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Spinal cord chimeras were constructed by orthotopic grafting of quail embryonal neutral folds, neural crest and neural tube into chicken embryos. The spinal cord xenografts were accepted for varying lengths of time, but most chimeras eventually rejected the quail transplant. This was associated with perivenular cuffing and demyelination with preservation of most neurons, as well as clinical neurological symptoms. Twenty-four chimeras were studied to delineate the time of first appearance of glial deposits of immunoglobulin and to identify the subpopulations of T cells in spinal cord infiltrates. The results suggested that deposits of immunoglobulins on glial elements preceded inflammatory cell infiltration. The perivenular cuffs consisted predominantly of T cells and showed a preponderance of CD8- over CD4-positive cells (CD4/CD8 ratios around 0.6). Further, CD4+ cells were found almost exclusively in the central portions of the infiltrate, with the periphery consisting almost only of CD8+ cells. The diffuse cellular infiltrate of the parenchyme contained T and plasma cells. The T cells were almost exclusively CD8+. Plasma cells were seen only at the outer borders of the cuffs and dispersed throughout the quail-derived spinal cord tissue. It seemed that rejection of quail-derived melanocytes in feathers ('quail-like feathers'), described by us earlier, often preceded neurological symptoms and showed a histopathological pattern comparable to spinal cord lesions, i.e., predominantly perivascular cuffing. In preliminary studies, enhancement of disease by immunization with quail organ suspension and decreased intensity of disease by combined immunosuppressive treatment with FK 506 and cycylophosphamide were suggested. The data presented here are compatible with the hypothesis that rejection of CNS quail tissue by chimeras is preceded in the periphery by rejection of melanocytes in segments of skin and in feathers, and that the spinal cord rejection relies on xenoantibodies and on cytotoxic as well as delayed hypersensitivity-type T cells. Finally, these data strengthen the analogy between the histopathologic presentation and immune effector composition of the xenograft rejection lesions in the chimeras and the plaques seen in patients with multiple sclerosis.

Quail-chicken spinal cord chimeras are a model for temporary acceptance followed by rejection of xenografts and also for demyelinating lesions of the central nervous system. The antiglobulin test with quail erythrocytes was employed to detect antibodies in sera of quail-chicken spinal cord chimeras. Sera of all 46 chimeras tested gave positive results. In virtually all instances, antibodies were detected within 10 weeks after hatching and they persisted for all the observation time up to 8 months. The antibodies detected in these tests were directed against species antigens of the quail. They were apparently identical with xenoantibodies described in a previous study, which were detected by indirect immunofluorescence with quail tissue sections; on the other hand, mixed agglutination tests with quail embryonal cell monolayers employed previously had detected a broader spectrum of antibodies that did the antiglobulin tests with quail erythrocytes. The antiglobulin test with quail erythrocytes seems the most cost-efficient and convenient test to monitor xenoantibody formation in this animal model.

The pathogenesis of streptococcus-induced nephritides (SIN) involves immune complex-mediated inflammation; however, specific mechanisms are still poorly understood. Using preparations of two strains of Streptococcus mutans (SM) in attempts to induce SIN in rabbits, one preparation was strongly and the other virtually not nephritogenic. The non-nephritogenic preparation provided a negative control for our studies. Streptococcal components were present in circulating immune complexes (CIC) as well as in tissue-bound immune complexes (TIC), especially early in the disease. CIC and TIC also contained rheumatoid factors (RF), which tended to predominate in late stages of the disease. The nephritogenic and the non-nephritogenic preparations of SM shared the same major tissue-binding components and induced similar titers of antimicrobial antibodies, but differed significantly in their ability to induce CIC and RF. It is proposed that kidney-binding microbial components, antimicrobial antibodies and high serum concentration of RF are necessary and sufficient determinants for the pathogenesis of SIN in this rabbit model.

