The subunit structure of non-specific cross-reacting antigen (NCA)

NCA was purified from normal human lung (L-NCA) and from liver metastases of colon adenocarcinoma (T-NCA), L-NCA and T-NCA had different carbohydrate compositions, but showed an immunological identity in double immunodiffusion with the use of anti-CEA and anti-L-NCA sera. In SDS-polyacrylamide gel electrophoresis L-NCA and T-NCA showed similar molecular weights of about 110,000 and 120,000, respectively. However, if the samples were heated for 3 min at 100 degrees C in the presence of 1% SDS before electrophoresis, their apparent mol. wt decreased to about 50,000, suggesting the dissociation of NCA molecules into subunits. The dissociation of NCA was independent of the presence of mercaptoethanol and did not destroy the ability of NCA to precipitate with anti-CEA and anti-NCA sera. Besides NCA (and CEA in tumor tissue), the presence of a lower molecular weight cross-reacting antigen was observed in lung and tumour. Double immunodiffusion showed that this additional antigen was not a dissociated form of NCA.     

Isolation and characterization of rat carcinoembryonic antigen

A rat tumor-associated antigen with properties similar to those of human carcinoembryonic antigen (CEA) has been detected with rabbit immune sera in extracts of transplantable rat colonic adenocarcinoma, RCA-1. This antigen, termed rat CEA, was also detectable by a monkey antihuman CEA serum and a rat monoclonal antibody to rat CEA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of perchloric acid extracts of RCA-1 tumor, followed by immunoblotting with the above-mentioned anti-CEA reagents, revealed that rat CEA activity resided in components with a molecular weight of approximately 350 kD. The glycoprotein nature of these components was indicated by positive staining with periodic acid-Schiff. Sephadex G-200 chromatography, as well as Sepharose 4B chromatography with and without sodium dodecyl sulfate indicated that the 350-kD components existed in the extracts as molecular aggregates. The 350-kD material, which had been purified by an affinity column containing rat monoclonal antibodies to rat CEA, reacted with the rabbit and monkey anti-CEA sera. This provided strong evidence that serological activity of rat CEA was confined to the 350-kD components.     

CEA and NCA in benign and malignant breast tumors

The involvement of Nonspecific Crossreacting Antigen (NCA) in the immunohistological demonstration of Carcino Embryonic Antigen (CEA) in 56 benign and 92 malignant lesions of the breast was analyzed. For this purpose, the authors utilized both polyclonal antisera and monoclonal antibodies. Polyclonal anti-CEA sera were used after absorption with normal tissue antigens, in order to remove crossreactivity, and without such an absorption. Ninety-three percent of breast carcinomas, 85% of mastopathic lesions not associated with a carcinoma, and 66% of fibroadenomas showed positive reactions with commercial unabsorbed polyclonal anti-CEA serum, which contained antibodies to NCA, whereas incubation with monospecific anti-CEA antiserum resulted in 42% positivity in carcinomas and negativity in mastopathic lesions and fibroadenomas. Forty-eight percent of breast cancer, 84% of mastopathic lesions, and 50% of the fibroadenomas contained NCA in different quantities. The staining pattern of carcinomas and fibroadenomas obtained with unabsorbed anti-CEA antibody and anti-NCA did not run parallel in all cases. Monoclonal antibodies against CEA and NCA confirmed the results obtained with polyclonal antiserum. This study suggests a cancer specificity of CEA in breast lesions.     

Antibody response to different determinants on carcino-embryonic antigen (CEA)

Studies on the diverse nature of the immune response of animals to different antigenic determinants on CEA indicate that the type of antiserum used is of prime importance in determining differences or identities between antigens isolated from tumour and normal tissues. One type of antiserum against CEA shows cross-reactions with a normal colon antigen (NCA) found in both malignant and normal tissues. This finding confirmed that a common determinant is shared by both CEA and NCA. A second type of anti-CEA did not cross react with NCA but reacted only with CEA, providing evidence for a unique CEA determinant. Yet a third and most common type of antiserum to CEA reacted equally well with CEA and NCA and was unable to distinguish between the two. This third type of anti-CEA was completely absorbed with NCA. Failure to recognize this type of anti-CEA would lead to erroneous conclusions regarding the existence of a unique antigenic determinant on CEA.     

