Malignant Glial Neoplasms: Definition of a Humoral Host Response to Tumor-Associated Antigen(s)

There is increasing evidence that human tumors possess tumor-associated neo-antigens. The host mounts an immunological response to these antigens, as evidenced by the detection of circulating humoral antibodies in a variety of human neoplasia.An indirect immunofluorescent antibody technique was employed to detect antibodies to tumor-associated antigens in the sera of patients with malignant gliomas. Viable single cell suspensions were used to demonstrate antibodies to surface contents of tumor cells and cell preparations were snap-frozen at −160° C to demonstrate antibodies to cytoplasmic components of tumor cells. After incubation with serum, the preparations were treated with polyvalent sheep antihuman globulin conjugated to isomer-1-fluorescein isothiocyanate, washed, and examined with a Leitz incident fluorescent microscope.Of the 17 sera from histologically proven malignant glial neoplasm patients, 2 (11%) were positive for an autologous surface antibody reaction. Five (23%) of 21 were positive for an autologus cytoplasmic antibody, however, 10 (47%) of 21 of the sera gave a positive reaction for cross-reacting cytoplasmic antibodies when tested with a battery of tumor cells obtained from different patients with malignant glial tumors.No reaction was observed with normal brain tissue. Absorption studies indicated the presence of a tumor-associated antigen.This study demonstrated that certain patients with malignant gliomas possess circulating antibodies to cytoplasmic components of their own tumor cells. The fact that a number of sera cross-reacted with tumor cells obtained from different patients suggests that antigenic cross-reactivity exists between malignant glioma cells from different patients. It is suggested that with further refinement, immunofluorescent detection of antibodies could evolve as a useful diagnostic adjunct in malignant glioma.     

Detection of in vitro and in vivo released antigens of diagnostic interest in Mycobacterium tuberculosis by immunoblotting

Identification of in vitro and in vivo released mycobacterial antigens are of considerable interest in diagnosis of Mycobacterium tuberculosis. Isolation of in vitro released antigen from M. tb excretory-secretory culture filtrate protein and in vivo released circulating tuberculous antigen from smear positive pulmonary tuberculosis sera by ammonium sulphate precipitation is reported. The antigens were resolved by SDS-PAGE and immunoblotting was performed using pooled serum of smear positive, smear negative pulmonary tuberculosis sera and normal sera to identify reactive tuberculous antigens. In vitro and in vivo released mycobacterial antigens showed reactivity at 100, 31, 43 and 20 kDa with smear positive and smear negative pulmonary tuberculosis patients. Further, the in vitro released antigen showed strong reactivity exclusively at 55 kDa antigen with smear positive and 24 kDa antigen with smear negative pulmonary tuberculosis sera. In vivo released antigen reacted exclusively at 170 and 16 kDa with smear positive and 19 kDa antigen with smear negative pulmonary tuberculosis patients. Antigens of 24 and 19 kDa which are reactive with sputum negative sera will be of diagnostic interest and need further study in patients with low bacillary load. The in vitro and in vivo released mycobacterial 100, 31,43 and 20 kDa antigens, reactive with patients sera are of diagnostic interest in tuberculosis.     

Clearance of false-positive antigen-antibody reactions of a diagnostic antigen produced inEscherichia coli with human sera

Although many pharmaceutically useful proteins are produced inE. coli expression system, it is very rare for the system to be used in the production of diagnostic antigen due to a major problem,i.e., false-positive reaction ofE. coli host-derived proteins contaminating purified diagnostic antigen with human sera. The N (nucleocapsid) protein of Seoul virus causing haemorrhagic fever with renal syndrome (HFRS) was produced inE. coli BL21 (DE3), and used for the detection of N protein-specific antibodies in human sera. Using the N protein as a diagnostic antigen of HFRS, the false-positive reaction was cleared by merely mixing the test sera with the extract ofE. coli host strain not harboring expression plasmid.     