A previous study described clinical and pathological manifestations observed in 49 chimeras produced by replacing a small fragment of the neural tube of a chicken embryo by a similar fragment from a Japanese quail embryo. Predominant antibodies in sera of these birds were directed against xenogeneic antigens which are devoid of organ specificity and which are present in quail tissues but absent from chicken tissues. Mixed agglutination test with quail cell mono-layers detected such antibodies in sera of all chimeras tested. In most instances, positive reactions were observed already in the 4th week after hatching; they persisted in all birds throughout the observation period of up to 8 months. Some of the detected antibodies were directed against saline-nonextractable surface antigens of quail cells. Enzyme immunoassay with quail organ extracts, agglutination of quail tissue particles, and indirect immunofluorescence test with quail organ sections were positive with sera of many, but not all, chimeras. CNS-specific antibodies, apparently autoantibodies, were detected in serum samples of only one chimera, using enzyme immunoassay with extract of chicken brain and indirect immunofluorescence test with sections of chicken spinal cord. Eluates from lesional spinal cord contained antibodies to non-organ-specific quail antigens but not to CNS-specific antigens. Cerebrospinal fluids of many chimeras had antibodies to quail antigens, but no evidence for antibody formation within the CNS was obtained. Cell-mediated immunity could be demonstrated in all chimeras tested by means of lymphocyte proliferation test after stimulation by quail organ extract. It was concluded that pathological events in the studied chimeras have been most likely mediated primarily by humoral immune responses to non-organ-specific quail antigens.

Ethanol-soluble, but saline-insoluble antigens were prepared as saline suspensions and studied in double diffusion reactions in a soft agarose gel. Positive reactions were observed with syphilis and SLE sera tested against the Kahn antigen as well as against commercial cardiolipin reagents. Also, ethanol-soluble brain antigen was studied for organ-specific reactions with rabbit immune sera. It was shown that double diffusion in gel can be employed as an analytical procedure for studies on reactions of saline suspensions of ethanol-soluble antigens.

Previous studies on the appearance in pathological human sera of antibodies to an antigen of normal mammalian organs were continued. In gel precipitation reactions, antibodies combining with saline extracts of mammalian organs were found in 20 of 63 cancer sera and in 4 of 15 syphilis sera, but only in 3 of 56 other pathological sera. Furthermore, an identical antigen was demonstrated in 5 of 58 pathological sera. The antigen under study did not belong to any group of known heterophile antigens and it was devoid of organ and tissue specificity.

Previous numerous papers by the senior author dealt with the human serum factor referred to as anti-antibody which is specifically directed against IgG antibodies that underwent molecular transformation in the course of the reactions with their corresponding antigens. The reactions of this serum factor could be conveniently detected by means of agglutination of Rh-positive erythrocytes sensitized by anti Rh antibodies. No precipitation tests could be developed.Most studies were conducted by means of double diffusion in gel precipitation.A rheumatoid arthritis serum, G, was noted that produced a strong reaction of double diffusion in gel precipitation with serum samples of a renal graft recipient, T. Further screening detected one more rheumatoid arthritis serum reacting with T; of 28 sera from renal graft recipients, 6 reacted in a similar way to T, but the reactions were considerably weaker and poorly reproducible. Evidence was presented that the precipitin in the two rheumatoid arthritis sera under study had properties of previously described anti-antibody.Sera with precipitating anti-antibodies may serve as exquisite reagents for detection of soluble immune complexes in human sera.

Agglutination tests with preparations of parenchymatous organs were developed. The tissue suspensions were dried at room temperature after they had been spread as a very thin layer on a glass plate, or otherwise, they were lyophilized. The dried preparations were pulverized and then prepared as stable suspensions in saline. The agglutination test was conducted on a slide by mixing one drop of the tested serum at a convenient dilution with one drop of tissue powder suspension. Agglutination in the form of readily discernible clumps could be assessed after 1-10 min. By means of this procedure, species-specific reactions were studied using suspensions of kidneys of various species. Organ-specific reactions were noted with suspensions of brain and thyroid. Agglutination of thyroid powder was observed with rabbit anti-rabbit thyroid sera as well as with many, albeit not all, sera of patients with Hashimoto's disease.