Cervical carcinoma antigen, carcinoembryonic antigen (CEA), and nonspecific cross-reacting antigen (NCA) in appraisal of uterine cervix smears

The distribution of cervical carcinoma antigens (AgCaCx), CEA, and NCA in different pathologic states of the uterine cervix was studied in cytologic smears by an immunofluorescence method (IF) using specific immune sera against perchloric acid (PCA) extract of cervical squamous cell carcinoma, anti-CEA, and anti-NCA. After excluding cross-reactivity with CEA and NCA, the presence of AgCaCx was demonstrated in the majority of cervical carcinomas, severe dysplasias, and only in one-fourth of squamous metaplasias, especially when accompanied by mild or moderate dysplasias. The intensity and percentage of IF-positive cells varied from case to case. The preparations of uterine cervix without pathologic changes usually were negative. Similar results were obtained with anti-CEA serum. NCA was present in the majority of smears independent of histologic diagnosis. The most intense fluorescence was observed in upper layers of the epithelium. NCA could be a differentiation antigen of stratified squamous cell epithelium.     

Isolation and Characterization of Porcine Mannan-Binding Proteins of Different Size and Ultrastructure

The authors report on the purification and characterization of mannan-binding proteins (MBP) isolated from porcine serum. The MBPs were purified by use of PEG precipitation, affinity chromatography on mannan-Sepharose, protein A- and anti-porcine IgM-Sepharose followed by gel filtration. The MBP proteins were collagenase sensitive and showed γ₁-γ₂-electrophoretic mobility. The MBP designated pMBP-28 had a molecular mass of 28 kDa when analysed on SDS-PAGE under reducing conditions and eluted corresponding to a molecular mass of approximately 700 kDa on gel filtration chromatography. Electron micrographs of pMBP-28 revealed an oligomeric protein similar to rodent MBP-A and human MBP but with a predominance of penta- and hexameric molecules. Another protein designated pMBP-27 was composed of peptides of 27 kDa and had an M_r of 300–350 kDa on gel filtration chromatography. Electron microscopy of pMBP-27 showed dimer and trimer molecules; the trimers without distinct stalk regions. The N-terminal 26 (pMBP-27) and 24 (MBP-28) amino acid residues showed 54% and 58% identity with human MBP. pMBP-28 showed a higher degree of sequence similarity to rat and mouse MBP-A (60% identity) than to mouse and rat MBP-C (41–45% identity). Both pMBPs exhibited Ca²⁺-dependent binding to D-mannose immobilized on agarose but no significant binding to N-acetyl-D-glucosamine- or fucose-agarose. The results further suggested the presence of a third pMBP which copurified with pMBP-27 but this protein was not sequenced.     

An Immunoglobulin M (IgM) Antibody to Carcinoembryonic Antigen (CEA)-like Antigen in Human Pancreatic Cancer

An immunoglobulin M (IgM) antibody to a carcinoembryonic antigen (CEA)-like antigen was isolated from the ascites fluid of a patient with pancreatic cancer by ammonium sulfate precipitation (25–55% saturation) and subsequent purification by gel filtrations on Sephadex G-200 and Sepharose 6B followed by protein A-Sepharose CL-4B chromatography. The IgM antibody was found to be in complex with a CEA-like antigen as revealed by the multiple precipitin lines obtained on immunoelectrophoresis and double diffusion with a mixture of antihuman IgM and anti-CEA. Dissociation of the complex with 0.2M glycine HCl buffer pH 2.5 and further chromatography on Sepharose CL-6B. followed by Sepharose 6B separated the IgM antibody from the CEA-like antigen. The IgM antibody formed an immunoprecipitate upon double diffusion with a homologous CEA cross-reactive antigen isolated from the liver metastasis of another patient with pancreatic cancer. The antigen (molecular weight 70,000 ± 15,000 daltons) which reacted with the IgM antibody was purified by Con A Sepharose affinity chromatography followed by gel filtration on BioGel A 1.5M. The IgM antibody reacted with tumor antigens from pancreatic cancer only. These results suggest that the human IgM antibody response to antigens may be specific to the tumor type involved.     

