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 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 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 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.     

Immunocytochemical studies of blood group A, H, I, and i antigens in gastric mucosae of infants with normal gastric histology and of patients with gastric carcinoma and chronic benign peptic ulceration.

Indirect immunofluorescence studies of blood group A, H, I, and i antigens were performed on the gastric mucosae and tumor tissues of patients with gastric carcinoma, on the mucosae of patients with chronic benign gastric ulceration, and on the mucosae of infants who had died of causes other than gastrointestinal disease. The following findings were of particular interest: (1) Normal 'secretor' type mucosae were distinguishable from 'non-secretor' type mucosae by the uniform staining of the A or H antigens at the surface and in the pits. Normal 'non-secretor' type mucosae showed little staining of the H or A antigens but, instead, there was staining with anti-I(Ma) antibody. Staining with anti-I(Step) and anti-i(Den) did not show a clear correlation with the 'secretor'/'non-secretor' status of the normal mucosae. (2) Apparently normal areas of gastric mucosae of patients with gastric carcinoma or the normal part of gastric mucosae of patients with benign gastric ulcer frequently showed focal areas of loss or gain of the blood group antigens as is often seen in gastric carcinoma tissues. (3) In the mucosae of patients with intestinal metaplasia there was marked loss of A/H antigens in 'secretors' and I(Ma) antigen in 'non-secretors'. (4) Staining characteristics of tissues from gastric carcinoma were:(a) Focal loss of the expected A/H or I antigens was observed with much variation in staining from area to area, but only a minority showed complete loss of the expected staining. (b) A majority of the carcinomas from 'secretors' showed foci of substantial staining with anti-I(Ma) in contrast to normal 'secretor' mucosae. This is probably due to incomplete biosynthesis of A/H determinants. (c) Incompatible A-like staining by a rabbit anti-A serum was observed in one out of nine adenocarcinomas from blood group B or O persons. (d) A few cases showed substantial i antigen staining. The aberrant expression of blood group A, H, I, and i antigens in neoplastic as well as in some areas of morphologically normal mucosa of patients with benign and malignant diseases of the stomach is discussed in the context of current biochemical knowledge.     

Blood group antigens A, B, H, Lea, Leb, and I(Ma) in resting and tetragastrin stimulated gastric juice of patients with non-neoplastic diseases of the stomach.

In view of the anomalous expression of blood group and related antigens in the gastric mucosae of patients with malignant and premalignant diseases of the stomach, and the potential clinical value of their measurement, a preliminary study has been performed on the blood group antigens A, B, H, Lea, Leb, and I(Ma) in glycoprotein rich extracts of the resting and tetragastrin stimulated gastric juice of patients without evidence of gastric cancer. The aim has been to assess whether the antigenic profiles known to distinguish the gastric mucosae of secretors from those of non-secretors are reflected in the glycoproteins of gastric juice. Antigenic profiles which distinguish secretors from non-secretors were observed in the stimulated rather than the resting gastric juice as follows: the A, B or H antigens but not I(Ma) were strongly expressed in the glycoproteins of secretors, while I(Ma) was the antigen characteristic of non-secretors. On the other hand, there was considerable overlap in the Lea and Leb antigen values in the resting and stimulated gastric juice of secretors and non-secretors. Among these antigens, I(Ma) is known to appear as a neo-antigen in the gastric mucosae of secretors with malignant and premalignant diseases of the stomach. Thus this antigenic determinant is potentially a clinically useful marker in the gastric juice of 75% of the population who are secretors. The clinical value of the levels of this antigen in the gastric juice now deserves investigation.     

Immunologic Mimicry Between Mouse Tissue and Enterobacterial Common Antigen

Organs of Swiss white albino and C57BL/6Ha mice were assessed for an antigen (CRA) which cross-reacts with common enterobacterial antigen (CA) of To this end, supernatant fluids (HKS) and ethanol-soluble fractions (ES) of heated homogenates of spleens, kidneys, and livers were examined for their capacities to react with CA hemagglutinins and to engender humoral and cellular events in the rabbit. The immunogenicity of CRA in the rabbit can not be predicted on the basis of CA hemagglutinin neutralization studies alone; although CRA was identified in the liver extracts of both mouse strains, according to this parameter, only the liver fraction of Swiss white albino mice elicited significant numbers of rosette-forming cells (RFC) in the spleens of rabbits. Also, kidney fractions, which primed the rabbits for booster with CA, were less effective in stimulating RFC in the spleens of the identical animals. Moreover, although extracts of mouse spleens failed to inhibit CA hemagglutination and did not prime rabbits for a CA hemagglutinin response, these same preparations clearly evoked RFC in rabbit spleens. Thus, the antigenicity and immunogenicity of CRA in target organs of mice reflect the mouse strain, extraction procedure, and testing method employed.     

