Antigenic relations of Rickettsia prowazekii and Rickettsia canada, established in the study of sera of patients with Brill's disease

Sera of patients with Brill's disease and of healthy persons with spotted fever in their past history were examined in the complement fixation reaction (CFR) to determine antigenic relations between R. prowazekii and R. canada. R. canada was found to have common antigenic determinants with R. prowazekii and R. mooseri. However, the antigenic determinants of R. canada differed from those of the mentioned rickettsiae. The titres of complement-fixing antibodies in the sera of patients with Brill's disease with the antigen of R. mooseri were lower than the titres with the homologous antigen within the range of 1-2 twofold dilutions of the serum. However, the oscillations of the titres with the antigen of R. canada in the study of the same sera were expressed in 1-5 twofold dilutions. In serological identification of canada rickettsiosis, antigens of rickettsiae of the spotted fever group should invariably be included in the investigation of the sera.     

Antibodies to Yersinia enterocolitica serotype 3 in autoimmune thyroid diseases

The prevalence of increased titres of antibodies to Yersinia enterocolitica (serotype 3) has been studied in sera from patients with various thyroid diseases. In contrast to the low prevalences of the antibodies in healty subject (24.3%), titres (greater than 10) of anti-Yersinia enterocolitica (anti-Yersinia) were found more frequently in patients with thyroidal disorders, especially in Graves' disease (70.0%). Furthermore, high titres of the antibodies (greater than or equal to 160) were found only in patients with Graves' disease. There was no significant correlation between the titers of anti-Yersinia antibodies and those of anti-TSH receptor antibodies in sera from patients with Graves' disease. In seven individual samples of sera, the anti-Yersinia antibody titer was high before treatment, but the decrease in the anti-TSH receptor antibody titer following treatment was associated with a simultaneous decline in anti-Yersinia antibodies in all of them. A highly positive and significant correlation between the titers of anti-TSH receptor antibodies and anti-Yersinia antibodies was obtained in each of them. These findings could be merely a reflection of the measurement of the cross-reaction of anti-Yersinia antibodies with anti-TSH receptor antibodies but the possibility of an association between Yersinia infection and the production of anti-TSH receptor antibodies in at least some patients with Graves' disease cannot be ruled out.     

Anti-immunoglobulins in multiple myeloma and benign monoclonal hyperglobulinemia]

Sera from 50 healthy adults, from 21 patients with multiple myeloma (MM) and from 11 patients with benign monoclonal hyperglobulinaemia (BMH) and from 28 patients with sarcoidosis were examined for the presence of anti-IgG-activity by passive haemagglutination technique. 62% of healthy adults (titre less than 2) and all of the sera from patients with MM and BMH (titres 32-512) as well as 42% of the sera from sarcoidosis patients were found to be anti-IgG positive. The anti-IgG positive sera showed also anti-(Fab)2-activity (with the exeption of 2 sera from sarcoidosis patients). No significant differences could be found between anti-Ig activity in sera from MM patients with BMH. There was also seen no correlation of anti-Ig with the clinical course of the disease. After column chromatography we could detect the partially simultaneous presence of anti-Ig with anti-Fc-specificity (MW ca. 900,000) and with (Fab)2-specificity (MW 150,000 or less than 90,000). Antibody dependent cytotoxicity (using melanoma cell lines as targets) was significantly inhibited by the isolated anti-Ig-fractions with low molecular sizes (MW less than 90,000). From these results it seems possible that anti-immunoglobulins may play a role in the clinical course of the disease.     

Formation of Paul-Bunnell antibodies by cultures of lymphocytes from infectious mononucleosis.

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 × 10⁷ 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.     

Study of interaction between IgG and IgM antibodies against rubella virus by the immunofluorescence method.

In immunoglobulin fractions or after elimination of IgG by absorption the immunofluorescence test for rubella IgM antibodies is more sensitive than in whole serum. Blocking of IgM activity by IgG antibodies was eliminated when the time of incubation of the serum with virus antigen was prolonged. After prolonged incubation higher titres of rubella antibodies were also obtained in the IgM immunoglobulin fractions. Protein A in Staphylococcus aureus suspension effectively absorbs antibodies of IgG class. The IgM antibody titres in absorbed sera of patients infected with rubella were in some cases 2 to 4 times higher than in unabsorbed sera.     