It was noted that many sera of patients with renal allograft produce distinct precipitation lines in gel diffusion tests with about 20% of infectious mononucleosis sera. The antibodies in infectious mononucleosis sera were of IgM isotope, but, interestingly, they could be removed by guinea pig kidney homogenate, which indicated that the reactions studied were of the Hanganutziu-Deicher rather than of the Paul-Bunnell type. This contention was strengthened by the fact that positive transplantation sera reacted also with standard serum with Hanganutziu-Deicher antibodies. Thus far, the presence of the antigen in the transplantation sera could not be related to the clinical status of the patients, however, the antigen was noted primarily in those sera that did not contain heterophile transplantation antibodies. It was proposed that the antigen detected in the transplantation sera was an altered tissue antigen released from the grafted organ. Besides, interactions between two serum samples from the same patient were noted in immunodiffusion tests. These reactions occurred very seldom and were unrelated to heterophile transplantation antigens or antibodies.

By means of double-diffusion precipitation tests in agarose gel with selected infectious mononucleosis (IM) sera, an antigen was detected in sera of patients with syphilis and leprosy. Of 378 syphilis sera tested 39 (10.3%) and of 36 leprosy sera 7 (19.4%) gave positive results. Sera of patients with various other diseases were negative. This antigen was demonstrable in sera of patients with both early and late syphilis and was shown to be unrelated to cardiolipin. Absorption studies of the IM sera with bovine and sheep erythrocytes and guinea pig tissues revealed that this antigen was identical with or closely related to the previously described B antigen of the Paul-Bunnell (P-B) antigenic complex and distinct from heterophile antigens of Hanganutziu-Deicher (H-D) and of Forssman specificity. However, 2 of 89 syphilis sera without the P-B antigen were shown to contain antigen(s) of H-D specificity. None of the syphilis or the leprosy sera contained P-B antibodies, but H-D antibodies were found in 13% of syphilis sera.

Cryostat sections of methylcholanthrene-induced murine tumors were previously shown to adsorb erythocytes sensitized by subagglutinating concentrations of anti-erythocyte antibodies. This type of hemadsorption was seen also with sections of normal murine spleen. It was shown in the present study that immune complexes composed of bovine serum albumin (BSA) and rabbit anti-BSA serum, or human IgG and mouse anti-human IgG serum inhibit adsorption of sensitized erythocytes by tumor and spleen sections. Maximum inhibition was produced by complexes formed at excess of antigen. In contrast, inhibition was not seen with complexes formed at equivalence. In addition, heat-aggregated human IgG, but not monomeric human IgG, inhibited hemadsorption by tumor tissue. Anti-BSA, antihuman IgG, BSA or human IgG alone did not produce significant inhibition. These results support the contention that murine tumor cells and lymphocytes have similar Fc receptors on their surface. The implication that the nonspecific attachment of immune complexes to the tumor cells and lymphocytes may alter the immunological response of the host to the tumor is discussed.

The heterophile antigen (Paul-Bunnell antigen, PBA) of infectious mononucleosis was isolated by extraction of an aqueous suspension of bovine erythrocyte stromata with chloroform-methanol (2:1). The upper aqueous layer contained gangliosides, PBA, and a high-molecular-weight glycoprotein. PBA and gangliosides were separated from the high-molecular-weight glycoprotein by extraction of lyophilized upper layer with chloroform-methanol solvents. Separation of PBA from gangliosides was carried out by chromatography on DEAE-cellulose with chloroform-methanol solvents. PBA appeared to be a minor glycoprotein component of the erythrocyte membrane and had both hydrophobic and hydrophilic properties. It was soluble in either organic or aqueous solvents. On SDS-polyacrylamide gel electrophoresis, it migrated as a single component that stained for protein with Coomassie blue, for carbohydrate with periodic acid-Schiff reagent, and for lipid with oil red 0; it had an apparent molecular weight of 26,000. It was composed of 62% protein with major amino acids; glutamic acid, proline, glycine, isoleucine, leucine, and threonine (158, 116, 98, 90, 85, and 82 residues per 1,000 residues, respectively). Carbohydrate content was 9.2% with major sugar constituents: sialic acid, galactosamine, and galactose. Serologic activity of PBA was destroyed by pronase but not by trypsin.