Kidney antigens active in isoimmunization: I. The antigenic diversity

Isoimmunization of rabbits with kidney extracts and Freund's adjuvant leads to the development of responses to a multiplicity of autoantigens. If a large number of kidneys is pooled, there is increased probability that responses directed toward isoantigens can also be detected. Such immunization thus results largely in antisera that possess multiple specificities, but the amount of antibody directed toward each specific antigen varies from one antiserum to another.

Advantage can be taken of these uneven responses by different animals, to enumerate the variety of responses to uniquely separable sets of determinants, by carefully selecting the antisera. In immunodiffusion studies, lines for such separable antigens can be sorted in an unambiguous manner. A total of five such antigens has now been found in kidney, three of which are kidney-specific and two of which are shared with liver. A sixth antigen, separable from liver, may be only a minor constituent of kidney, and was not directly detected in kidney extracts. It is important that fresh concentrated extracts of kidney homogenates be used. The detection of isoantigens requires concentrated extracts prepared from individual kidneys.

     

Cell-Surface Regulation of the Human Alternative Pathway of Complement

When incubated in normal human serum, rabbit erythrocytes are haemolysed as a result of activation of the alternative pathway of complement (APC), but sheep erythrocytes do not spontaneously activate the human APC under physiological conditions. The mechanism for this difference has been attributed to differences in the relative affinity of membrane-bound C3b for its natural ligands, factor B and factor H, that favour the formation and stability of the APC C3 convertase on rabbit erythrocytes and inhibit convertase activity on sheep erythrocytes. Previous studies have also suggested that factor I inactivated C3b on sheep erythrocytes more effectively than on rabbit erythrocytes. Further, sheep erythrocytes have recently been shown to have a membrane protein that associates non-covalently with cell-bound C3b, but rabbit erythrocytes lack a predominant C3b binding protein. Together, these results suggested the possibility that sheep but not rabbit erythrocytes have a membrane constituent with factor I cofactor activity. To investigate this hypothesis, rabbit and sheep erythrocytes bearing radiolabelled C3b were treated either with factor I or with factor I and factor H, and conversion to iC3b was assessed by autoradiography. Factor I caused a concentration-dependent conversion of C3b to iC3b on sheep erythrocytes; however, only trace amounts of C3b on rabbit erythrocytes were degraded even when high concentrations of factor 1 (83 μg/ml) were used. While C3b on rabbit erythrocytes was converted to iC3b by the combination of factor H and factor I, much less factor H was required for the same degree of conversion of C3b on sheep erythrocytes. Treatment of sheep erythrocytes with neuraminidase had no effect on either factor I-dependent cofactor activity or the capacity of factor H to serve as a factor I cofactor. Sheep erythrocytes did not manifest decay accelerating activity, however, suggesting that the factor I cofactor constituent is a functional analogue of the human membrane cofactor protein.     

Increased sensitivity for detecting avian influenza-specific antibodies by a modified hemagglutination inhibition assay using horse erythrocytes

The hemagglutination inhibition (HI) assay is a widely used serological method to measure the levels of protective antibody responses against influenza viruses. However, the traditional HI assay which uses chicken erythrocytes is not sufficiently sensitive for detecting HI antibodies specific to avian influenza viruses. Previously, it was demonstrated that employing an assay using horse erythrocytes was able to increase the sensitivity of HI assay. The current report describes further optimization of this modified HI assay. It was shown that this method was able to increase detection of HI activities in rabbit sera immunized with H5 HA antigens, and proved that this increased sensitivity is useful in dissecting the strain specificity of HI antibody responses. In addition, the modified HI assay using horse erythrocytes increased the sensitivity of detecting HI antibodies specific for three major serotypes of avian influenza viruses, H5, H7 and H9, in people who may have asymptomatic infection with avian influenza viruses. Based on these results, the optimized use of horse erythrocytes should be standard practice for detecting HI activities against avian influenza viruses.     

Cell-surface regulation of the human alternative pathway of complement. Sheep but not rabbit erythrocytes express factor I-dependent cofactor activity.