Hanganutziu-Deicher antibodies in infectious mononucleosis and other diseases

An enzyme immunoassay (EIA) was developed for the detection of heterophile, Hanganutziu-Deicher (H-D) antibodies in sera of patients with infectious mononucleosis (IM) and various other diseases. The EIA with a high m.w. glycoprotein (HMWGP) isolated from bovine erythrocyte stromata was shown to detect H-D antibodies directly, in spite of higher titers of Paul-Bunnell (P-B) antibodies in the IM sera. Absorption and inhibition studies of IM sera demonstrated H-D specificity of the antibodies combining with HMWGP in the EIA. The H-D antibodies were found in sera of 56% Caucasians and 27% of Japanese suffering from IM. The vast majority of the H-D antibodies in IM sera was of IgM class. Sera of patients with various other diseases also gave positive results: rheumatoid arthritis, 22%; syphilis, 19%; cancer of the gastrointestinal tract, 13%; and lepromatous leprosy 9.7%. The incidence of positive results in control sera from apparently healthy subjects was less than 4%. Results of this study confirmed our previous observation that whereas P-B antigens appear in immunogenic form in only IM, the H-D antigen is expressed as an immunogen in various diseases including IM.     

A Study of Tuberculosis Antibodies by Bentonite Flocculation

Bentonite particles sensitized with concentrated human Old Tuberculin were used for detection of circulating antibodies to M. tuberculosis in human sera.There was no sharp distinction between the titres of patients with active and inactive tuberculosis, but progressively higher antibody titres accompanied increased severity of infection. The sera of 95.3% of the patients studied had a titre of 1:8 or higher. This level was considered to be the minimal significant titre indicative of the presence of metabolically active bacilli in old and new lesions. Tuberculin-negative controls and syphilitic sera were negative. A common zone of incidence in the 1:8-1:32 range was found where differentiation between tuberculin-positive healthy people and patients with tuberculosis was not possible; however, titres of 1:64 or more were found only in tuberculous patients. The antibody under study is heat-labile.     

Characteristics of in vitro colony formation by cells from haemopoietic tissues

Summary The characteristics of stimulation of colony formationin vitro from cells of mouse haemopoietic tissues has been briefly reviewed. Mouse kidney or embryo feeder cells, media conditioned by the cells from these tissues, normal or leukemic mouse sera, sera from leukemic or infectious mononucleosis patients, human urine and mouse embryo extracts are all sources of colony stimulating activity and their properties have been described. All sources of colony-stimulating activity produce clones of cells of the granulocyte series. In tritiated thymidine treated mice injection of preparations rich in colony-stimulating activity has been shown to produce a neutrophil leucocytosis and accelerate the rate of accumulation of labelled neutrophils in the blood. It is suggested that thein vitro assay can detect factors capable of stimulating granulocyte development.     

The titration of tetanus antitoxin III. A comparative evaluation of indirect haemagglutination and toxin neutralization titres of human sera

The tetanus antitoxin titres of 174 serum samples from healthy adults were determined by a standardization indirect haemagglutination test (IHA) and the conventional toxin neutralization (TN) test. The serum samples were titrated by the IHA test using glutaraldehyde-fixed and toxoid sensitized sheep erythrocytes before and after the treatment of the sera with 2-mercaptoethanol (2-ME). The IHA method has been found to be very sensitive and specific for the estimation of tetanus antitoxin in human sera. The IHA titres before the treatment of the sera with 2-ME were generally about four times higher than the TN titres and the correlation coefficient between these titres was 0.94. The IHA titres after the treatment of the sera with 2-ME were in good agreement with the TN titres and there was no statistically significant differences between the titres by the two methods. The tetanus antitoxin titres of 50% of the sera were below the minimum protective titres of tetanus antitoxin (0.01 IU/ml). In 19.5% of the sera the antitoxin level (IU/ml) ranged from 0.01 to 0.1, in 20.1% from 0.1 to 1.0 and in 10.4% from 1.0 to 10.0.     

The serodiagnosis of human hydatid disease: 1. The routine use of latex-agglutination and complement-fixation in diagnosis

The combined use of complement fixation (CF) and latex agglutination (LA) tests is reported on sera from 6328 patients with suspected hydatid disease; 191 were confirmed positive at operation ('known positives'). Results by LA are related to CF titres. Both tests were negative in 90% of specimens. Nine patients were subsequently found infected of whom 3 became positive in tests after operation. Of sera positive in both tests, 75% were from 'known positives'. The remainder were almost certainly from infected patients. Half the patients whose sera were LA positive/CF less than or equal to 1/4 were follow-up 'known positives' in whom CF titres had waned; 2 were early infections. Only 3% of the cases with an LA negative/CF titre of greater than or equal to 1/16 were 'known positives' and 6% where the CF titre was 1/8. The remaining CF results in the group were false positives and accounted for 1.2% of all sera tested. Findings show that a CF titre greater than or equal to 1/8 with positive LA indicates past or present infection; a negative CF test with positive LA usually indicates past infection; rarely, infection is present when a CF titre is greater than or equal to 1/8 and LA is negative. A rising CF titre and positive LA indicates current infection; reliable prognosis following treatment is given by CF.     