It has been shown that a TAA termed rat CEA had the tissue distribution and physico-chemical properties similar to those of human CEA. In addition, it has been demonstrated that these two antigens shared antigenic determinants. These findings supported our contention that rat CEA and human CEA are analogous moieties. The production of CEA-specific autoantibodies and the induction of resistance to CEA-positive rat tumors after immunization of rats with extracts containing rat CEA raises the possibility that human CEA may be immunogenic in man. Treatment with heat, proteolytic enzymes and periodate oxidation revealed that rat CEA, similar to human CEA contained both carbohydrate and protein epitopes. The epitopes shared by rat and human CEA that were detectable by the monkey anti-human CEA serum appeared to be carbohydrate, whereas the epitopes on rat CEA with which the rat mAb combined appeared to be protein, and those detected by the rabbit anti-rat CEA serum appeared to be carbohydrate, as well as protein. These studies also indicated that, although the rat, rabbit and monkey produced antibodies specific for rat CEA, the epitopes detectable by antibodies from one species appeared to be distinct from those detectable by antibodies from the other two species. These observations have important implications in studies on human CEA. Its use as a reliable diagnostic marker for malignancy hinges on the detection of tumor-specific epitopes on the molecule. Such epitopes have not yet been clearly identified by antibodies produced in foreign species. Indeed, our finding that the rat mAb to rat CEA bound only to tumor extracts and not to extracts of normal tissues, including intestinal tissues, suggests that human beings would be the most likely source of a tumor-specific antibody to human CEA. In future studies, the role antibodies play in immunity against CEA-positive tumors will be explored and attempts will be made to determine whether all of the serologically detectable epitopes on rat CEA can induce tumor resistance, or whether this activity is limited to epitopes detectable only by rat antibodies. This information could have important implications in the use of human CEA in the immunotherapy of malignancies.

An inhibition test of agglutination of sensitized erythrocytes by human or rabbit anti-antibody was established for the detection of circulating immune complexes. Anti-antibody is a previously described IgM serum factor which combined specifically with homologous IgG antibodies that underwent molecular transformation in reaction with their corresponding antigen. In testing in vitro -formed immune complexes by human or rabbit IgG antibodies, it was found that this test is capable of detecting immune complexes at antigen concentration as low as 2–3 μg/ml. After the addition of corresponding antigens, all 14 serum-sickness sera and all 9 chronic thyroiditis sera which contained precipitating antibodies inhibited human anti-antibody. IgG of normal human sera, heataggregated IgG, and repeatedly frozen and thawed normal sera gave negative inhibition tests. By means of this inhibition test, immune complexes were demonstrated in 35% of the systemic lupus erythematosus sera, 69% of the rheumatoid arthritis sera, 54% of the malaria sera, 22% of the postrenal transplantation sera, 22% of the liver disease sera, 32% of the leukemia sera, and 35% of the lymphoma sera. All 19 of the leprosy sera from India also gave positive results. None of the sera of 36 healthy staff members and only 3% of the sera of random blood donors gave positive results.

Serological interactions among sera of human renal graft recipients in double diffusion in gel were observed by chance. Antigen was detected in six of 127 recipients and antibody in two of 30 recipients tested. One of the six recipients carrying the antigen had also antibody in one serum sample. On the basis of the pattern of reactions observed, the hypothesis was expressed that the described antigen-antibody system had "pan" rather than "allo" character.

Short-term cultures of peripheral blood lymphocytes obtained from 20 infectious mononucleosis patients 2–4 weeks after the onset of the disease were studied for formation of heterophile antibodies. In studying pooled supernatant fluids of lymphocytes from three patients cultured for 3–20 days, lytic antibodies for red blood cells of bovine (BRBC) and sheep (SRBC) origin were demonstrated. These hemolysins were shown to be of IgM nature and Paul-Bunnell specificity. Subsequently, plaque-forming cell (PFC) assays were performed with lymphocyte cultures of 15 patients. Significant numbers (60–750/2 × 10 7 cells) of PFC secreting antibodies against BRBC were demonstrated in lymphocyte cultures of 12 patients. The number of PFC apparently reached its peak after 5 to 10 days of culturing. No or a very few PFC were observed in the lymphocytes that were not cultured or in lymphocytes cultured for 3 weeks or longer. Lymphocyte cultures prepared in a similar fashion from normal individuals or patients suffering from sore throat and submandibular lymphadenopathy of other than infectious mononucleosis origin did not produce PFC. Production of lytic zones by antibodies to BRBC secreted by PFC was inhibited by preincubation of lymphocytes of infectious mononucleosis patients with solubilized Paul-Bunnell antigen but not with other heterophile antigens, indicating that antibodies involved in the PFC formation are of Paul-Bunnell specificity. An increased number of PFC against BRBC were obtained in two of three lymphocyte cultures after cultivation with BRBC or solubilized Paul-Bunnell antigen.