Nude mice injected with DNA released by antigen stimulated human tlymphocytes produce specific antibodies expressing human characteristics

Nude mice were injected with DNA purified from the nucleoprotein complex released by T lymphocytes previously exposed in vitro to inactivated herpes or poliovirus. After five days the serum of these mice was tested for its virus neutralizing activity. Results show that injected nude mice synthesize antiherpetic or antipolio antibodies depending on the antigen used to sensitize the T lymphocytes in vitro. These antibodies were not found in the serum of uninjected control mice or mice injected with inactivated herpes or polio viruses. Mice injected with DNA release by human T cells produced antibodies carrying human allotypes since they could be neutralized by anti-allotype sera. Moreover their antiviral activity was inhibited by anti-human IgM or IgG. However, the mice which were injected with DNA released by antigen stimulated murine T lymphocytes produced antiviral antibodies which were not neutralized by anti-human allotype sera.     

Hanganutziu-Deicher antigen and antibody in pathologic sera and tissues.

Heterophile, Hanganutziu-Deicher (H-D) antigen was studied in pathologic sera by means of inhibition of agglutination of bovine erythrocytes by H-D antibodies. H-D antigen was demonstrated in 38% of random cancer sera, 25% of lymphoma or leukemia sera, 25% of leprosy sera, 8% of infectious mononucleosis sera, 6% of rheumatoid arthritis sera, and 27% of synovial fluids of rheumatoid arthritis patients. None of 134 normal human sera gave positive results. Some of the inhibition-positive cancer sera formed precipitation lines with H-D antibody-containing sera. Over 50% of various extracts of cancer tissues as well as spleens of lymphoma or leukemia patients were shown to contain H-D antigen by means of the inhibition test.     

Precipitation Reactions in Agar Between Swine Serum and Homologous Pancreas Extracts

Hyperimmune anti-hog cholera and nonimmune swine sera yielded approximately 50% more precipitation reactions in agar-gel diffusion tests with pancreas extracts from SPF noninfected swine than with extracts obtained from swine experimentally infected with virulent hog cholera virus. The pancreas-reacting property of swine serum was determined to be relatively heat stable, withstanding 68 C for 30 minutes. Of various swine serum fractions tested, the only one that reacted with pancreas extracts contained gamma, beta and alpha-globulins. In the absence of alpha-globulin, precipitation reactions were not observed. Sera of newborn SPF piglets, containing 50% alpha-2 globulin, formed more intense precipitation lines with swine pancreas extracts than were formed by the sera of their dams with the same extracts. The pancreas-reacting activity of swine sera was completely removed by absorption with pancreatic tissue. This property was not removed by absorption with guinea pig kidney, or beef, swine or human erythrocytes. Maceration of pancreatic tissue released reactive substances in a polydispersed form. This was demonstrated by the ability of almost all supernates and sediments from differential centrifugation of such preparations to form precipitation lines with swine sera. Reactive substances from swine pancreas were found to be relatively heat labile, being inactivated in one hour at 56C. No evidence was obtained in this study to indicate that the observed precipitation reactions were related to hog cholera virus and its corresponding antibody. The reactions are believed to have resulted from the interaction of protein-related substances present in normal swine pancreas with a relatively heat stable component, possibly alpha-globulin, in swine serum.     

Evaluation by the skin prick test of Anisakis simplex antigen purified by affinity chromatography in patients clinically diagnosed with Anisakis sensitization

Anisakis simplex crude extracts (CE) (IPI, ASAC and ALK-ABELLO), A. simplex larval antigens purified using a column of IgG anti-A. simplex (PAK) or a column of IgG anti-Ascaris suum (PAS), antigen eluted from columns of IgG anti-A. suum (EAS) and an A. suum adult CE were assayed by the skin prick test. Thirty percent of assayed patients showed a negative reaction in the Anisakis skin prick test. Of 70% positives, two patients had a weal greater than that produced by histamine with the A. simplex extract from ABELLO and IPI. The A. suum skin prick test was positive in 35% of patients, with a lower reaction than that observed with the A. simplex extract from IPI in 57% of the sera and a higher reaction in 28% of the sera. All patients with positive reactions with the crude extract also showed positive weals with the two purified antigens, PAK and PAS. All patients, except three, with a reaction to A. suum antigen, were positive to the EAS antigen. In five patients the weal size produced by PAS was greater than that observed with PAK, whereas in another six patients the contrary was observed. Only one of these six patients did not react to EAS antigen, coincident with the patient showing only a slight increase (7%) in the weal size induced by PAK vs. PAS. When the EAS antigen was tested on patients positive to both PAK and PAS, six patients presented a weal size of >30% and only three patients who were positive to PAS did not react to the EAS antigen. These three patients were also negative against the A. suum CE. Purification by affinity chromatography eliminates from the PAS antigen the proteins responsible for producing cross-reactions with Ascaris (present in the EAS antigen).     

