Analysis Of Surface Antigens Research Articles

Genetic and biochemical analysis of erythrocyte-stage surface antigens belonging to a family of highly conserved proteins of Babesia equi and Theileria species

Erythrocyte-stage Babesia equi expresses a 34-kDa immunodominant antigen recognized by antibody from persistently infected horses worldwide. This erythrocyte-stage surface protein, equi merozoite antigen-1 (EMA-1) is encoded by a single copy gene, and was previously shown to share 33% amino acid identity with similar sized proteins of Theileria sergenti and T. buffeli. A mean homology of 31% amino acid identity extends to similar sized proteins of T. parva, T. annulata and T. mutans. Genomic and cDNA copies of a second B. equi gene, ema2 were cloned. The single copy ema2 gene encodes a 30-kDa protein (EMA-2) that shares 52% amino acid identity with EMA-1. EMA-2 also shares a mean amino acid identity of 31% with proteins of similar molecular mass from Theileria species. EMA-1 and EMA-2 each contain a glycosylphosphatidylinositol anchor. These unique erythrocyte-stage surface proteins of B. equi and Theileria species lack antigenic repeats, and excluding the signal peptide, contain one or no cysteines. Consistent with the hypothesis that this family of proteins interacts with the erythrocyte surface, the T. species proteins possess a basic isoelectric point. The B. equi proteins have acidic isoelectric points, but 24-mer peptides within them have strongly basic net charges.

A simple perfusion technique for isolation of maternal intervillous blood mononuclear cells from human placentae

A noninvasive perfusion method for the recovery of maternal placental (intervillous) blood for use in immunologic assays is described. 60% of the perfused blood samples tested for fetal red blood cell (RBC) contamination were found to be pure maternal blood; in the remainder, fetal RBC contamination, with a single exception, was less than 6%. The intervillous mononuclear cells (IVBMC) isolated from this blood were of predominantly maternal origin as demonstrated by a polymerase chain reaction-based DNA typing technique. The number of IVBMC obtained was within the range of 9 to 55×10 6 cells. Phenotypic analysis of IVBMC surface antigens revealed that 61% of the cells were CD3+ T-cells and 18% were CD19+ B-cells. The CD4+ and CD8+ T-lymphocyte subsets accounted for 28 and 26% of the IVBMC, respectively. The IVBMC were functionally competent as evidenced by in vitro lymphoproliferation and cytokine production in response to mitogen and PPD stimulation. This technique allows for rapid and safe isolation of large numbers of IVBMC which are functionally active up to 12 h post-delivery, thus representing a significant improvement over previously described methods. It should facilitate more vigorous research in the study of uteroplacental immunity and infectious disease research, particularly in field settings where sample collection and laboratory facilities are distant.

Verotoxigenic Escherichia coli Infection: U.S. Overview

Escherichia coli O157:H7 remains a public health problem in the United States despite a dramatic increase in the awareness of, and concern about, foodborne infections since the 1993 multistate E. coli O157:H7 epidemic. Although surveillance data can be difficult to interpret, the incidence of endemic disease caused by this organism is probably not increasing, and might be decreasing, at least in selected populations. With increased recognition of E. coli O157:H7 infection has come the investigation of increasing number of outbreaks, leading to the recognition of many “new” vehicles, including some foods not traditionally associated with enteric infections, such as dry-cured salami and lettuce. Molecular fingerprinting techniques are being used to track the transmission of E. coli O157:H7 through human populations. Analysis of DNA encoding virulence factors and surface antigens suggests that diarrheagenic E. coli have evolved by acquiring large DNA fragments, with subsequent chromosomal recombination. Some Shiga toxin–producing E. coli other than E. coli O157:H7 are no doubt pathogens, but the majority of these toxigenic strains found in food are probably not virulent. More research is needed to define the characteristics that render selected Shiga toxin–producing organisms harmful to humans.

Chronic administration of dexamethasone results in Fc receptor up-regulation and inhibition of class I antigen expression on macrophages from MRL/lpr autoimmune mice.