Specificity of antibodies to Victoria strain of Newcastle disease virus (NDV) found in infectious mononucleosis (IM) and other pathologic sera was investigated by agglutination of NDV-modified human·red blood cells, as well as by immunodiffusion and enzyme immunoassay with various preparations of the virus. These studies clearly demonstrated that the NDV antibodies are distinct from P-B or H-D antibodies. The unexpected observation that guinea pig kidney (GPK) tissues absorbed NDV antibodies allowed their classification into a group of ‘GPK-positive’ heterophile antibodies. The simultaneous occurrence of the NDV antibodies and H-D antibodies in IM and other diseases suggests the possibility that multiple new antigenic determinants, especially those of carbohydrate nature, may appear due to the alteration of self-antigens as a result of various pathologic processes.

Antibody titers to varicella-zoster (V-Z), herpes simplex (HSV) and vaccinia viruses were determined in sera collected in Buffalo, NY, from patients with multiple sclerosis (MS), patients with other diseases, and normal individuals. The mixed agglutination test employing cell cultures infected with one of the above viruses as a source of antigen was used to determine antibody titers. The geometric mean titer (GMT) for V-Z in 59 MS cases was significantly higher than that observed in patients with other diseases and in normal individuals. The GMT for vaccinia was also higher in MS patients but the difference was not as great as for V-Z. No difference was observed in the titer of antibodies for HSV. Similar results were obtained for 51 MS patients compared to healthy controls matched for sex and age. Higher antibody titers for V-Z and HSV were observed in cerebrospinal fluids from MS cases than in those from non-MS CNS patients in Baltimore, MD. Antibodies to V-Z, HSV and vaccinia were detected in washing fluids from brain homogenates of MS cases.

Two cell lines secreting Paul-Bunnell antibodies were established by means of Epstein-Barr virus-induced immortalization of B lymphocytes from patients with infectious mononucleosis. Progressive loss of antibody production, most probably due to a shutdown of antibody secretion by individual cells was, however, observed. The first of the established cell lines lost its secretion ability after 10 weeks, and the second after 33 weeks of culture. Further improvements of the technique are necessary to stabilize the antibody secretion.

Thirty-eight human hematopoietic cell lines were studied for the expression of heterophile Paul-Bunnell (P-B) antigen by means of indirect immunofluorescence tests. Of 5 normal B-cell lines of Oriental origin, 3 showed very high and 2 moderate increase in number of cells stained for IgM after preincubation with infectious mononucleosis (IM) sera. In contrast, of 9 normal B-cell lines of Causasian origin, none showed high and only 2 showed moderate increase of cells stained for IgM. The remaining 7 cell lines did not show any such increase. Of 24 cell lines from patients with proliferative diseases of hematopoietic cells, 17 Caucasian, 6 Negro, and 1 Oriental, only 3 myeloma cell lines, all of Caucasian origin, showed significant increase of stained cells. Absorption experiments as well as mixed agglutination studies showed that antibodies in the IM sera combining with cell lines are of P-B specificity and not of any other heterophile specificity.

Double diffusion tests in gel were employed for studies of reactions between infectious mononucleosis sera and extracts of bovine, sheep and equine erythrocyte stromata. The extracts were obtained by ultrasonication of stromata prepared from trypsin-digested erythrocytes. The reaction with bovine stroma extract was composed, in many instances, of two lines. A single line was observed in reactions with sheep and equine stroma extracts. This line merged into a reaction of partial or complete identity with one of the lines formed with bovine stroma extract. Evidence was obtained that some infectious mononucleosis sera may contain heterophile (Paul-Bunnell) antibodies belonging to IgG class in addition to those of IgM nature.

