erythrocytes

Extracts of stromata of human adult and newborn (cord) erythrocytes were prepared with n-butanol. The aqueous phase and butanol phase extracts were examined for the presence of blood group I, i, HI and H substances in inhibition of agglutination experiments. The recovery of HI activity in both the aqueous and butanol phase extracts of adult red cells but only in the aqueous phase of cord cells is reported here for the first time. The i specificity was present in the aqueous phase but not in the butanol phase of cord erythrocytes, also not previously reported. Although the recovery of I but not i substance in aqueous phase extracts of adult cells had been shown by other workers, examination of the butanol phase extracts for I and i substances had not been documented. In the present study, Ii substances were not demonstrable in any of the butanol phase extracts of adult and cord erythrocytes.     

The I,i, and HI Blood Group Antigens in Extracts of Human Erythrocytes

Extracts of stromata of human adult and newborn (cord) erythrocytes were prepared with n-butanol. The aqueous phase and butanol phase extracts were examined for the presence of blood group I, i, HI and H substances in inhibition of agglutination experiments. The recovery of HI activity in both the aqueous and butanol phase extracts of adult red cells but only in the aqueous phase of cord cells is reported here for the first time. The i specificity was present in the aqueous phase but not in the butanol phase of cord erythrocytes, also not previously reported. Although the recovery of I but not i substance in aqueous phase extracts of adult cells had been shown by other workers, examination of the butanol phase extracts for I and i substances had not been documented. In the present study, Ii substances were not demonstrable in any of the butanol phase extracts of adult and cord erythrocytes.     

The H, HI, I and i blood group antigens in butanol extracts of rabbit tissues

Aqueous and butanol phase extracts of rabbit erythrocytes and the tissues gastric mucosa, liver, lung and kidney were examined by inhibition of agglutination for the presence of H, HI, I and i blood group substances. Of the four specificities, only I substance was recovered from erythrocytes and only in the aqueous phase extracts. Both aqueous and butanol extracts of gastric mucosae contained H substance, but HI and I were present only in the aqueous extracts of this tissue. Extracts of rabbit lungs only had H and I activity and only in the aqueous phase. No H, HI, I or i substance was detected in any of the extracts of liver and kidney. Reduced levels of H substance were found in aqueous extracts of gastric mucosae of 'A-like' rabbits. Three H specificities were demonstrated in the butanol phase extracts of gastric mucosae of rabbits. These specificities were similar to those previously shown in extracts of gastric mucosae and erythrocytes of humans.     

A FAMILY CASE OF BLOOD GROUP B VARIANTS

Carriers of weak B variant were found in two generations of a family. The saliva of the father contained a small amount of B substance, but the saliva of his two daughters contained only H but not B. The erythrocytes of the propositus reacted only with potent anti-B and anti-(A + B) sera, and though the erythrocytes were not agglutinated by the weaker anti-B sera, the eluates showed that they combined with anti-B antibody. The eluates from normal human B and AB erythrocytes, and guinea-pig's erythrocytes which had adsorbed human anti-B agglutinin, agglutinated normal B erythrocytes but not the weak B. The eluates from the weak B erythrocytes and rabbit's erythrocytes both of which had adsorbed anti-B, agglutinated the weak B erythrocytes as well as the normal B erythrocytes. These weak B erythrocytes showed agglutinability as strong as that of O erythrocytes against anti-H eel serum. The sera of these weak B variants contained normal anti-A agglutinin and a weak anti-B cold agglutinin which was reactive at 5°C. As the saliva of the propositus contained as much H substance as was found in group O secretor saliva, but not B substance, her blood group was denoted by B_x. Her sister has same characters of B_x. in the erythrocytes and in the saliva. Her father's blood group was AB, and his erythrocytes showed weak B antigenicity, and his saliva contained rather weak B substance as well as the normal amounts of A and H substances.     

Inhibition of delayed hypersensitivity by pre-immunization without complete adjuvant

Guinea-pigs were immunized by injections of blood group substance with incomplete adjuvant, followed after an interval of approximately 2 weeks, by intracutaneous immunization with the same antigen and Freund's adjuvant containing M. tuberculosis. This treatment inhibited the appearance of delayed skin reactions, while circulating antibody production took place as in controls which had received complete adjuvant only with blood group substance, and had delayed skin reactions. The inhibition of the skin reaction was found to be antigen-specific with regard to unrelated antigens, but showed cross-inhibition for serologically different human blood group substances. The first immunization had to be given more than 2 days before the immunization with complete adjuvant. A similar phenomenon was seen with ovalbumin as antigen. In addition to inhibition of the delayed skin reaction, there appeared to be less γ₂-antibody to ovalbumin than in ovalbumin plus complete adjuvant-only controls. Passive administration of antibody did not affect the development of a delayed hypersensitivity state in complete adjuvant-immunized animals with blood group substance or ovalbumin as antigen. Present evidence favours an explanation of the phenomenon in terms of temporary paralysis on the part of some of the antibody-producing cells—viz. those concerned with delayed hypersensitivity and γ₂-antibody production.     