Analysis of circulating immune complexes (CIC) in tuberculosis: levels of specific antibody and antigens in CIC and relationship with serum antibody

Eighty sera from tuberculosis (TB) patients, 16 Indian and 10 American control sera were analyzed by ELISA for relative titres of antibody against mycobacterial antigens. Levels of specific antibody and mycobacterial Ag in circulating immune complexes (CIC) isolated from these sera were also studied. All these parameters were found to be elevated in TB sera as compared to control sera. Maximum increase was however noted in CIC specific antibody titres. A good correlation was observed between serum and CIC levels of specific antibody (r = 0.72) and between specific antigen (Ag) and antibody (Ab) levels within CIC (r = 0.64). In a few of the TB sera examined, CIC specific Ab contributed less than 1% to the Ab titres in sera. In order to examine the differences between different subgroups within TB patients, a statistical analysis of variance was performed. Sex of the patients had no effect on any parameter. Sputum-positive patients had significantly higher levels of CIC Ag and Ab than the sputum-negative patients, although no significant difference occurred in respect to serum Ab. All three parameters were significantly higher in patients on chemotherapy as compared to fresh untreated cases. The relevance of these observations to the development of a CIC-based immunodiagnostic assay for TB is discussed.     

Paul-Bunnell antigen in lymphoma and leukemia spleens.

Lymphoid cells obtained from spleens of patients with lymphomas or leukemias were studied for the presence of heterophile (Paul-Bunnell (P-B)) antigen. A mixed agglutination (MA) test was established utilizing monolayers of cells attached to poly-L-lysine-coated wells of plastic U plates. After incubation of the monolayers with infectious mononeucleosis (IM) sera, indicator cells, sheep, or trypsinized bovine erythrocytes were added. The results were assessed according to sedimentation patterns of the indicator cells on the monolayers. Positive MA reactions were shown to be due to specific binding of P-B antibodies to the corresponding antigens on the spleen cells. Positive results were obtained with 15 of 37 spleens from patients with Hodgkin's disease, 5 of 8 lymphoma spleens, 4 of 15 chronic myelocytic leukemia spleens and 2 of 4 chronic lymphocytic leukemia spleens. Only 2 of 25 spleens from patients with various other diseases and 1 of 26 apparently normal thymus specimens gave positive results. This study confirmed demonstration of P-B antigen in lymphoma and leukemia by means of absorption experiments, which was reported previously.     

The amphipathic membrane proteins associated with gangliosides: The Paul-Bunnell antigen is one of the gangliophilic proteins

Two amphipathic protein fractions soluble in organic solvents as well as in water have been isolated from the ganglioside fraction of bovine erythrocyte membranes by successive chromatography in chloroform-methanol mixture on DEAE-Sephadex, silicic acid, and α-hydroxypropylated Sephadex G50 (LH60) columns. These two fractions contained a similar low molecular weight protein but with distinctively different amino acid composition. One of these proteins has been characterized by having a strong Paul-Bunnell antigen activity and had a binding affinity to ganglioside. A similar protein without Paul-Bunnell antigen activity was isolated as the major ganglioside-associated protein.     

Studies of inhibitors of bone marrow colony formation in normal human sera and during a viral infection

Various methods are compared for removing inhibitors of bone marrow colony stimulating activity from the serum of normal individuals. The best method proved to be chloroform treatment of sera, which is easily performed on a large scale. Chloroform treatment had no effect in itself on the colony-stimulating factor (CSF) and no additional inhibitors were detected after such treatment. Inhibitors were characterized by preparative ultracentrifugation. After fractionation, most of the inhibitory activity was recovered in the light (LDL) and very light density lipoprotein (VLDL) fractions. Treatment with specific anti human β-lipoprotein serum removed most of the inhibitory activity. Increased CSF levels have been reported to accompany the acute phase of infectious diseases. Application of the chloroform treatment to sera from patients with mumps showed that the mean CSF in these sera did not differ significantly from that found in sera from normal individuals. High CSF levels in acute infections therefore seem to reflect variations in inhibitor levels rater than a true increase in CSF.     