A nonimmunoglobulin precipitin to tissue extracts in pathological human sera

An alpha-globulin component was noted in pathological human sera, which produced gel precipitation reactions with extracts of human and animal liver. The highest incidence of the precipitin was found in malaria (95%), renal graft rejection (81%), and rheumatoid arthritis (57%). The precipitinogen was thermostable and ethanol soluble; of two precipitation lines formed by this component, one merged into identity reaction with a line produced by commercial lecithin of bovine origin. The possible diagnostic application of the reactions noted was considered.     

Antibodies in the sera of patients with bronchogenic carcinoma that react with antigen from a tumour cell line

Summary Antigenic material was isolated from a human squamous cell bronchogenic carcinoma tissue culture line A549 that was found to react with antibodies in the serum of patients with lung cancer in an enzyme-linked immunosorbent assay (ELISA), while it did not react with sera from normal individuals. The antigen was tested with a panel of sera from a variety of patient groups by means of the ELISA. Results showed significantly higher numbers of sera from patients with lung cancer, particularly those of squamous cell origin, reacting with the antigen than of sera from 173 normal individuals or patients with breast and gynaecological cancers or melanomas.     

Use of donor-specific T-cell lines for monitoring of human allograft recipients. I. Demonstration of IgG binding to autologous TCL

Donor-specific and highly cytotoxic T-cell lines (TCL) as well as lectin-induced TCL were established from pretransplant lymphocytes of 6 cadaveric renal allograft recipients. These TCL were used in the 125I-staphylococcus protein A assay to detect IgG antibodies in pre- and posttransplant sera of these patients preferentially binding to autologous donor-specific TCL. Such antibodies were detected in pretransplant sera from 4 of these 6 allograft recipients. Antibody levels in these 4 patients and in 1 additional case who became positive after transplantation further increased during acute cellular rejection episodes. They disappeared after successful treatment but remained elevated until transplantectomy for treatment of irreversible rejection in 1 case. IgG antibodies binding to autologous lectin-induced TCL were detected in only 1 patient and exhibited a pattern clearly different from those binding to donor-reactive TCL. Although attempts to define the antigenic specificity of the autoantibodies binding to donor-specific TCL by genetical and biochemical means has remained unsuccessful so far, the demonstration of their relationship to in vivo expansion of donor-reactive immune cells deserves further attention.     

Human monoclonal antibodies reactive with antigens of the group A Streptococcus and human heart

Human mAb were produced from tonsillar or PBL of normal individuals or patients infected with group A streptococci. Lymphocytes were purified on Ficoll-Hypaque gradients and stimulated in vitro with purified group A streptococcal membranes or M protein extracts. The mAb were selected for study based on their reaction with group A streptococci, pep M5 protein, and/or M6 Escherichia coli protein. Further analysis by Western immunoblot or competitive inhibition ELISA revealed that there were two types of antibodies: one type that reacted with myosin and DNA and the other type that reacted with myosin, keratin, and/or actin. The specificities of these human mAb are similar to specificities observed in our previous studies of murine mAb reactive with group A streptococci and heart Ag. For comparison, anti-myosin antibodies were affinity purified from the sera of infected or acute rheumatic fever patients and were shown to react with myosin and DNA as well as with group A streptococci and M protein. To affinity purify these antibodies from normal sera, five times the amount of sera was required to obtain detectable quantities. These data suggest that the human mAb reactive with group A streptococci and myosin reflect the antibodies seen in sera from infected patients or acute rheumatics and that the B lymphocyte clones capable of producing these cross-reactive antibodies are also present in normal individuals.     

Renal graft dysfunction during infection with cytomegalovirus: association with IgM lymphocytotoxins and HLA-DR3 and DR7.