Further Evidence that Carbohydrates are the Immunodeterminant Structures of Blood Group M and N Specificities

Terminal β-δ-galactopyranosyl (Gal) groups are implied in blood group N but not M specificity by the following findings: (a) Rabbit anti-asialoganglioside sera specific for terminal β-Gal-(1→3)-GalNac agglutinate human group 0 M and N erythrocytes, the latter to a significantly higher titer, while rabbit anti-ganglioside G_MI sera, where sialic acid modifies the antibody specificity, do not. The agglutination score with N erythrocytes was about twice that with M red blood cells. The asialoganglioside antibodies were readily absorbed by group 0 N erythrocytes, which were up to 10 times more efficient than group 0 M erythrocytes. Erythrocyte agglutination by the anti-asialoganglioside sera was inhibited by M and N antigen preparations isolated from group 0 red cell ghosts. N antigen was a better inhibitor. Asialoganglioside effectively inhibited the red cell agglutinations by anti-asialoganglioside serum. Ganglioside G_MI did not inhibit, (b) Horse anti-pneumococcus Type XIV serum, which has anti-β-Gal specificity, precipitated highly active N but not M substances. This precipitation was specifically inhibitable by oligosaccharides with terminal β-Gal. (c) Beta galactosidase specifically inactivated native N and 'acid-produced' N substances with the release of about three moles Gal per subunit of N antigen. It did not affect M antigen.     

Pollen‐derived low‐molecular weight factors inhibit 6‐sulfo LacNAc+ dendritic cells' capacity to induce T‐helper type 1 responses

Background Evidence is accumulating that the pollen exsudate contains an array of non‐allergenic, pro‐inflammatory and immunomodulatory substances acting on the innate and adaptive immune system. In this context, pollen‐associated E₁‐phytoprostanes (PPE₁) were shown to licence human monocyte‐derived dendritic cells for T‐helper type 2 (Th2) polarization of naïve T cells. Objective This study aims at analysing the impact of pollen‐associated lipid mediators on cytokine secretion and maturation of 6‐sulfo LacNAc⁺ dendritic cells (slanDCs), the most abundant native dendritic cell (DC) in human peripheral blood, and further dissecting the biologically active substance(s) within aqueous pollen extracts. Results Aqueous birch pollen extracts dose‐dependently inhibited the lipopolysaccharide (LPS)‐induced IL‐12 p70 production, while the levels of IL‐6 remained unaffected. PPE₁ inhibited secretion of both IL‐12 p70 and IL‐6. Aqueous pollen extracts, but not PPE₁ or F₁‐phytoprostanes significantly reduced the LPS‐induced surface expression of the maturation markers CD80, CD83, CD40 and CCR‐7, an effect that was independent of proteins and that was still present in a 3 kDa cut‐off fraction of the pollen extract. These effects were observed irrespective of the atopy status of the donors. Finally, slanDCs exposed to aqueous pollen extracts were impaired in eliciting an IFN‐γ response in naïve CD4⁺ T cells. Conclusion Our data show that slanDCs, a subset of human blood DCs with constitutively high potency to induce Th1 responses, are susceptible to the Th2 polarizing effect of low molecular weight, non‐protein factors derived from pollen. Cite this as: S. Gilles, D. Jacoby, C. Blume, M. J. Mueller, T. Jakob, H. Behrendt, K. Schaekel and C. Traidl‐Hoffmann, Clinical & Experimental Allergy, 2010 (40) 269–278.     