When incubated in normal human serum, rabbit erythrocytes are haemolysed as a result of activation of the alternative pathway of complement (APC), but sheep erythrocytes do not spontaneously activate the human APC under physiological conditions. The mechanism for this difference has been attributed to differences in the relative affinity of membrane-bound C3b for its natural ligands, factor B and factor H, that favour the formation and stability of the APC C3 convertase on rabbit erythrocytes and inhibit convertase activity on sheep erythrocytes. Previous studies have also suggested that factor I inactivated C3b on sheep erythrocytes more effectively than on rabbit erythrocytes. Further, sheep erythrocytes have recently been shown to have a membrane protein that associates non-covalently with cell-bound C3b, but rabbit erythrocytes lack a predominant C3b binding protein. Together, these results suggested the possibility that sheep but not rabbit erythrocytes have a membrane constituent with factor I cofactor activity. To investigate this hypothesis, rabbit and sheep erythrocytes bearing radiolabelled C3b were treated either with factor I or with factor I and factor H, and conversion to iC3b was assessed by autoradiography. Factor I caused a concentration-dependent conversion of C3b to iC3b on sheep erythrocytes; however, only trace amounts of C3b on rabbit erythrocytes were degraded even when high concentrations of factor I (83 micrograms/ml) were used. While C3b on rabbit erythrocytes was converted to iC3b by the combination of factor H and factor I, much less factor H was required for the same degree of conversion of C3b on sheep erythrocytes. Treatment of sheep erythrocytes with neuraminidase had no effect on either factor I-dependent cofactor activity or the capacity of factor H to serve as a factor I cofactor. Sheep erythrocytes did not manifest decay accelerating activity, however, suggesting that the factor I cofactor constituent is a functional analogue of the human membrane cofactor protein.     

Hemagglutination with avian infectious laryngotracheitis virus

Summary Avian infectious laryngotracheitis virus grown in primary chicken kidney cell cultures was tested for hemagglutination (HA) with erythrocytes of a variety of species at 4°C, 22°C, and 37°C. HA was observed at all temperatures with mouse erythrocytes but not with cattle, sheep, goat, swine, rabbit, guinea pig, chicken, and goose erythrocytes. A strain variation between mice in the agglutinability of their erythrocytes necessitated selection of mice to obtain erythrocytes. The HA reaction was inhibited by specific antiserum. Some factors involved in the HA and HA-inhibition (HI) were investigated and standard HA and HI tests were established. HI antibody titers of individual chicken sera had a significant positive correlation with their neutralizing antibody titers.     

Hemagglutination with ovine rotavirus

Summary The virus was grown in MA 104 cells, a stable cell line derived from embryonic rhesus monkey kidney, and tested for hemagglutination (HA) with erythrocytes of a variety of species at 4°C, room temperature and 37°C. HA was observed at all temperatures with chicken, sheep, rabbit, guinea pig and human erythrocytes but not with horse, cattle, goat, swine and goose erythrocytes. HA reaction was inhibited by specific antiserum. Some factors involved in the HA and HA-inhibition (HI) were invetigated and standard HA and HI tests were worked out. The HI test as well as neutralization test clearly distinguished ovine rotavirus from bovine, human, simian, equine, porcine and lapine rotaviruses.With 2 Figures     

Role of the Helicobacter pylori virulence factors vacuolating cytotoxin, CagA, and urease in a mouse model of disease.

The pathogenic role of Helicobacter pylori virulence factors has been studied with a mouse model of gastric disease. BALB/c mice were treated orally with different amounts of sonic extracts of cytotoxic H. pylori strains (NCTC 11637, 60190, 84-183, and 87A300 [CagA+/Tox+]). The pathological effects on histological sections of gastric mucosae were assessed and were compared with the effects of treatments with extracts from noncytotoxic strains (G21 and G50 [CagA-/Tox-]) and from strains that express either CagA alone (D931 [CagA+/Tox-]) or the cytotoxin alone (G104 [CagA-/Tox+]). The treatment with extracts from cytotoxic strains induced various epithelial lesions (vacuolation, erosions, and ulcerations), recruitment of inflammatory cells in the lamina propria, and a marked reduction of the mucin layer. Extracts of noncytotoxic strains induced mucin depletion but no other significant pathology. Crude extracts of strain D931, expressing CagA alone, caused only mild infiltration of inflammatory cells, whereas extracts of strain G104, expressing cytotoxin alone, induced extensive epithelial damage but little inflammatory reaction. Loss of the mucin layer was not associated with a cytotoxic phenotype, since this loss was observed in mice treated with crude extracts of all strains. The pathogenic roles of CagA, cytotoxin, and urease were further assessed by using extracts of mutant strains of H. pylori defective in the expression of each of these virulence factors. The results obtained suggest that (i) urease activity does not play a significant role in inducing the observed gastric damage, (ii) cytotoxin has an important role in the induction of gastric epithelial cell lesions but not in eliciting inflammation, and (iii) other components present in strains which carry the cagA gene, but distinct from CagA itself, are involved in eliciting the inflammatory response.     