The MRL/lpr mouse develops, after approximately 8 weeks of age, a severe autoimmune syndrome with many features resembling human systemic lupus erythematosus, including autoantibodies against DNA and basement membranes resulting in immune complexes, vasculitis, and multiorgan disease. While this murine model of lupus has been used for the identification of therapeutics with potential efficacy in human autoimmune disease, the long-term impact of chronic immunosuppressive therapy on macrophage function in this paradigm is not understood. To this end, MRL/lpr mice were treated prophylactically with dexamethasone at 0.01, 0.1, and 1 mg/kg of body weight for 20 weeks or were allowed to develop autoimmune disease and, at 15 weeks of age, treated therapeutically with 1-mg/kg dexamethasone for 8 additional weeks. Analysis of surface antigens on resident peritoneal macrophages demonstrated a progressive loss in class I expression with a concomitant increase in Fc receptor expression. Neither phagocytosis nor CD11b expression was modulated with chronic steroid treatment. Furthermore, dexamethasone treatment was associated with a reduction in anti-DNA antibodies and total immunoglobulin G and yet an elevation in serum cholesterol due to an increase in high-density lipoproteins. Therefore, the MRL/lpr mouse serves not only as a small-animal model of autoimmune disease but also as one in which the negative and positive sequelae associated with chronic immunosuppression can be further understood.

Effects of retinoic acid on a new human erythro-megakaryocytic cell line AP-217

We have characterized a new human cell line AP-217, derived from the peripheral blood of a patient with chronic myeloid leukemia in blastic crisis.The analysis of cell surface antigens and ploidy showed that AP-217 was an erythro-megakaryocytic cell line.The effects of inducers of differentiation were studied and focused on retinoic acid (RA).Uninduced AP-217 cells produced a low level of hemoglobin (Hb) that showed a moderate but significant dose-dependent increase after 13 cis-RA induction (four times above the control at 10 −5M).To outline this effect, AP-217 cells were cloned at limiting dilution.A subclone (clone 2) was isolated which expressed glycophorin A on 12% of cells, and showed a marked sensitivity to RA.After a 4 day induction with increasing concentrations of RA (1−10 × 10 −6M) Hb production by clone 2 cells was enhanced 12 times over the control at the highest concentration (10 −5 M). No effect of RA on the Hb production of K-562 and HEL was observed. This increased Hb production occurred simultaneously with a growth inhibition in clonogenic cultures (20% reduction) associated with a drastic reduction of the colony size.Moreover, we demonstrated the expression of mRNA for the β globin gene in clone 2 and AP-217-cells.This is the first report of a positive effect of RA on the erythroid differentiation of a human leukemic cell line.

Flow cytometric analysis of cell surface antigen recognized by monoclonal antibody (MSN-1) in normal, hyperplasia, and carcinoma of endometrial cells: Its diagnostic value for endometrial carcinoma

A monoclonal antibody, MSN-1, was used for flow cytometric analysis of cells from normal endometrium, endometrial hyperplasia, and endometrial carcinoma. The 90th percentile of the specimen control was used as a threshold. Reactivity was defined by the percentage of stained cell about the thresholds, adjusted for the expected percentage. A sample was considered positive if the reactivity exceeded 10%. The positivity rate for normal endometrium, endometrial hyperplasia, and endometrial carcinoma specimens was 9.2%, 19.2%, and 84.6%, respectively. The mean (+/-SD) reactivity rate of MSN-1 for normal endometrium, endometrial hyperplasia, and endometrial carcinoma was 3.3 +/- 6.2%, 7.4 +/- 13.8%, and 34.4 +/- 24.2%, respectively. There was a significant difference of the reactivity rates between normal endometrium and endometrial hyperplasia, and between endometrial hyperplasia and endometrial carcinoma (P < 0.01). In the subgroup of endometrial carcinoma patients with confined muscular invasion, the reactivity and the positivity rates were also analyzed. There was no relationship between the depth of muscular invasion and the reactivity rate or the positivity rate. In endometrial carcinoma patients who simultaneously underwent endometrial cytology, the relationship of the results of cytology and the positivity rate was also analyzed. When the results of cytology and flow-cytometric analysis were combined, the positivity rate of endometrial carcinoma was 100% (30/30). In conclusion, flow-cytometric analysis of endometrial cells employing MSN-1 could be a useful supplementary diagnostic method for endometrial carcinoma.

