Heavy and light chain allotypic markers on rabbit homocytotropic antibody.

Evidence has been presented that rabbit homocytotropic antibody prepared against ovalbumin bears allotypic markers of both group a (heavy chain) and group b (light chain). This was shown by specific removal of activity in passive cutaneous anaphylaxis assays by anti-allotype immunoadsorbents. The homocytotropic antibody has properties which indicate that it belongs to a new class of rabbit immunoglobulin. These results demonstrate that this newly described class possesses the allotypes of both groups a and b, as has been previously shown for other rabbit immunoglobulin classes.

Allotype exclusion in uniform rabbit antibody to streptococcal carbohydrate.

Rabbit antibodies to streptococcal polysaccharide are described which show selectivity of expression of the allotypic specificities on both the heavy (H) and light (L) chains. One of these antibodies binds weakly to Sephadex. A purification method based on this binding has yielded antibody completely lacking any group a allotypic marker on its H chains.

Diverse expression of group a allotype specificities on the heavy chains of homogeneous rabbit antibodies.

Variations in the ability of homogeneous antibodies to absorb group a allotype antisera suggest that these V(H) region allotypes comprise a spectrum of specificities. While no homogeneous antibody tested would completely absorb antisera to the group a specificities, the total IgG preparations from individual rabbits was capable of doing so, including preimmune IgG from the rabbits that produced the homogeneous antibodies. Differences in the serologic reactivities of homogeneous antibodies indicate that each possesses only a portion of the spectrum of the allotypic specificities expressed by the total IgG.

Purification of carcinoembryonic antigen by removal of contaminating mucopolysaccharides.

The fraction of carcinoembryonic antigen preparations not bound by concanavalin A was studied. A significant portion of this nonbound fraction of low antigenic activity was shown to be mucopolysaccharides by gas chromatography-mass spectrometry, cellulose acetate strip electrophoresis, and depolymerization by bovine testicular hyaluronidase.

The binding of carcinoembryonic antigen by antibody and its fragments.

In order to assess the potency of antigenic fragments of carcinoembryonic antigen (CEA) in the radioimmune assay; it is necessary to know whether the high affinity of goat anti-CEA antibody (which makes possible the detection of as little as 10--11M CEA) is due to bivalent binding of the CEA molecule. Immunoglobulin G and the F(ab')2 and Fab fragments derived from it were prepared from an anti-CEA serum and tested for their abioity to bind CEA. Equivalent concentrations of binding sites of the bivalent F(ab)2 and univalent Fab fragments of anti-CEA were identical to the immunoglobulin G fraction in the standard inhibition curve. Fragments of CEA obtained by trypsin digestion produced equivalent inhigition curves when tested with either immunoglobulin G, F(ab')2, or Fab". This, increased avidity due to bivalent binding to a single antigen molecule cannot be invoked to explain the sensitivity observed in the CEA assay. This high sensitivity implicates the protein rather than the carbohydrate as an important part of the antigenic determinant(s) of CEA.

Demonstration of elevated anti-Lewis antibodies in sera of cancer patients using a carcinoembryonic antigen-polyethylene glycol immunoassay.

The serum levels of carcinoembryonic antigen (CEA) and CEA-binding proteins of 68 controls and 170 cancer patients were determined. The CEA levels were determined by a double-antibody radioimmunoassay using radiolabeled CEA, and the CEA-binding proteins were determined by an immunoassay utilizing radiolabeled CEA and polyethylene glycol. The major CEA-binding proteins in serum were anti-blood group antibodies as demonstrated by differential binding of serum proteins from A, B, or O positive individuals to radiolabeled CEA's which were previously shown to carry specific blood group determinants. No statistically significant differences were observed for the binding of control versus cancer patient sera to CEA-A (carrying blood group A1), except, as expected, A negative (O or B positive) individuals gave high binding to CEA-A, while A positive individuals gave low binding to CEA-A. Statistically significant differences were observed for controls versus cancer patients for binding to CEA-Lewisab (CEA-Leab). CEA-Leab-binding activity was higher in females and smokers in the control group, but this distinction was not found in the cancer patients. The high levels of anti-Leab antibodies may be explained in females by exposure to fetal antigens during pregnancies and in smokers or cancer patients by exposure to precursors to blood group substances. The sera of 7% of the patients with colonic carcinoma, 18% of the patients with breast carcinoma, and 23% of the patients with bronchogenic carcinoma bound more CEA-Leab than did the serum of the highest male control. A correlation between CEA-Leab-binding activity and the levels of serum CEA was found for patients with colonic carcinoma but was not significant for the groups with other cancers or the control group. Serial determinations of CEA-Leab-binding activity for 21 patients with bronchogenic carcinoma changed congruently with the serum levels of CEA in 12 cases. The significance of these results in terms of expression of immature blood group antigens, the subsequent production of antibodies against them, and the prognostic value of this response is discussed.