Purification and characterization of Par o I, major allergen of Parietaria officinalis pollen.

Par o I, a major allergen of Parietaria officinalis, was purified from the pollen extract. The purified allergen was obtained by ultrafiltration, Sephadex gel filtration and DE-52 ion exchange chromatography: the purified preparation yields a single band in polyacrylamide gel isoelectric focusing (PAG-IEF), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, a single immunoprecipitation arc in crossed immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis (CRIE) and a single peak in size exclusion high-performance liquid chromatography (HPLC). Par o I is a glycoprotein with a protein to carbohydrate ratio of 100:21. The molecular weight, determined by SDS-PAGE, Sephadex G-50 gel filtration and size exclusion HPLC, varied between 13.5 and 14.5 kDa according to the method employed. The isoelectric point was 4.6. The amino acid composition and the sequence of the first twelve N-terminal residues were determined. The allergenicity was assayed in vivo and in vitro. 29/29 Parietaria-allergic patients were skin positive to Par o I and possessed high level of specific serum IgE antibody as it determined by radioallergosorbent test (RAST). Par o I contained dominant epitopes for human IgE as inhibited to 85% the pollen extract RAST performed with a pool of sera of allergic patients. The RAST inhibitory activity was not abolished by deglycosylation.     

Gel filtration of carcinoembryonic antigen (CEA) and non-specific crossreacting antigen (NCA) on the Superose 12HR column: effect of guanidine hydrochloride

CEA and NCA, which behave as dimeric molecules in SDS-polyacrylamide gel electrophoresis, were submitted to gel filtration on the Superose 12HR column in phosphate-buffered saline (PBS) pH 7.0 or in 6 M guanidine hydrochloride. The elution volumes of these antigens were compared with those of protein markers. The reference proteins showed almost linear relation between log Mr and elution volume, but they were eluted distinctly faster in guanidine that in PBS. The elution volumes of NCA corresponded to Mr approx. 110,000 in PBS and 50,000 in guanidine. These values were accordant with those obtained by other methods and suggested that guanidine dissociates NCA into subunits. The apparent molecular weights of CEA (migrating as 180 kDa molecule in SDS-electrophoresis) estimated by gel filtration on Superose were approx. 700,000 in PBS and 160,000 in guanidine. These results also suggested the dissociation of CEA by guanidine, but due to anomalous elution of CEA from the Superose column they are less convincing than in the case of NCA.     

Antigenic Differences of CEA in Colonic Cancer, Gastric Cancer and Fetal Tissues

CEA samples were prepared individually from liver metastases of gastric and colonic cancer, and purified with gel filtrations, block electrophoresis and Con A affinity chromatography (the latter was not performed in all cases). Anti-CEA antisera were produced in 7 and 4 guinea pigs against CEA of gastric cancer and colonic cancer respectively. Upon extensive absorptions with PCA extracts of normal tissues (rich of NCA) and NCA-2, their anti-CEA activities were examined on micro-Ouchterlony plates and by immunofluorescence techniques.
There were two types of anti-CEA antibodies, as observed in our previous study [9], in which crude samples of CEA were utilized as immunogens: a type that showed identical reaction to CEA of adenocarcinomas of the digestive system, squamous cell carcinomas of endodermally derived organs, medullary carcinoma of the thyroid as well as tissues of fetal gastro-intestinal tract but not to others, while the other type was proven to be specific to gastric or colonic cancer depending on cancer tissues from which the immunized CEA was extracted. In immunofluorescence study, no reaction of these organ-specific anti-CEA antibodies to fetal tissues was observed.
Further, CEA-M (Con A binding fraction) and CEA-P (Con A non-binding fraction) separated by Con A affinity chromatography, showed identical reaction to any types of anti-CEA antibodies, though CEA-P was apparently less antigenic.     