Flow cytometric analysis of cell surface antigen recognized by monoclonal antibody (MSN‐1) in normal, hyperplasia, and carcinoma of endometrial cells: Its diagnostic value for endometrial carcinoma

Flow cytometric analysis of cell surface antigen recognized by monoclonal antibody (MSN‐1) in normal, hyperplasia, and carcinoma of endometrial cells: Its diagnostic value for endometrial carcinoma

胸腺腫肺転移巣内リンパ球表面抗原の解析 胸腺腫内Ｔリンパ球分化の可能性

胸腺腫肺転移巣内リンパ球表面抗原の解析 胸腺腫内Ｔリンパ球分化の可能性

Flow cytometric analysis of surface antigens on human conjunctival epithelial cells.

We analyzed the levels of expression of different surface adhesion molecules on normal human conjunctival epithelial cells with cytology and flow cytometry. The levels of beta 1 (CD29), VLA-1 (CD49a), VLA-2 (CD49b), and VLA-3 (CD49c) integrins were high, whereas those of VLA-5 (CD49e) and VLA-6 (CD49f) were faint, and that of VLA-4(CD49d) was undetectable. Among the beta 2 integrins, the levels of Mac-1 (CD11b) and p150.25(CD11c) were positive, while LFA-1 (CD11a) was not detectable. HLA class I and class II were also expressed. However, CR2 (CD21), CD57, CD44 and ICAM-1 (CD54) were not detected. Our study suggests that these techniques may aid in identifying changes in the expression levels of various surface antigens on conjunctival epithelial cells that could occur in ocular surface disease.

Development and Characterization of v-myc/v-raf-Transformed Murine Fetal Thymocyte Cell Lines

Transformed murine fetal thymocyte cell lines were derived by incubating fetal thymic organ cultures with a v-myc/v-raf-containing retroviral construct in order to model developmental stages within the early triple negative (CD3−CD4−CD8−) thymocyte population. The resulting 10 cell lines had a lymphoid morphology, were all CD44+, CD90+, and were triple negative by surface antigen analysis. The cell lines, however, were distinguishable by differences in the expression of T cell-associated and T cell-specific genes. The CD3 genes were observed to be discoordinately expressed, in that CD3γ chain gene expression was detected in 2 cell lines in the absence of CD3δ and ϵ expression. Expression of the CD3γ chain gene was observed in cell lines without the expression of other T cell-specific genes or T cell receptor rearrangement and may be one of the earliest T cell-specific genes to be expressed. The transcription factor Ikaros was expressed in all 10 cell lines, whereas the transcription factor TCF1α was expressed only in the 2 most differentiated lines. In 8 cell lines, expression of partial TCR β and/or TCRα transcripts was observed by Northern blot. In several lines, expression of rearranged TCRα transcripts in the absence of TCRβ transcripts was demonstrated; however, TCRβ DJ rearrangements were observed by Southern blot in all but 1 of these cell lines. Thus, these cell lines, ordered based on the general pattern of additive gene expression observed, may reflect various stages of triple-negative thymocyte differentiation and provide anin vitromechanism to elucidate some of the molecular events involved in early thymocyte development.

Comparative analysis of cell surface antigens expressed by cell lines derived from human germ cell tumours

The pattern of cell surface antigen expression of a set of cell lines derived from human germ cell tumours and corresponding to various cell phenotypes found within these tumours was studied using immunofluorescence. Twenty-two different antibodies were used. Many of these antibodies have been noted to recognise epitopes that are either preferentially expressed by embryonal carcinoma (EC) cells, or by more differentiated cell types. Using scatter plots and rank correlations, 6 groups of antibodies were distinguished with respect to their staining patterns on the cell lines tested. Several antibodies showed a specific staining pattern in relation to the differentiation state of the cells. Two groups of antibodies included those recognising high m.w. glycoproteins (antibodies TRA-1-60, TRA-1-81, GCTM2, 3-177, K4 and K21) and the ganglioseries glycolipid antigens SSEA-3 and -4 (antibodies MC631 and MC813-70). These antibodies mostly stained EC cells but not other cell types, confirming previously published data. However, one of these groups, comprising antibodies K4 and MC631, was more exclusively associated with the EC cell phenotype than was the other group. Antibodies recognising the liver isozyme of alkaline phosphatase (TRA-2-49 and TRA-2-54) also reacted strongly with most EC cell lines, although they reacted significantly with a number of other cell lines as well, whereas antibodies to the placental isozyme tended to react only weakly with EC cells. The antibodies recognising the ganglioseries glycolipids GD2 and GD3 (VIN2PB22 and VINIS56) preferentially stained cells with neuroectodermal characteristics. Other antibodies showed a heterogeneous staining pattern for the cell lines with different phenotypes. The data obtained from the cell lines were, in general, similar to data obtained from immunohistochemical studies on tissue sections of primary germ cell tumours of the adult testis, including carcinoma in situ.