Further evidence that carbohydrates are the immunodeterminant structures of blood group M and N specificities

Terminal beta-D-galactopyranosyl (Gal) groups are implied in blood group N but not M specificity by the following findings: (a) Rabbit anti-asialoganglioside sera specific for terminal beta-Gal-(1 leads to 3)-GalNac agglutinate human group 0 M and N erythrocytes, the latter to a significantly higher titer, while rabbit anti-ganglioside GMl sera, whereas sialic acid modifies the antibody specificity, do not. The agglutination score with N erythrocytes was about twice that with M red blood cells. The asialoganglioside antibodies were readily absorbed by group 0 N erythrocytes, which were up to 10 times more efficient than group 0 M erythrocytes. Erythrocyte agglutination by the anti-asialoganglioside sera was inhibited by M and N antigen preparations isolated from group 0 red cells ghosts. N antigen was a better inhibitor. Asialoganglioside effectively inhibited the red cell agglutinations by anti-asialoganglioside serum. Ganglioside GMl did not inhibit. (b) Horse anti-pneumococcus Type XIV serum, which has anti-beta-Gal specificity, precipitated highly active N but not M substances. This precipitation was specifically inhibitable by oligosaccharides with terminal beta-Gal. (c) Beta galactosidase specifically inactivated native N and "acid-produced' N substances with the release of about three moles Gal per subunit of N antigen. It did not affect M antigen.     

Reactions of monoclonal antibodies to human MN blood groups with red blood cells of subhuman primates

All 12 samples of chimpanzee erythrocytes tested were agglutinated by the commercial polyclonal anti-M reagent of rabbit origin, but none was agglutinated by monoclonal anti-M reagent of murine origin. This, as well as results of absorption experiments, showed that the polyclonal and monoclonal anti-M reagents detect different M epitopes. Polyclonal anti-N reagent of rabbit origin and monoclonal reagent of murine origin obtained by immunization with N blood group substance did not react with any chimpanzee erythrocyte samples. On the other hand, monoclonal anti-N reagent originating from immunization with M blood group substance agglutinated two of the 12 tested chimpanzee erythrocyte samples. This and other experimental results strongly suggested that the latter monoclonal anti-N reagent combined with "N" antigen, i.e., an antigen present on glycophorin B of human M as well as N erythrocytes; apparently erythrocytes of some chimpanzees contain also this antigen.     

Reactions of Monoclonal Antibodies to Human MN Blood Groups with red Blood Cells of Subhuman Primates

All 12 samples of chimpanzee erythrocytes tested were agglutinated by the commercial polyclonal anti-M reagent of rabbit origin, but none was agglutinated by monoclonal anti-M reagent of murine origin. This, as well as results of absorption experiments, showed that the polyclonal and monoclonal anti-M reagents detect different M epitopes. Polyclonal anti-N reagent of rabbit origin and monoclonal reagent of murine origin obtained by immunization with N blood group substance did not react with any chimpanzee erythrocyte samples. On the other hand, monoclonal anti-N reagent originating from immunization with M blood group substance agglutinated two of the 12 tested chimpanzee erythrocyte samples. This and other experimental results strongly suggested that the latter monoclonal anti-N reagent combined with “N” antigen, i.e., an antigen present on glycophorin B of human M as well as N erythrocytes; apparently erythrocytes of some chimpanzees contain also this antigen.     

Catfish antibodies to blood group substances: III. Further studies with sera from fish injected with human group O secretor fluids

An antiserum prepared in the freshwater catfish Tandanus tandanus by the injection of O secretor seminal plasma was fractionated into anti-H reagents showing different specificities by absorption with A₁B erythrocytes and by absorption and elution from A₁B cells. Although purified human and hog H blood group substances inhibited the haemagglutination of O erythrocytes by both the eluate from A₁B cells and the serum remaining after absorption with A₁B cells, all of the simple sugars tested, except 2′-fucosyl-lactose, failed to inhibit either sample. The H-substances inhibited the A₁B-eluate at dilutions which were significantly higher than those required to inhibit the A₁B-absorbed serum. Inconsistent with this result was the finding that 2′-fucosyl-lactose, a trisaccharide with a structure similar to the terminal H-active groupings on the type 2 chains of the ABH macromolecules, was a more active inhibitor of the absorbed than of the eluted serum. Seventeen different samples of O secretor saliva either failed to inhibit the A₁B-absorbed serum, or produced inhibition at very low dilution. These same saliva samples inhibited the A₁B-eluate in high dilution.     