Sensitive detection of carbohydrate determinants on carcinoembryonic antigen preparations by lectin and antibody binding using polyethylene glycol.

Blood group determinants have been found in five carcinoembryonic antigen (CEA) preparations with a lectin and antibody-binding assay using polyethylene glycol 6000 to separate free from bound radiolabeled antigen. The assay described gives excellent sensitivity for the binding or inhibition of binding of various lectins or antibodies to CEA. One of the CEA preparations investigated has an A1 determinant, another has a B determinant, and all have H, Lea, Leb, and MN blood group determinants. In addition, all of the preparations tested bound concanavalin A. These findings are consistent with the idea that incomplete or unexpected glycosylation patterns occur in glycoproteins produced by tumor cells. Since antibodies directed against blood group substances cross react with carbohydrate determinants on CEA, clinical determinations of CEA or anti-CEA levels in serum may be adversely affected.

Amino-terminal sequence of a carcinoembryonic antigen-like glycoprotein isolated from the colonic lavages of healthy individuals.

A glycoprotein has been isolated from the colonic lavages of healthy individuals that is immunologically equivalent to carcinoembryonic antigen purified from tumor tissue. The NH2-terminal sequence of the glycoprotein from normal colon lavages is Lys-Leu-Thr-lle-Glu-Ser-Thr-Pro-Phe-(Asn)-Val-Ala-Glu-Gly-Lys-Glu-Val-(Leu,lle)-(Leu,lle)-(Leu,lle)-Val-(His,Arg?)-?-(Leu,lle). This is homologous to the NH2-terminal sequence of 23 of the first 24 amino acids of carcinoembryonic antigen isolated from tumor tissue.

Isolation and immunochemical and chemical characterization of carcinoembryonic antigen-like substances in colon lavages of healthy individuals.

Material with carcinoembryonic antigen (CEA)-like activity, isolated from colon lavages of healthy individuals, has been chemically characterized. The CEA-like activity in these lavages was purified by gel filtration on Sepharose 6B and Sephadex G-200 and by affinity chromatography on concanavalin A linked to Sepharose. The purified material migrated in polyacrylamide-sodium dodecyl sulfate electrophoresis as a single diffuse band with mobility identical with that of tumor CEA. The material with CEA activity from tumor tissue and from normal colon lavages gave lines of identity in double diffusion experiments and had similar inhibition curves with esssentially the same specific activities in a radioimmune assay for tumor CEA. The amino acid and carbohydrate compositions were similar to that observed for CEA tumor preparations. Lysine was the single amino terminus. Methylation analysis demonstrated that the monosaccharide linkages were similar to those in CEA isolated from tumor tissue.

Methylation analysis of the carbohydrate portion of carcinoembryonic antigen.

The carbohydrate structural units of carcinoembryonic antigen samples isolated from four different tumors were quantitated using gas chromatography-mass spectrometery after methylation and subsequent conversion to their alditol acetates. Different carcinoembryonic antigen preparations showed some quantitative but no qualitative differences in the structural units present. The results indicate that a large portion of the fucose residues in the glycoprotein were linked to N-acetylglucosamine and that most of the branching mannose residues were probably linked to three N-acetylglucosamine residues.

Physical characterization and structural studies of the carcinoembryonic antigen.

Knowledge of the structure of carcinoembryonic antigen (CEA) is essential if we are to understand the relationship of this molecule to similar, possibly cross-reacting, molecules present in nonmalignant states. Electron microscopy shows that at neutral pH, CEA particles consist of homogeneous, morphologically distinctive, twisted rod-shaped particles, about 9 X 40 nm. The carbohydrate structure of CEA has been studied by periodate oxidation. All the N-acetylneuraminic acid and fucose and a portion of the galactose and mannose were destroyed by the first periodate treatment without altering immunological activity. N-Acetylneuraminic acid was shown to be linked to galactose since its prior removal with neuraminidase led to an equivalent increased destruction of galactose by one treatment with periodate. Significantly, even after 100% of the fucose and N-acetylneuraminic acid, 75% of the galactose, and 50% of the N-acetylglucosamine and mannose were destroyed by serial periodate oxidation (Smith degradation), the remaining portion of the CEA molecule lost no more antigenic activity than did control samples where periodate was omitted. No carbohydrate was lost or destroyed in the control reaction.