Sera from patients who had received renal allografts were studied for the presence of circulating immune complexes by using platelet aggregation technique combined with density gradient centrifugation. A simple and highly reproducible modification of the platelet aggregation technique, employing the use of relatively small amounts of blood from pretested donors as the source for platelets, is described. Immune complexes were detected in post-transplantation sera from 3 out of 16 patients. The development of a persistent immune complex state as a consequence of grafting was concluded in one patient.

New Zealand rabbits were injected three times a week with 2 mg of mercuric chloride (HgCl2) (MC) to test the hypothesis that toxic agents release autologous antigens, generating antibody response and tissue injury. After 2 weeks of injections the rabbits developed anti-basement membrane antibodies binding to renal and extrarenal basement membranes and to the peri- and endo-mysium of skeletal muscles. Glomerular histology appeared normal. Membranous glomerulonephritis developed 1–2 months later with granular subepithelial deposits containing rabbit IgG and C3. Similar deposits were present in the basement membranes of other organs. Raji cell tests for immune complexes in the sera were negative in the initial stages and positive in the late stages of the disease. Radioactive MC was found in the cytoplasm of renal tubular, intestinal, and hepatic epithelial cells but not in glomerular deposits. Renal eluates and selected sera reacted with the basement membranes and the collagen matrix in normal or collagenase-digested sections of renal or extrarenal normal rabbit tissues. This reactivity was reduced or abolished by washing tissue sections in PBS or by absorption of renal eluates with whole kidney homogenates. The findings are consistent with the hypothesis that MC induces a biphasic disease characterized first by the production of antibodies to basement membranes and the extracellular collagen matrix and subsequently by antigen-antibody complexes formed in situ and in the circulation and presumably containing soluble polysaccharide components of the collagen matrix.

The multiple nature of heterophile, Paul-Bunnell (P-B) antigen has been suggested by our previous studies as well as those of others. In the present study, two distinct antigens of P-B specificity were defined by means of hemagglutination and immunodiffusion tests in agarose gel; one antigen (BS) was shared by bovine (BRBC) and sheep red blood cells (SRBC) and another antigen (B) was limited to BRBC. Both anti-BS and anti-B antibodies were shown to be present in sera of 106 patients with infectious mononucleosis (IM) whereas they were virtually absent from the sera of the vast majority of patients with diseases other than IM and normal sera. Absorption and agglutination inhibition tests demonstrated the presence of BS and B antigens on horse erythrocytes. Goat erythrocytes were also shown to possess BS antigen, however, B antigen was found on erythrocytes of some but not all individual goats. By means of the previously established procedure, BS antigen was extracted from stromata of BRBC, SRBC and erythrocytes of horse and goat, and B antigen from BRBC and erythrocytes of some goats. BS and B antigens were also extracted from bovine lymphocytes and murine thymus and spleen tissues but not murine erythrocytes.

In a previous communication, carcinoembryonic antigen (CEA) of rat, which is an analogue to human CEA, was demonstrated in gastrointestinal adenocarcinomas induced in inbred Fischer rats by injection of 1,2-dimethylhydrazine. Antibodies detectable by enzyme immunoassay were elicited in Fischer rats by immunization with a perchloric acid extract of the CEA-containing rat tumor, RCA-1, incorporated into Freund’s complete adjuvant. Specificity studies showed that activity of the rat antisera could be virtually abolished by inhibition with the tumor extract used at a concentration of 25 μg/ml. Inhibition by newborn rat tissues required extract at a concentration of 250 μg/ml. Extracts of normal adult tissues did not inhibit at these concentrations, but did inhibit at a concentration of 2,500 μg/ml. The results showed that rat CEA, though present in low concentration in normal adult rat tissue, is capable of eliciting an immune response in rats.