Characterization of a new polyomavirus (Polyomavirus papionis-2) isolated from baboon kidney cell cultures

Summary Viruses with papovavirus morphology were seen in fluids from baboon kidney cell cultures on three separate occasions (isolates A, B, and C). The size of the virions, 47.9 nm, placed the virus in the polyomavirus genus. It grew well in baboon kidney and Vero cells and less well in human embryo lung (HEL) fibroblasts.The virus could not be identified as the previously described baboon polyomavirus, SA 12, or as any of the other known primate polyomaviruses BK, JC or SV 40, the non-primate viruses mouse polyoma, K, rabbit kidney vacuolating virus (RKV) or bovine polyomavirus (FRKV) by immunofluorescence, immune electron microscopy or hemagglutination inhibition (HI) tests. A rabbit antiserum to the new virus (isolate A) reacted only with the three isolates and not with the other primate polyomaviruses studied.Thirteen percent of 118 wild-caught baboons (Papio anubis) had HI antibody to the new polyomavirus and 21 percent were seropositive for SA 12; only two baboons had antibody to both viruses. These results suggest that in baboons there are two antigenically distinct polyomaviruses which circulate independently. The two viruses may also be distinguished by their hemagglutinating properties: SA 12 agglutinated erythrocytes from a wider range of species but only the newly recognized polyomavirus agglutinated baboon erythrocytes.We propose that the two baboon viruses, SA 12 and the new virus, should be namedPolyomavirus papionis-1 andPolyomavirus papionis-2 respectively.     

The data showing antigenic relationship between salmonella typhi and human leucocytes

Summary As a result of the immunization of 20 rabbits with alcohol typhoid Vi-vaccine (strain Ty₂ Glatt) the authors obtained in all cases antisera containing complement-fixing leucoantibodies (against the leucocytes of man). The sera of 26 nontreated rabbits did not show the presence of such antibodies. Additional investigations proved that the anti-Ty (Vi)-sera bind (in the complement fixation test) not only extracts of human and rabbit leucocytes — thus showing the presence of hetero- and iso-leucoantibodies — but also bind (at lower titers) extracts of human, rabbit and guinea pig erythrocytes, showing the presence of hetero- and iso-erythroantibodies. These results are considered as a further evidence of the possible antigenic relationships between some cell and microbe antigens.(Presented by Active Member of AMN USSR, N. N. Zhukov-Verezhnikov) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 52, No. 10, pp. 89–92, October, 1961     

Antigenically variable Borrelia burgdorferi isolated from cottontail rabbits and Ixodes dentatus in rural and urban areas.

Spirochetes were isolated from 71 subadult Ixodes dentatus removed from cottontail rabbits captured in Millbrook, N.Y., and in New York, N.Y. Spirochetes were also cultured from kidney tissues of six rabbits. While all isolates reacted with monoclonal antibody H9724, which identifies the spirochetes as borreliae, more than half did not bind with antibody H5332 and even fewer reacted with H3TS, both of which were produced to outer surface protein A of Borrelia burgdorferi. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles of three isolates differed from one another and from all previously characterized B. burgdorferi strains from humans, ticks, and wildlife in North America. The 12 periplasmic flagella that originated subterminally from each pointed end of a rabbit Borellia isolate contrasted with the 11 or fewer flagella for B. burgdorferi reported previously from North America. Although DNA homology and restriction endonuclease analysis also revealed differences among a rabbit kidney isolate, an I. dentatus isolate, and B. burgdorferi B31, similarities were sufficient to lead us to conclude that the borreliae in rabbits and I. dentatus are B. burgdorferi. Enzyme-linked immunosorbent assay titers of sera from humans with diagnosed Lyme disease to rabbit tick B. burgdorferi were often similar to one another and to those recorded for a reference B. burgdorferi strain.     

Expression of the Major Histocompatibility Complex Antigens on Different Liver Cellular Components in Rat and Man

We hint analysed the distribution of the major histocompatibility complex (MHC) antigens on different liver cellular components in rat and man. Two types of antisera were used: monospecific rabbit antisera raised against isolated molecules of class I and class II and ordinary hyperimmune alloantisera directed to their allelic specificities. A modified Staphylococcus aureus rosette test, enabling morphological identification of the rosette-forming cell type, was used. In both species the passenger cells, especially the Kupffer cells, reacted strongly with rabbit anti-class I and II The passenger erythrocytes reacted with anti-class I in rat but not in man. Human liver vascular endothelial and bile duct cells displayed a distinct reaction with anti-class I and II, whereas the hepatocytes reacted very weakly, if at all. In rat liver parenchymal component the expression of these antigens was different and weaker. Rat bile duct cells reacted with anti-class I and II in approximately 50% of the testings, whereas rat vascular endothelial cells and rat hepatocytes were essential non-reactive. The alloantiserum reactivity (tested only in the rat) followed closely that of heterologous antisera, and the reactivity of anti-β₂m that of class I alloantisera. Although these differences are probably quantitative rather than qualitative, the results suggest considerable variation in the expression of the MHC antigens on different liver cellular components. These differences partially explain why liver allografts behave differently than, e.g., renal allografts upon transplantation.