Serum levels of carcinoembryonic antigen and a tumor-extracted carcinoembryonic antigen-related antigen in cancer patients.

Levels of carcinoembryonic antigen (CEA) and a tumor-extracted CEA-related antigen (TEX) were determined in sera from patients with carcinomas of the breast, colon, lung, head and neck, and a number of miscellaneous categories. The purpose of this study was to compare the levels of each antigen at various stages of malignant disease. Competitive radioimmunoassays developed in our laboratory were shown to be sufficiently specific to detect either of these two antigens independent of each other. The results indicate that: (a) with our specific assays, CEA was not significantly elevated in smoker controls, but TEX was elevated in 29% of smoker controls; (b) TEX was equivalent to CEA as a tumor marker for colonic cancer. TEX was better than CEA as a marker for lung cancer and, based on limited data, there is a possibility that TEX is a significantly better tumor marker than is CEA in early lung cancer; (c) TEX was superior to CEA as a tumor marker for breast and head and neck cancers; (d) there is a strong indication that serial determinations of TEX can be used as effectively as CEA in the monitoring of disease progress. These preliminary results must be confirmed by increasing the number of cancer patients and including nonmalignant disease controls.

Effects of a beta-human chorionic gonadotropin subunit immunogen administered in aqueous solution with a novel nonionic block copolymer adjuvant in patients with advanced cancer.

The clinical and immunological effects of a vaccine consisting of CTP37, a synthetic peptide corresponding to the COOH-terminal peptide (CTP) of beta-human chorionic gonadotropin (beta-hCG) conjugated to diphtheria toxoid, combined with CRL 1005, a novel synthetic nonionic block copolymer adjuvant, were examined. Twenty-one patients with metastatic, nontrophoblastic cancers received up to four immunizations by i.m. injection of a fixed dose of CTP37 and escalating doses of CRL 1005. Doses of CRL 1005 adjuvant as high as 75 mg were administered with 1 mg of CTP37 without evidence of significant local or systemic toxicity. Immunizations resulted in the production of IgG antibody to beta-hCG. CRL 1005 doses of 3-25 mg appeared to be optimal for antibody induction. Immunizations also resulted in increases in the cellular response of peripheral blood mononuclear cells (PBMCs) to the unconjugated CTP, hCG, and diphtheria toxoid. Responding PBMCs specifically secreted the TH1-associated cytokines IFN-gamma and interleukin (IL)-2 as well as the TH2-associated IL-5 and IL-10. Increased expression of IFN gamma and IL-5 mRNAs by PBMCs 4 h after immunization was also observed. CRL 1005 administered with CTP37 in aqueous solution is well tolerated. The CTP37-CRL 1005 subunit vaccine has the capacity to stimulate potentially beneficial humoral and cellular immune responses in patients with advanced cancer.

Simultaneous deletion of the intervening sequences from the human interferon-gamma gene by oligodeoxynucleotide-directed mutagenesis.

Oligodeoxynucleotide (oligo)-directed mutagenesis was used to simultaneously delete the three intervening sequences (IVS) from the human interferon-gamma (IFN-gamma) gene. Prior to the deletion experiment, the two largest IVS of the native gene were shortened by restriction enzyme digestion and subsequent ligation (IVS-1 was reduced from 1239 bp to 399 bp and IVS-3 from 2425 bp to 183 bp). This modified gene was cloned into vector M13mp9. Three oligos [21-25 nucleotides (nt) long] were synthesized, one to bridge each of the three introns. A different set of three oligos was made for use as hybridization probes to identify colonies containing the correct deletion. A study was made of enzymatic reaction and primer annealing conditions needed to optimize intron deletions. The efficiency of the simultaneous deletions was higher than the product of the efficiencies of the deletions of the individual IVS, suggesting that the deletion events are not independent. The gene created by the simultaneous deletion was cloned into an Escherichia coli plasmid expression vector and IFN-gamma activity was produced.

Increasing the immunogenicity of a trivalent influenza virus vaccine with adjuvant-active nonionic block copolymers for potential use in the elderly.