Immune complexes (IC) present in the glomeruli of rabbits with chronic serum sickness (CSS) and in patients with systemic lupus erythematosus (SLE), idiopathic membranous nephropathy (IMN), and acute poststreptococcal glomerulonephritis (PSGN) were analyzed by incubation with antigenic preparations. The efficacy of these preparations to dissolve IC was assayed by comparison of results of direct immunofluorescence tests performed with the kidney tissues before and after incubation with antigenic preparations. The FITC-conjugated antisera used in these tests were specific for IgG, C3, and – in the case of CSS – for the eliciting antigen, bovine serum albumin (BSA). During the acute proteinuric phase of CSS in rabbits, incubation of tissue sections with BSA alone led to complete dissolution of IC. In many rabbits with late phase proteinuria, however, tissues had to be incubated with both BSA and aggregated fraction II of rabbit serum. In all biopsy specimens from patients with IMN, and in some specimens from patients with PSGN and SLE, aggregated fraction II of human serum resulted in complete or incomplete dissolution of IC. On the other hand incubation of tissues with excess DNA in SLE or with streptococcal antigens in PSGN did not lead to dissolution of IC. These studies suggest significant participation of antibodies to aggregated immunoglobulins (i.e., rheumatoid factors or rheumatoid-like factors) in IC found in the above-mentioned diseases. Other antigen-antibody systems, however, may also contribute to the deposits in the glomerulonephritides studied.

IgG rheumatoid factors were demonstrated by enzyme immunoassay using, as antigen, goat antibodies to human serum albumin in the form of immune complexes. Elevated levels of IgG rheumatoid factor were noted in the majority of patients with seropositive rheumatoid arthritis but also relatively often in normal blood donors. Reactivity of IgG rheumatoid factor was in most instances inhibitable by IgG from various species, including man. Exceptionally, restricted specificity towards IgG from bovidae, was recognized.

By means of agglutination inhibition test, Paul-Bunnell (P-B) antigen was demonstrated in sera of patients with cancer (5%), lymphomas or leukemias (12.5%) and rheumatoid arthritis (RA) (1.4%) as well as in synovial fluids from RA patients (27%). In contrast, none of 124 normal human sera gave positive results. P-B antigen was also demonstrated in 3 of 12 perchloric acid extracts of cancer tissues and 2 of 11 saline extracts obtained at 100°C from spleens of lymphoma-leukemia patients. Extracts of apparently normal tissues from 16 individuals gave negative results.

A classification of heterophile antibodies is proposed, which is based on interactions with guinea pig kidney homogenate. The major groups of antibodies combining with guinea pig kidney encompass Hanganutziu-Deicher antibodies, Forssman antibodies, and antibodies to Newcastle disease virus. Antibodies which fail to combine with guinea pig kidney are primarily those of Paul-Bunnell variety.

Thermostable ethanol insoluble antigens (BE antigens) were identified that occur in all human tissues and human dispersed cells, but which are absent from normal human serum. In contradistinction to previously described organ-specific BE antigens, these antigens were referred to as non-organ-specific tissue antigens (NOST). Antisera obtained by immunization of rabbits with BE preparations of human organs had been selected for being devoid of any organ specificity and had been absorbed by BE preparations of pooled human serum. Such antisera could be used as reagents for detection of NOST BE antigens in pathological human sera. Inhibition of enzyme immunoassay proved to be a convenient procedure for these studies. Inhibition of 35% or more was only exceptionally noted in studying sera of normal subjects 20-40 years old, but inhibition exceeding 35% (positive results) was noted in 48% of sera from subjects at the age of 70 years or more. A high incidence of positive results was also encountered in sera of patients with end-stage renal disease (30%), renal graft recipients (18%), and in patients with lymphoma or leukemia (44%), but interestingly enough, no positive tests were noted in patients with lepromatous leprosy.

Prolonged survival of C57BL/6 (B6) mice bearing syngenic EL-4 leukemia cells resulted from immunization with irradiated EL-4 cells on the day of inoculation of live leukemia cells. No prolongation of survival was observed if the irradiated cells were injected 6 or 13 days after live cell inoculation. Protection also was observed in EL-4-bearing mice that were treated with cyclophosphamide (CY) rather than by immunization, however, the protective effects were observed only when CY treatment was instituted 6 days after inoculation of live leukemia cells. No protection was seen when CY was administered on the day of, or 13 days after inoculation of live leukemia cells. In fact, administration of CY on the day live EL-4 cells were inoculated appeared to enhance the lethal effects of the tumor. In mice that underwent combined treatment, i.e., immunization and CY, prolonged survival was seen only in the group that received combined treatment 6 days after inoculation of live leukemia cells. No protection was seen in mice receiving combined treatment on the day of or 13 days after inoculation of live leukemia cells. The role that suppressor T cells might play in the observed results is discussed.