Sedative and Anxiolytic Effects of the Extracts of the Leaves of Stachytarpheta Cayennensis in Mice

The leaves are used ethnomedicinally in Nigeria and other parts of the world for insomnia and anxiety among other uses. The investigations sought scientific evidence for the ethnomedicinal use of the leaves for the management of insomnia and anxiety as well as the neural mechanisms for the activities. The sedative and anxiolytic effects of the extracts of the leaves of Stachytarpheta cayennensis were examined in this study. The methanolic extract (5–50 mg/kg, i.p.) as well as the ethylacetate (10–50 mg/kg, i.p.), butanol and aqueous fractions (5–50 mg/kg, i.p.) of the extract were examined. Sedation was assessed as reduced novelty-induced rearing (NIR), reduced spontaneous locomotor activity (SLA) and increased pentobarbitone-induced sleeping time (PIST) in mice. The anti-anxiety effect (methanol 2.5–5.0; butanol 5.0; aqueous 20.0; ethylacetate 25.0 mg/kg, i.p.) was assessed using an elevated plus maze. LD₅₀ was calculated for the extract and the fractions after the intraperitoneal route of administration using the Locke method. The methanolic extract, the butanol and the aqueous fractions inhibited rearing and spontaneous locomotion but prolonged pentobarbitone induced sleep. The ethylacetate fraction however increased both rearing and locomotion and decreased pentobarbitone sleeping time. The butanol and aqueous fractions, but not the methanol extract showed indices of open arm avoidance consistent with anti-anxiety effect. Naltrexone (2.5 mg/kg, i.p.) reversed the inhibition of rearing, locomotion and prolongation of pentobarbitone sleep due to the aqueous fraction of the extract. Flumazenil (2mg/kg, i.p.) abolished the effects of both methanolic extract and the butanol fraction on rearing, locomotion, pentobarbitone sleep and anxiety model. The methanolic extract, the butanol and aqueous fractions possess sedative activity while the ethylacetate fraction possesses stimulant property. The anxiolytic effect was found in both the aqueous fraction and the butanol fraction but not in the main methanol extract and also not in the ethylacetate fraction. Flumazenil, blocked the effect of the leaves of Stachytarpheta cayennensis on rearing, locomotion and elevated plus maze suggesting that GABA receptors are involved in the observed sedative and anxiolytic activities. This study also found opioid receptors involved in the sedative activity of the leaves of Stachytarpheta cayennensis. The rationale for the ethnomedicinal use of the leaves for the management of insomnia and anxiety were confirmed scientifically in this study.     

Detection of blood group P1 activity in F. hepatica and F. gigantica (author's transl)]

Four lyophilizated liver flüke antigens and one of hydatid fluid were examined for the presence of blood group like antigens P1, A, B, M and N by an hemagglutination inhibition test with hyperimmune and natural hemagglutinating sera. P1 or P1-like substance was found in the extracts of "Fasciola hepatica" and "F. gigantica" in a lower concentration than in echinoccocal fluid. This presence may explain the occurrence of anti-P1 antibodies in patients infected with liver flüke and belonging to the P2 blood group. However biochemical studies will be necessary to determine if the P1 activity is due to the blood group P1 substance of the red cell itself or due to a P1 like substance.     

Inhibition Effects of Scorpion Venom Extracts (Buthus Matensii Karsch) on the Growth of Human Breast Cancer MCF-7 Cells

Background

To observe the inhibition effects of the Buthus matensii Karsch (BmK) scorpion venom extracts on the growth of human breast cancer MCF-7 cells, and to explore its mechanisms.

Methods

Two common tumor cells (SMMC7721, MCF-7) were examined for the one which wasmore sensitivity to scorpion venom by MTT method. Cell cycle was determined by flow cytometry. Immunocytochemistry was applied to detect apoptosis-related protein Caspase-3 and Bcl-2 levels, while the expression of cell cycle-related protein Cyclin D1 was shown by Western blotting.

Results

Our data indicated that MCF-7 was the more sensitive cell line to scorpion venom. The extracts of scorpion venom could inhibit the growth and proliferation of MCF-7 cells. Furthermore, the extract of scorpion venom induced apoptosis through Caspase-3 up-regulation while Bcl-2 down-regulation in MCF-7 cells. In addition, the extracts of scorpion venom blocked the cells from G₀/G₁ phase to S phase and decreased cell cycle-related protein Cyclin D1 level after drug intervention compared with the negative control group.

Conclusions

These results showed that the BmK scorpion venom extracts could inhibit the growth of MCF-7 cells by inducing apoptosis and blocking cell cycle in G₀/G₁ phase. The BmK scorpion venom extracts will be very valuable for the treatment of breast cancer.     