MORPHOSPECIES VS. GENOSPECIES IN TOXIC MARINE DINOFLAGELLATES: AN ANALYSIS OF GYMNODZNIUM CATENATUM/GYRODINIUM IMPUDICUM AND ALEXANDRIUM MINUTUM/A. LUSITANICUM USING ANTIBODIES, LECTINS, AND GENE SEQUENCES1

ABSTRACT Morphological features are the predominant criteria used to define species of marine dinoflagellates. Taxonomic problems with some toxic groups has led to the implementation of molecular taxonomy techniques and development of a genospecies concept. As a result, the relationships between “morphospecies” and “genospecies” has been questioned. In this study, the genetic differentiation between two sets of closely related morphospecies, Gymnodinium catenatum Graham/Gyrodinium impudicum Fraga and Alexandrium minutum Halim/Alexandrium lusitanicum Balech, were analyzed. The extent of morphological differentiation existing within these two groups is of the same order of magnitude. Analysis of cell surface antigens detected by preadsorbed serum, cell surface glycan moieties detected by lectins and sequencing of the D9 and D10 domains of the Large‐subunit ribosomal RNA gene, showed that the extent of genetic differentiation existing between the dinofagellates Gymnodinium catenatum/Gyrodinium impudicum is substantial. Therefore, both morphological and genetic criteria resolve these organisms as two distinct entities. In contrast, Alexandrium minutum/Alexandrium lusitanicum were indistinguishable using the some suite of molecular markers. The findings demonstrated that classifications based on morphological criteria may be incongruous. On a practical level, molecular taxonomy provides useful tools to distinguish between morphologically similar microalgal species and furthermore can prevent misidentification of species such as Gymnodinium catenatum/Gyrodinium impudicum, a frequent occurrence when samples are fixed with Lugol's or formaldehyde solution.

Surface antigen analysis of group B Neisseria meningitidis outer membrane by monoclonal antibodies: Identification of bactericidal antibodies to class 5 protein

Twenty-four monoclonal antibodies (mAbs) against group B Neisseria meningitidis surface antigens were analyzed by immunoenzymatic assays and by a bactericidal test. Two mAbs were specific to polysaccharide B and one to lipopolysaccharide. The others were specific to polysaccharide B and one to lipopolysaccharide. The others were directed against outer membrane proteins ranging in molecular mass from 25 to 200 kDa. The outer membrane protein epitopes recognized by the mAbs were not conformational and were located on the outer surface of the microorganism. Linear epitopes on the class 5 protein, exposed on the surface of the membrane, were able to induce bactericidal antibodies to the homologous strain. The susceptibility of Neisseria meningitidis to these antibodies was unchanged when this organism was cultivated under conditions of iron depletion. These results demonstrate that peptides derived from class 5 proteins are potentially important in synthetic peptide or in recombinant protein vaccines containing linear bactericidal epitopes.

Intestinal cellular immunity after primary rotavirus infection

Infection of neonatal gnotobiotic lambs with a bovine strain of rotavirus was used to characterize the kinetics of the primary cellular intestinal immune response to this agent. At 2-3 days after infection virus was first detected in the faeces and increased numbers of CD45R+ cells were observed in peripheral blood. These cells persisted in significantly increased numbers in the circulation until 7-8 days after infection. At this time, virus was no longer detectable in the faeces. The increase in CD45R+ cells preceded the appearance of virus-neutralizing antibodies in the serum at 1 week after infection. Maximal antibody titres were reached 2 weeks after infection. Virus-primed cells were first observed 1 week after infection in the jejunal and ileal Peyer's patches, mesenteric lymph nodes and peripheral blood, and persisted in the mesenteric lymph nodes and jejunal Peyer's patches for a further 4 weeks. Analysis of lymphocyte surface antigens indicated that different sub-populations of lymphocytes were responding in the various lymphoid tissues; a majority of CD4+ cells was observed in the mesenteric lymph nodes, whereas B cells predominated in the ileal Peyer's patches.