Of 121 consecutive adult recipients of cadaver renal transplants who were treated with low dose steroids and azathioprine, 23 developed active cytomegalovirus infections. These 23 patients were divided into three groups on the basis of their symptoms related to the infection: five patients had no renal, respiratory, or haematological abnormalities; seven had renal dysfunction; and nine had renal dysfunction plus respiratory or haematological abnormalities. Two patients were regarded as a separate group because their infections occurred two to four weeks after graft nephrectomy. All but three of the patients produced IgM or IgG lymphocytotoxins during their infections. In the patients with mild infections and in control patients without infections, however, these lymphocytotoxins were predominantly IgG antibodies that were not precipitated by 3.5% macrogol (polyethylene glycol). In contrast, 12 of the 16 patients with renal dysfunction during their infections had broadly reactive IgM lymphocytotoxins. These IgM lymphocytotoxins lysed T as well as B lymphocytes at 22 degrees C and were precipitated by 3.5% macrogol, suggesting that they were circulating as immune complexes. Rheumatoid factors were found in sera from nine patients with cytomegalovirus infections, seven of whom developed leukopenia or pneumonia, or both, in addition to renal dysfunction. Some of these immune responses associated with cytomegalovirus infection in transplant recipients may be genetically controlled since 10 of 11 patients positive for HLA-DR3 or DR7 produced IgM lymphocytotoxins.     

Serotyping for Homotransplantation. XXII,Specificity of Cytotoxic Antibodies Developing after Renal Transplantation

Cytotoxic antibodies against allogeneic lymphocytes were detected in 13 patients after removal of a renal allograft. Antibodies were not detected in seven patients who had grafts in situ for less than two months, but were detected in 13 out of 17 patients who had grafts in situ for longer than two months. The HL-A specificity of these positive sera was compatible with the HL-A antigen mismatch of the donor-recipient pairs in most instances, providing further evidence that such antibodies result from direct sensitization by the allograft and may play some part in renal allograft rejection.     

Immunoblotting reactivity of human sera from various sources against purified epstein-barr virus

The immunoblotting technique was used to analyse polypeptides of purified Epstein-Barr virus reacting with antibodies present in sera from clinically healthy individuals, from patients with infectious mononucleosis (IM) or AIDS, and from renal transplant recipients with molecular sizes in the range of 40–290 kDa were detected.The 47- and 160-kDa nucleocapsid polypeptides, as well as the 72-, 74-, 140-, 220- and 290-kDa membrane polypeptides were the major viral proteins detected in the sera. Sera from clinically healthy individuals contained antibodies directed against all EBV membrane and nucleocapsid antigens. Sera from renal transplant recipients, from patients with IM and from patients with AIDS failed to react with certain nucleocapsid and membrane antigens; in particular, sera from AIDS patients and renal transplant recipients did not react with the 220-kDa polypeptide, one of the major membrane antigens, while sera from subjects with IM and from healthy individuals did.A high proportion of sera from patients with IM (38% vs 5% of clinically healthy individuals and 0–5% of the AIDS patients and renal transplant recipients) reacted with a 42-kDa polypeptide, suggesting its possible role in acute EBV infection.     

Plasmodium berghei: Specific stimulation of rat lymphocytes by soluble antigens released in vitro from infected erythrocytes

Soluble components released in vitro from rat erythrocytes parasitized with Plasmodium berghei have been shown to stimulate nonadherent spleen lymphocytes from rats convalescent from homologous infection. The active product is released from the infected erythrocytes either spontaneously during incubation, or as the result of artificial rupture of infected erythrocytes by freezing and thawing or sonication. This antigen produced precipitation lines with antiplasmodial antibodies from sera of convalescent and hyperimmune rats and from immunized rabbits.     

Hepatitis B virus polypeptide X: expression in Escherichia coli and identification of specific antibodies in sera from hepatitis B virus-infected humans.

Sequence analysis of the hepatitis B virus (HBV) genome revealed the presence of an open reading frame (ORF X) which has the potential to encode a 154-amino acid polypeptide. A fusion protein containing 145 of the amino acids encoded by ORF X and 8 amino acids of beta-galactosidase was expressed and characterized in bacterial extracts. Immunoprecipitations with the ORF X fusion protein as a radioactively labeled antigen were performed to screen sera of humans infected with HBV for the presence of antibodies against ORF X-encoded determinants (anti-X). Such antibodies were identified in 9 samples from a set of 26 sera characterized as positive for HBV surface antigen but were not found in 16 normal human sera. The data reported here demonstrate that sera from some patients with markers of HBV infection contain antibodies directed against the polypeptide encoded by ORF X. As such, these findings represent evidence that ORF X constitutes a gene, or a portion of a gene, which is expressed during HBV infection. Although there does not appear to be a direct relationship between anti-X and any individual markers of HBV infection, our data suggest that anti-X is more prevalent in HBV-positive sera containing antibodies to HBe3 antigen (anti-HBe3).     