Antigenic phenotype of malignant mesotheliomas and pulmonary adenocarcinomas. An immunohistologic analysis demonstrating the value of Leu M1 antigen.

To evaluate the usefulness of an immunohistologic approach to the differential diagnosis of mesothelioma and pulmonary adenocarcinoma, the authors studied paraffin-embedded, fixed tissue sections from 50 primary adenocarcinomas of the lung and 28 mesotheliomas of the pleura by using a panel of monoclonal antikeratin, antihuman milk fat globule (HMFG-2), anti-Leu M1, and monoclonal anticarcinoembryonic antigen (CEA) antibody; we also used a conventional heterologous anti-CEA antiserum with and without prior absorption with spleen powder to remove antibodies to nonspecific cross-reacting antigen (NCA). Keratin was present in both mesotheliomas and adenocarcinomas and did not help in distinguishing between these two neoplasms. HMFG-2 was detected in 48 (96%), and Leu M1 was positive in 47 (94%) of the adenocarcinomas, but not in any of the mesotheliomas. By using conventional rabbit antiserum, the authors detected CEA in the majority of adenocarcinomas (96%), but also in two cases of mesothelioma. When the anti-CEA antiserum was absorbed with NCA, the number of positively reacting adenocarcinomas decreased considerably to 76%; however, after this treatment, none of the mesotheliomas gave positive reactions. The monoclonal anti-CEA antibody was reactive in 36 of the adenocarcinomas (72%), but in none of the mesotheliomas. Our results indicate that, in addition to HMFG-2 and CEA, the expression of Leu M1 antigen by most primary pulmonary adenocarcinoma (94%) and its absence in mesothelioma could be used as a valuable marker for primary adenocarcinoma of the lung that involves the pleura and permits its differentiation from mesothelioma.     

Carcinoembryonic (CEA)--like antigen in adenomas and cancers of the breast in human patients.

By using specific anti-CEA serum and indirect immunofluorescence, a substance was detected in breast cancers which reacted similarly to CEA demonstrable by the same method in cancers of gastrointestinal tract. However, the double diffusion test in agar gel and additional absorption analysis showed that it was a glycoprotein different from CEA, although possessing common or similar antigenic determinants with it. This antigen also differs from another antigen called "nonspecific cross-reacting antigen NCA" and from alpha 1-acid glycoprotein of human serum.     

Blood markers of early and late airway responses to allergen in asthmatic subjects. Relationship with functional findings

We evaluated the relationship between blood markers of mast-cell (plasma histamine and serum level of heat-stable neutrophil chemotactic activity [NCA]) and eosinophil (serum eosinophil cationic protein [ECP]) activation during early airway response (EAR) and late airway response (LAR) to allergen inhalation in 24 asthmatic subjects. After EAR, 14 subjects showed significant LAR (FEV₁ fall: 25%), while 10 subjects showed equivocal LAR (FEV₁ fall: 15–20%). A significant increase from baseline value was observed in plasma histamine and in serum NCA during both EAR and LAR, while serum ECP significantly increased only during LAR. The sensitivity of different markers to detect significant FEV₁ fall during EAR and LAR was low, except for NCA. Changes in blood mediators were similar in both groups with significant and equivocal LAR. There was a significant relationship between the increase in NCA during EAR and the severity of LAR. Stepwise regression between changes in different blood markers showed a significant relationship between histamine increase during EAR and ECP increase during LAR. Thus, serum NCA is a more sensitive marker of EAR and LAR than plasma histamine and serum ECP, and its increase during EAR seems predictive of the severity of the subsequent LAR.     