Intracellular and surface distribution of CD9 in human eosinophils.

Expression of CD9 is a feature of both eosinophils and platelets. We have investigated the CD9 expression on resting and activated eosinophils with regard to possibly interacting platelets. Mixed leukocytes were obtained from the platelet-containing (PC) and platelet-depleted (PD) peripheral blood of healthy donors. A cell membrane permeabilization technique, the FOG method, enabled us to detect the eosinophils as a separate population and permitted flow cytometric analysis of both surface and intracellular antigens. Monoclonal antibodies against CD61 were used to identify platelets. The CD9/CD61 ratio indicated that CD9 on resting eosinophils originates mainly from eosinophils and not from adhered platelets. No difference in CD9 expression was obtained between resting eosinophils in PC and PD blood. However, the expression of CD9 was decreased (p < 0.05) on eosinophils in PMA-activated PD blood but increased (p = 0.001) in PC blood, probably due to interacting platelets since CD61 increased simultaneously. In addition, we were able to detect an intracellularly stored pool of CD9 in eosinophils which decreased after activation with PMA. Together these results indicate a translocation of intracellularly stored CD9 to the cell membrane upon activation, probably followed by a subsequent shedding.

A simple assay for examining the effect of transiently expressed genes on programmed cell death

Programmed cell death (PCD) has been observed in a wide variety of cell types in response to physiologic signals or types of stress. How these stimuli trigger PCD, and whether there is a common PCD signal transduction pathway, is not clear. As more genes are described that may participate in or regulate PCD, an assay system in which gene products can easily be introduced and/or modulated would be of great value. To avoid the generation and screening of multiple individual stable cell transfectants, a simple transient transfection death assay has been developed. 2B4.11, a murine T cell hybridoma, was transfected by electroporation with a constitutively active β-galactosidase reporter gene and the cells were incubated in culture medium or with a PCD-inducing stimulus. The amount of β-galactosidase activity remaining in the intact cells at the end of the culture period represented only viable transfected cells. Bcl-2 was chosen to examine whether this system would be useful to study the effect of transiently transfected genes since it blocks PCD in a number of experimental systems. Consistent with data obtained using stable transfectants, transient expression of Bcl-2 in 2B4.11 completely protected cells from glucocorticoid- and cytotoxic agent-induced PCD. This protection from death was confirmed at the individual cell level by the transient co-expression of a class I L d surface antigen and flow cytometric analysis. Some of the advantages of the transient transfection death assay described here are; (1) the simple and sensitive β-galactosidase assay, (2) the rapidity of the assay, (3) the ability to perform conventional viability assays to monitor treatment-induced cytotoxicity, (4) multiple gene products can be tested alone, and in combination, (5) antisense or dominant negative approaches can be used, and (6) the adaptability of this assay system to other cell types, transfection techniques, or reporter and expression vectors. The transient transfection death assay should make it easier to identify and order important steps in the PCD signal transduction pathways.

Analysis of Cell Surface Antigens Using Anti-rat Hepatocyte Monoclonal Antibodies, Particularly HAM 1

The expression of antigens on rat hepatocytes was examined by immunoelectron microscopy using monoclonal antibody HAM 1. The antigen recognized by HAM 1 was expressed mainly on both sinusoidal and bile-canalicular faces and only sparsely on the contiguous face. All rat hepatoma cell lines examined were intensely labeled with HAM 1, but poorly labeled with HAM 3, HAM 4, and HAM 5, as revealed by flow cytofluorometry and radioimmunoassay. Expression levels of HAM 1 antigen on the AH 44 hepatoma cell line similar to the degree seen in normal liver were demonstrated by radioimmunoassay. HAM 1 antigen was also expressed weakly on lymphocytes, thymocytes, and some bone marrow cells, but not on red blood cells, and differed from the MHC class I antigen recognized by HAM 2 and OX 18. Significant amounts of cell surface antigens recognized by all the monoclonal antibodies (HAM 1-HAM 5) were confirmed by radioimmunoassay on the cell surface of primary cultured hepatocytes. These results suggest that the rat hepatoma cell lines employed are different from normal hepatocytes, that primary cultured hepatocytes are more similar to normal hepatocytes in the degree of expression of their cell surface antigens, and that HAM 1 antigen appears to be a significant antigen on both normal and transformed hepatocytes.