In a patient with biopsy-proved membranous nephropathy (nephrotic syndrome) acute fatal renal failure suddenly developed. Autopsy revealed rapidly progressive glomerulonephritis superimposed on the previous membranous changes. IgG eluted from the kidney was shown by immunofluorescence technic to bind to the glomerular basement membrane of normal human and monkey kidneys, and was capable of producing an acute anti-glomerular-basement-membrane nephritis in two monkeys. The IgG antibody, by radioisotopic methods, was specifically directed against primate kidney. It is postulated that immune complexes responsible for the original membranous nephropathy induced release of antigenic glomerular-basement-membrane fragments into the circulation, stimulating formation of antibodies to the membrane and producing fatal acute glomerulonephritis. (N Engl J Med 290:1340–1344, 1974)

Enterobacterial common antigen (ECA) has attracted considerable interest since the original publication by Kunin in 1962. In the present study we demonstrated this antigen directly in the urine from patients with urinary tract infections (UTI) elicited by enterobacteria. Sheep erythrocytes were incubated with UTI urine; this resulted in their coating with ECA, which was studied by means of hemagglutination by anti-ECA serum. Test tube hemagglutination and the more simple slide hemagglutination were employed and with both procedures similar results were obtained. Positive results were observed in 94–99% of urine specimens from enterobacterial UTI collected in The Buffalo Children’s Hospital in the 1960s. ECA in urine could also be demonstrated by hemagglutination inhibition. In this test, antibodies in anti-ECA serum were neutralized as a result of incubation of this serum with urine, and agglutination by the antiserum of sheep erythrocytes coated with a standard ECA preparation was prevented or reduced. By means of this latter test, ECA could be demonstrated in 67–88% of urine specimens from enterobacterial UTI. The possible diagnostic application of these tests has been discussed.

Gangliosides that possess the same sugar sequence but differing in the type of sialic acid (N-acetyl- or N-glycolylneuraminic acid) were compared for their reactivity with Hanganutziu-Deicher (‘serum sickness’) antibodies by double-diffusion gel precipitation tests. Only N-glycolylneuraminic acid containing gangliosides formed precipitation lines with Hanganutziu-Deicher antibodies, thus suggesting that Hanganutziu-Deicher antigens are gangliosides that contain N-glycolylneuraminic acid.

Sera of 198 patients with infectious mononucleosis (IM), which were obtained during the 3rd or 4th week of the disease, were studied for the presence of antibodies to BS and B antigens of the Paul--Bunnell (P-B) antigenic complex. Six of these IM patients had anti-B antibodies without or with very low-titer anti-BS antibodies and the remaining patients had both types of antibodies at high titers. These 6 IM patients would have been misdiagnosed as seronegative if the traditional P-B tests with sheep erythrocytes had been employed. BS antigen was demonstrated in a large amount in chloroform-methanol extracts of peripheral blood buffy coat and of erythrocytes obtained from 1 of the 6 patients during the 3rd week of the disease and right after recovery.

Cytolytic antibodies were tested in sera of C 3 H/HeHa mice grafted twice with solid syngeneic sarcoma or immunized with an MC-sarcoma converted into an ascitic form. These sera reacted with syngeneic tumor as well as with normal and malignant allogeneic cells. Absorption experiments suggested that these antisera contained two populations of antibodies. One population combined with antigen(s) shared by the MC-sarcomas of C 3 H/HeHa mice, by normal and malignant cells of C 3 H/St mice, and by normal and malignant cells of C 57 BL/ 6 mice. The other antibody population failed to react with cells of C 57 BL/ 6 mice but it reacted with C 3 H/HeHa MC-sarcoma and with normal and malignant cells of C 3 /St origin.