Carcinoembryonic antigen like antigen in granular cell myoblastomas

Summary A series of granular cell myoblastomas (GCM) and other benign and malignant tumours of soft tissue were examined for cytoplasmic content of carcinoembryonic antigen (CEA) by the two-layer conjugated immunoperoxidase technique. Using a commercial rabbit anti-CEA serum only granular cell myoblastomas showed positive cytoplasmic reaction. Pretreatment with periodic acid made this reaction less intense, but when the commercial rabbit anti-CEA serum was absorbed with tissue powder from normal human spleen the positive reaction was totally abolished. It is concluded that the positivity of GCM for CEA using commercial rabbit anti-CEA serum is due to the content of non-specific cross-reacting antigen (NCA) and maybe other cross-reacting glycoproteins in this tumour, and not to CEA as claimed in a previous study.     

Biochemical and Allergenic Properties of the House Dust Mite Extract, Dermatophagoides pteronyssinus

A Further study has been made on the house dust mite extract, Dermatophagoides pteronyssinus , with emphasis on gel-filtrated fraction 2 (F₂). The crude mite extract showed at least nine discs on polyacrylamide disc electrophoresis and contained less than 0.25 % sialic acid and less than 0.5 mM hexosamine and no detectable uronic acid. From gel filtration of the crude extract a No. 2 fraction (F₂) with allergenic activity showed at least five components on SDS disc electrophoresis covering a molecular weight range of between 15,000 and 70,000. The major allergenic activity of F₂ dissolved in pH 7–8 and 4–5 on an isoelectric focusing column. Affinity chromatography of lectins showed that allergenic activity did not relate to structures of N -acetyl-D-glucosamine or N -acetyl-D-galactosamine. Allergenic activity of the crude extract was not affected by peptic digestion and the mite digest prepared by trypsin and pronase showed a similar fraction-action and activity profile as crude extract.     

Extra activation component of calcium release in frog muscle fibres

In addition to activating more Ca²⁺ release sites via voltage sensors in the t-tubular membranes, it has been proposed that more depolarised voltages enhance activation of Ca²⁺ release channels via a voltage-dependent increase in Ca-induced Ca²⁺ release (CICR). To test this, release permeability signals in response to voltage-clamp pulses to two voltages, –60 and –45 mV, were compared when Δ[Ca²⁺] was decreased in two kinds of experiments. (1) Addition of 8 m m of the fast Ca²⁺ buffer BAPTA to the internal solution decreased release permeability at –45 mV by > 2-fold and did not significantly affect Ca²⁺ release at –60 mV. Although some of this decrease may have been due to a decrease in voltage activation at –45 mV – as assessed from measurements of intramembranous charge movement – the results do tend to support a Ca-dependent enhancement with greater depolarisations. (2) Decreasing SR (sarcoplasmic reticulum) Ca content ([Ca_SR]) should decrease the Ca²⁺ flux through an open channel and thereby Δ[Ca²⁺]. Decreasing [Ca_SR] from > 1000 μ m (the physiological range) to < 200 μ m decreased release permeability at –45 mV relative to that at –60 mV by > 6-fold, an effect shown to be reversible and not attributable to a decrease in voltage activation at –45 mV. These results indicate a Ca-dependent triggering of Ca²⁺ release at more depolarised voltages in addition to that expected by voltage control alone. The enhanced release probably involves CICR and appears to involve another positive feedback mechanism in which Ca²⁺ release speeds up the activation of voltage sensors.     