Positive Fluorescent Treponemal Antibody Reactions in Diabetes

In a survey of 129 diabetic patients and 142 normal individuals, a significantly higher percentage of positive reactions in the fluorescent treponemal antibody-200 (FTA-200) test was found among diabetic patients than in the normal population. Absorption of all FTA-200-reactive sera with an extract of Reiter's treponeme eliminated most of the positive reactions in sera from diabetic patients, and three of the five positive reactions detected in sera from apparently normal subjects. On immunoelectrophoresis, precipitin bands developed most frequently between the Reiter sorbent and sera from diabetic patients positive in the FTA-200 test. Serum components responsible for FTA reactivity and precipitin reactions against the sorbent were resistant to treatment with mercaptoethanol, suggesting antibody of the IgG class. Cross-reacting antibodies produced in response to normal treponemal flora, and perhaps acquiring enhanced reactivity by means of nonspecific interacting substances in sera peculiar to the altered physiological state of diabetes, are suggested as possible causes of positive reactions of unabsorbed sera. No correlation could be made between age of the diabetic patient, treatment or duration of the disease, and FTA or precipitin reactivity of the patient's serum.     

Anomalous reactions of mouse alloantisera with cultured tumor cells. I. Demonstration of widespread occurrence using reference typing sera.

M0use alloantisera produced against different specificities of the K, I, and D regions of the H-2 gene complex reacted as immunogenetically anticipated with normal lymphoid target cells of different haplotypes in cytotoxicity and indirect immunofluorescence tests. These same alloantisera, however, produced anomalous positive reactions when tested on cultured MCA-induced sarcoma cells from B10 background H-2 congenic mice. Absorption experiments demonstrated that the anomalous activity in these sera was directed against a tumor membrane antigen(s) which was distinct from H-2 region specificities against which the reference alloantisera were produced, and which was shared in common by multiple cultured sarcoma lines. Similar anti-tumor antibody activity could be demonstrated in the serum of older (greater than 12 weeks) but not younger normal unimmunized mice of the strains used as recipients for alloantiserum production. It is suggested that the observed anamalous anti-tumor activity in these alloantisera may be due to the presence of antibodies reactive with envelope antigens of murine leukemia virus which are expressed on sarcoma cells maintained in culture.     

Antibodies to the C epitope of Neisseria gonorrhoeae are present in patients with gonorrhoea and absent in normal sera

We have previously described a surface oligosaccharide antigen (epitope C) present in fresh isolates of Neisseria gonorrhoeae and in variants grown in subcutaneous chambers, but poorly formed by variants repeatedly subcultured in vitro. We have now investigated the presence of antibodies to epitope C in sera from normal individuals and from patients with gonorrhoea. Sera were analysed by Western blotting and ELISA, and compared with a pool of sera from normal individuals with no known history of gonorrhoea. Antigenic extracts and monoclonal antibody to the C epitope were used for competition and inhibition studies. Only the sera from patients contained antibodies to epitope C. Antibodies to several other gonococcal antigens were found in sera from patients, and also in normal sera. Collectively, the results indicate that epitope C is expressed in humans, that patients with gonorrhoea develop antibodies to it, and that such antibodies are absent in sera of normal individuals.     

Substrate specificity for the detection of autoantibodies to anterior pituitary cells in human sera

Using the indirect immunofluorescence test for the detection of pituitary autoantibodies in human serum, the results obtained with human fetal and non-human pituitary antigens were compared. Of the sera that were positive on human fetal substrate, 4% were recovered with adult baboon, 0% with fetal cymologous, 20% with porcine, 11% with bovine, 11% with ovine, and 7% with rat tissue. The rate of heterophilic antibodies to the above animal substrates was 5% to 14%. In contrast to human adult pituitaries, normal human sera did not bind to Fc receptors on ACTH-cells of human fetal pituitaries. This allowed the demonstration of ACTH-cell antibodies. The specificity of the reaction was proven by absorption studies with purified Fc fragments. These results suggest that human fetal tissue is the best source of antigen for the detection of pituitary autoantibodies. The use of animal tissue including non-human primate pituitary yields results that bear no clinical significance.