Analysis of cytoplasmic and surface antigens in childhood T-cell acute lymphoblastic leukaemias: clinical relevance of cytoplasmic TCR beta chain expression.

Expression of surface and cytoplasmic antigens on the blasts from 42 cases of childhood T-cell acute lymphoblastic leukaemia (T-ALL) were analysed. All with childhood T-ALL, except for one case expressing cytoplasmic TCR delta chain, were classified on the basis of differential expression of cytoplasmic CD3 (cCD3), TCR beta chain (cTCR beta) and surface CD3 (sCD3) into the following three groups: group I (cCD3+, cTCR beta-, sCD3-), eight cases (19.5%); group II (cCD3+, cTCR beta +, sCD3-), 23 cases (56.1%); group III (cCD3+, cTCR beta +/-, sCD3+), 10 cases (24.4%). Each group defines the stepwise maturational stage of the CD3/TCR complex along the intrathymic T-cell differentiation. Group I had the lowest initial WBC count among the three groups (P < 0.05) and showed significantly (P < 0.05) a higher event-free survival (0.75) than those of group II (0.33). There was no significant difference in both the initial WBC count and the event-free survival between groups II and III. Thus, the absence of cTCR beta in sCD3-negative T-ALL appears to be a good prognostic factor, suggesting that this classification provides a useful tool to predict the prognosis of childhood T-ALL. This is the first report, to our knowledge, studying the relationship between the expression of cytoplasmic CD3/TCR antigens and the clinical features in T-ALL.

Characterization of human hepatocyte lines derived from normal liver tissue

Four separate continuous lines of human hepatocytes (HH01, HH02, HH09, HH25) were developed from normal liver tissue by subjecting cocultures of human hepatocytes with rat liver epithelial cells in a highly enriched medium to frequent subculturing. The addition of conditioned medium from either the human hepatoma line Hep G2 or one of these stable human hepatocyte lines (HH09) appeared to facilitate establishment of line HH25. These human hepatocyte lines have been in continuous culture for 2 to 5 yr and consist of approximately 95% human cells by analysis of cell surface antigens. Cytogenetic analysis also confirmed the human origin of these cells and showed clonal origin with abnormal ploidy. Cells in these human hepatocyte lines retain morphological features of hepatocytes by both light and electron microscopy. They also retain glucose-6-phosphatase activity and secrete proteins characteristic of hepatocytes, such as albumin, alpha-fetoprotein and transferrin. After incubation with 13 mumol/L dibenz(a,h) anthracene for 24 hr, each line had detectable activity of aryl hydrocarbon hydroxylase, ethoxyresorufin O-deethylase and methoxyresorufin O-demethylase. Thus, these human hepatocyte lines retain important differentiated characteristics of hepatocytes. Derived from normal liver tissue, they appear to be immortalized. They provide a new model system for studying human hepatocellular drug metabolism. These lines may also be useful for studying the regulation of synthesis of albumin, alpha-fetoprotein and other proteins in human hepatocytes, determining the effects of cytokines and growth factors and designing systems to effect gene transfer into human hepatocytes for the purpose of gene therapy.

Site-directed mutagenesis of cysteine residues of hepatitis B surface antigen. Analysis of two single mutants and the double mutant.

The structure of hepatitis B surface antigen (HBsAg) is mainly maintained by an intricate disulfide network responsible for most of its structural and antigenic properties. Characterization of three cysteine-replacement mutants of HBsAg has been performed by both structural and immunological methods. Replacement of Cys121 or Cys124 with serine results in mutant proteins that show diminished binding titres to both monoclonal antibodies and to a polyclonal serum, indicating that a structural change has taken place. Circular dichroism analysis shows that the substitution of either of these two residues also diminishes the helical content of the protein. However, the double mutant, in which both cysteine residues have been simultaneously changed, reverts the properties of the single mutations, and shows similar behaviour to the wild-type protein. Both the single and double cysteine mutants are efficiently glycosylated and secreted from Chinese hamster ovary cells and, in all cases, the mutant proteins assemble into spherical particles of similar buoyant density to both the wild-type and serum derived HBsAg.