Solubilization of Species-Specific Antigens and Murine Alloantigens by Pulsed High Frequency Sonic Energy

Membrane-associated species-specific antigens (monkey) and alloantigens (mouse) were solubilized by pulsed high frequency sonic energy. 15 to 25% of the total antigenicity of the donor cells was recovered. The product was highly water-soluble, essentially protein, and stable during extensive ultrafiltration or storage for one year. Gel filtration and electron microscopic analyses of soluble species-specific antigen suggested molecular heterogeneity with a major active component in the 50,000 molecular weight region. Soluble antigen was effectively bound to immunoadsorhents but only 10 to 15% was desorbed in active form.
Murine alloantigen was purified 20 to 25 times, the soluble preparations contained approximately 500 ID₅₀/ml or 1ID₅₀/25–30 μg protein, and the specificity ratio was 20 to 40. Extraction of murine cells with 3M KC1 yielded substantially lower soluble antigen activity. Solubilized murine alloantigen showed activity in both in vitro and in vivo tests.     

An approach to the routine estimation of circulating carcinoembryonic antigen immune complexes in patients with carcinomata of the gastrointestinal tract.

The carcinoembryonic antigen (CEA) distribution obtained after fractionation of thirty sera of patients with gastrointestinal tumours by gel filtration in Sephadex G200 was compared with that resulting from extraction of the sera with perchloric acid. In all cases gel filtration yielded a fraction of free CEA and in twenty-three cases an additional fraction of CEA immune complexes. The amounts of free CEA corresponded fairly well to the fractions of CEA which could be extracted by perchloric acid. The CEA content of the fraction containing CEA immune complexes was comparable to the CEA content of the perchloric acid precipitates. The presence of CEA immune complexes in the perchloric acid precipitates could be demonstrated by gel filtration under dissociating andnon-dissociating conditions and after pre-incubation with 125I-CEA by radioimmuno-double-diffusion using monospecific rabbit antisera against human IgM and IgG.     

Effects of inflation on the coupling between the ribs and the lung in dogs

The coupling between the ribs and the lung in dogs increases with increasing rib number in the cranial part of the rib cage and then decreases markedly in the caudal part. The hypothesis was raised that this non-uniformity is primarily related to differences between the areas of the lung subtended by the different ribs, and in the current study we tested this idea by assessing the effects of passive lung inflation. Thus, by causing a descent of the diaphragm, inflation would expand the area of the lung subtended by the caudal ribs and improve the coupling between these ribs and the lung. The axial displacements of the ribs and the changes in airway opening pressure (Δ P _ao) were measured in anaesthetized, pancuronium-treated, supine dogs while loads were applied in the cranial direction to individual rib pairs at functional residual capacity (FRC) and after passive inflation to 10 and 20 cmH₂O transrespiratory pressure. In agreement with the hypothesis, inflation caused an increase in Δ P _ao for ribs 9 and 10. The most prominent alteration, however, was a marked decrease in Δ P _ao for ribs 2–8; at 20 cmH₂O, Δ P _ao for these ribs was only 30% of the value at FRC. Additional measurements indicated that this decrease in Δ P _ao results partly from the increase in diaphragmatic compliance but mostly from the reduction in outward rib displacement. This alteration in the pattern of rib motion should add to the decrease in muscle length to reduce the lung expanding action of the external intercostal muscles at high lung volumes.     

Antigenic Determinants of Carcinoembryonic Antigen

Radioimmunoassays for carcinoembryonic antigen (CEA) have been established using a variety of different antisera raised in goat, monkey, rabbit, guinea pig and rat. The extent to which different antigens compete for antibodies in the sera has been estimated. The antigens used were: CEA, asialo CEA, Smith-degraded CEA (50% carbohydrate removed), CEA treated with an extract of Trichomonas foetus (70% carbohydrate removed), reduced-alkylated CEA (disulphide bonds ruptured), a peptic digest of CEA and CEX. The results demonstrated that there are at least five chemically distinct antigenic determinants: three are on protein—one, sensitive to conformation, a second, sensitive to conformation and to periodate, a third, insensitive to conformation—and two on carbohydrate, one common to CEA and CEX and another on CEA alone.