High molecular weight nonionic block copolymers consisting of a large hydrophobic core made from repeat oxypropylene units and smaller hydrophilic blocks of oxyethylene repeat units were evaluated as adjuvants in experimental influenza virus vaccine formulations. The goal was to identify a copolymer that would increase the immunogenicity of the commercial Fluogen trivalent influenza virus vaccine. Vaccine experiments done using BALB/c mice provided data that allowed us to identify a copolymer that increased both antibody titers specific for total virus proteins as well as antibodies with hemagglutination inhibition (HAI) activity. This copolymer, termed CRL1005, increased the production of IgG1, IgG2a and IgG2b which suggested it increased the activity of both Type-1 and Type-2 T-helper lymphocytes. The CRL1005 copolymer was tested further in rhesus monkeys with similar results. Levels of antibodies specific for total virus protein preparations were increased as were HAI antibody titers following vaccination with the copolymer-supplemented Fluogen vaccine. Thus, the CRL1005 copolymer adjuvant appears to be compatible for use with the current generation of inactivated viron-based influenza vaccines and useful for increasing the immunogenicity. A more potent influenza virus vaccine could well be more efficacious in the aged segment of our population than current vaccines.

Acid treatment enhances IL-2 activity of conditioned medium.

Although conditioned medium (CM) from human lymphocytes or mononuclear cells is the most readily available source of interleukin-2 (IL-2) for human T cell culture, its IL-2 activity in our experience is inconsistent. It is likely that this is, at least in part, due to the presence of toxic substances in the CM. Using CM from TPA/SEA induced human mononuclear cells, we have found that acid treatment (pH 2.0, greater than 30 min) significantly improves its ability to promote T cell growth. It is postulated that the selection process which occurs in cultures of mitogen stimulated T cells may result in cells which are sensitive to mitogen-induced lymphotoxins and that these are inactivated by the acid treatment. Since acid treatment did not similarly improve the T cell growth promoting ability of PHA induced, lectin-free commercial IL-2, there must be other differences between it and our CM, which play a role in T cell growth.

Production of mitogen-free immune interferon and T-cell growth factor by human peripheral blood lymphocytes induced with biotin-labeled staphylococcal enterotoxin A.

A method for the facile removal of mitogens or inducers of lymphokine production from cell culture medium of stimulated cells is described. The technique is based on the covalent attachment of biotin to mitogen or inducer and the removal of the biotinylated products from stimulated cell culture medium using immobilized avidin. Using this procedure, biotin-labeled staphylococcal enterotoxin A (SEA-B) was shown not to differ significantly from unmodified SEA in its capacity to stimulate mitogenesis and induce production of immune interferon (IFN) and T-cell growth factor (TCGF) in cultures of human mononuclear cells from peripheral blood. SEA-B was also shown not to differ from SEA in its binding to SEA antibodies. Results of mitogenicity studies and competitive radioimmune assay (RIA) measurements indicate that SEA-B is essentially completely removed from stimulated cell culture medium by absorption with avidin coupled to Sepharose 4B.

Immune responses during human schistosomiasis mansoni. VII. Further analysis of the interactions between patient sera and lymphocytes during in vitro blastogenesis to schistosome antigen preparations.

The lymphocyte blastogenic responses of chronic schistosomiasis mansoni patients were tested in vitro in medium supplemented with either normal human serum or patients' serum (either autologous or third party). As expected, when patients' lymphocytes were cultured in patient sera, many of them (75--78%) displayed reduced responsiveness to schistosome antigens (derived from either the cercariae, adult worms or eggs of Schistosoma mansoni), but not to Candida albicans extract. For decreased blastogenesis to be manifest, a combination of both suppressive sera and suppressible cells was required; however, some patients had nonsuppressible cells and not all sera were suppressive. In an attempt at classification, four categories of patient responsiveness concerning serosuppression are proposed. The categories depend on the suppressive capabilities of patient sera and the response of patient lymphocytes to suppressive sera. By individually testing the capabilities of each patient's lymphocytes and sera in relationship to each antigenic preparation, we were able to assign the majority of patient responses to a given category. It is hoped that by using these categories, a better understanding of the mechanisms concerning serosuppression will be obtained.

Association between household triatomine density and incidence of Trypanosoma cruzi infection during a nine-year study in Castro Alves, Bahia, Brazil.

We studied the association between human incidence of Trypanosoma cruzi infection and household infestation density of Panstrongylus megistus in Castro Alves, Bahia, Brazil. During a 9-year period, 19 persons seroconverted; 17 were children, 17 lived in nonplastered houses, and 13 lived in houses infested with triatomines. Although 6 seroconverting persons lived in houses where triatomines could not be found, the risk of seroconversion was significantly greater in infested houses and 16 times greater in densely infested houses (greater than 15 bugs/person-hour of search). The highest rate of seroconversion (6/100 person-years exposure) occurred in houses containing the greatest number of bugs infected with T. cruzi (greater than 6 infected bugs/person-hour). These observations suggest that vector control measures could have a dramatic impact on transmission of T. cruzi by P. megistus.