Surface antigens on Schistosoma mansoni. II. Adsorption of a Forssman-like host antigen by schistosomula

A study was made of the nature of mouse (host) antigens adsorbed by schistosomula of Schistosoma mansoni. Using the mixed antiglobulin test, extracts of a number of individual mouse tissues were tested for their ability to coat schistosomula. All were effective to some extent, with the greatest activity being found in extracts of the lung and spleen. Antibodies against the schistosomulum-coating antigen as well as surface host antigens of adult Schistosoma mansoni were removed by absorbing with erythrocytes from a number of Forssman-positive but not Forssman-negative animal species. These antibodies were also absorbed by Forssman-positive guineapig kidney extract and methanol soluble (Forssman-positive) but not insoluble fractions of sheep erythrocyte stromata and mouse lungs. Schistosomula could be coated in vitro with methanol soluble fractions of mouse lung and erythrocytes and sheep erythrocytes. Though both mouse and sheep coating antigens reacted with anti-mouse and anti-sheep antibodies, reactions were stronger with the homologous antiserum. It was concluded that schistosomula of Schistosoma mansoni adsorb from mice an antigen similar but not identical to the Forssman antigen of sheep erythrocytes, and that this antigen is also found on the surface of adult worms.     

HLA antigens: rabbit antisera reacting with all A series or all B series specificities

Papain-solubilized HLA antigens giving only two bands of 34 000 mol.wt. and 11 000 mol.wt. by sodium dodecyl sulfate (SDS) gel electrophoresis and isolated from the cultured human lymphoblastoid cell line RPMI 4265, have been used to prepare antisera in rabbits. Antisera were raised against soluble products of the A (or 1st) series of HLA, bearing the determinant HLA-A2 (A2 substance), or against a mixture of products of the B (or 2nd) series of HLA, bearing the determinants HLA-B7 and HLA-B 12 (B7-12 substances). Rabbit antisera to A2 substance reacted primarily with A2 substance on Ouchterlony analysis, showing an apparent spur of cross-reactivity with B7-12 substances. Rabbit antisera to B7-12 substances reacted primarily with B7-12 substances, giving a spur of cross-reactivity with the A2 material. Neither antiserum precipitated β-₂microglobulin. Both types of sera reacted with membrane molecules of 43 000 mol.wt. and 11 000 mol.wt. by immune precipitation and gel electrophoresis in SDS of detergent-solubilized radiolabeled membranes from cultured cell lines, as predicted for sera directed towards HLA antigens. F(ab′)₂ fragments of the antibodies blocked the complement-mediated cytotoxicity of all HLA alloantisera tested for human peripheral blood lymphocytes. After absorption with B7-12 substances, F(ab′)₂ fragments of antisera to A2 substance only blocked the cytotoxicity of HLA alloantisera to A series specificities. After absorption with A2 substance, F(ab′)₂ fragments of rabbit antisera to B7-12 substances only blocked the cytotoxicity of HLA alloantisera to B series specificities. The results prove the existence of shared antigenic determinants between all members of the same series. These findings support the genetic evidence that A series HLA antigens are allelic products of a single locus, while B series HLA antigens are allelic products of a separate locus, by establishing some invariance of structure, presumably amino acid sequence, between members of the same series. The apparent cross-reactivity between A2 substance and B7-12 substances, and the ability of the unabsorbed F(ab′)₂, preparations to block the cytotoxicity of all HLA alloantisera, suggests that some determinants are common to both HLA loci. This may be considered to support the hypothesis that the two loci arose by gene duplication.     

Antibacterial efficacy of new commercially manufactured disinfectant substances against Salmonella typhimurium

The antibacterial effect of 19 new commercially manufactured disinfectant substances on a Salmonella typhimurium strain was studied. The substances tested represent 9 quaternary ammonium salts (QAT) and 11 combinated QAT with other ingredients. The antimicrobial efficacy was characterized by influencing the growth and reproduction of bacterial cells expressed either by MIC and ED₅₀ values (ED50 values represent concentration of substance in μ/ml which cause inhibition of growth by 50%), as well as by the inhibition of incorporation rate of [¹⁴C] adenine and [¹⁴C] leucine. The disinfectants are divided into three groups according to their efficacy. The first group comprises substances with strong inhibitory effect (MIC 0.04–0.19 μ/ml) such as Neoquat S, Antibacteric P, Divoquat forte, Sokrena and Diesin forte (sole from the group belonging to multicomponent substances). QAT except Antibacteric P interfere with energy metabolism (R values ～1). The second group represents substances with good antibacterial efficacy (MIC values up 1.56 μg/ml), and the third group substances with MIC values up 12.5 μg/ml. Cutasept G was found ineffective also in the concentration 100 μg/ml. The method of inhibition of [¹⁴C] precursors is suitable as one from possible criterion in evaluation of antibacterial efficacy of various synthetic substances.