Hematopoietic factors in graft-versus-host reaction

Graft-versus-host (GVH) reaction has a curious unsolved area in the immunopathogenesis and pathophysiology of the immunohematopoietic system, and GVH disease remains one of the major obstacles in clinical allogeneic bone marrow transplantation. T lymphocytes and T lymphocyte subpopulations are now recognized to be initiators of this GVH reaction and disease. Also, T lymphocytes are known to be accessory cells in the regulation of hematopoiesis, and produce a variety of lymphokines relevant to hematopoiesis. Admittedly, remarkable hematopoietic changes can be found in GVH reaction, but the cellular mechanisms underlying these changes are so complex they have yet to be fully elucidated. In fact, elevated serum levels of myeloid and erythroid colony-stimulating activities were found in mice suffering from GVH disease in which marked granulopoiesis and suppression of erythropoietic differentiation were seen. In addition, each granulocyte/macrophage colony-stimulating factor (GM-CSF) or burst-promoting activity (BPA) could be detected in sera from patients with GVH disease following allogeneic bone marrow transplantation. There seems to be at least two mechanisms involved in the control of hematopoiesis with either humoral or local environmental factor, probably via the T lymphocytes or T lymphocyte subpopulations activated by alloantigens or autologous non-T cells.     

Isolation of salmon pancreas disease virus (SPDV) in cell culture and its ability to protect against infection by the 'wild-type' agent

A Scottish salmon pancreas disease virus (SPDV) has been isolated and its optimum growth conditions determined. Although several fish cell lines have been tested, successful culture was achieved only with CHSE-214 cells. Cytopathic effects were observed after 5 days. The highest virus titres, calculated by microtitration assay, were reached at 15 degrees C. After 7-9 days post-inoculation, CHSE-214 cell supernatants contained between 10(7)-10(5) TCID50 ml(-1) The cultured isolate is chloroform- and pH 3.0-sensitive, and virions are 50-60 nm in diameter. These characteristics are similar to the Irish SPDV isolates. The culture isolate induced typical pancreas disease (PD) lesions in experimentally infected Atlantic salmon and convalescent fish were resistant to experimental infection with PD-infective kidney homogenates obtained by serial in vivo passages from a PD-infected farmed salmon (termed wild-type SPDV). Furthermore, fish immunised with the inactivated cultured virus were protected against a cohabitation challenge with the wild-type virus. Immunised fish sera showed virus-neutralising activity before challenge (7 weeks post-immunisation) and from 3-6 weeks post-challenge, when sera from non-immunised fish did not neutralise the virus. At 6 weeks post-cohabitation challenge, previously immunised fish had neutralising titres of up to 1:65. Following intraperitoneal (i.p.) challenge, immunised fish showed neutralising titres as high as 1:226 at 8 weeks post-challenge. Non-immunised fish injected i.p. with the wild-type virus developed serum-neutralising activity against the cultured isolate when sampled 8 weeks after infection, confirming an antigenic relationship between the wild-type and cultured virus. The results demonstrate that the tissue culture-adapted isolate of SPDV could be successfully used to protect against challenge by the wild-type virus and could therefore have potential use as an inactivated vaccine against PD.     

High titres of antibody to murine leukaemia virus in lymphocyte (Ly) alloantisera

The radioimmune precipitation (RIP) assay was used to examine the antibody titres against endogenous AKR murine leukaemia virus (MuLV) in a number of antisera to lymphocyte (Ly) alloantigens. The sera from normal donor and unimmunized recipient mice used in raising the alloantisera were also examined for anti-MuLV activity. It was found that all the antisera had high anti-MuLV titres and that in all but one case alloantigen immunization augmented the anti-viral titres. The degree of augmentation did not appear to be related to the anti-MuLV titre in the donor strain sera. Three I-region antisera were also examined for anti-MuLV antibodies and were found to have lower anti-viral titres than the Ly antisera even though immunization to I-region products greatly augmented the anti-viral titre. These results caution against the use of Ly antisera in characterizing the phenotype of lymphoid tumour cells without prior virus absorption.     

Heterophile antibodies to rabbit erythrocytes in human sera and identification of the antigen as a glycolipid

Normal human sera contain heterophile hemagglutinins to rabbit erythrocytes which are different from anti-B isoantibody and other heterophile antibodies such as Hanganutziu-Deicher antibody or Paul-Bunnell antibody. The antigen to this antibody was purified from rabbit erythrocyte stroma, and identified as pentaglycosyl ceramide, Gal(alpha 1-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)Glc-Cer.     

Smooth muscle autoantibodies in normal rabbit sera. Cross-reaction with human platelets as demonstrated by immunofluorescence.

27 normal rabbit sera were tested by indirect immunofluorescent technique. All sera stained smooth and skeletal muscle of human and mammalian tissues and human washed platelets. Two sera had ANA activity. These autoantibodies had low titres. Lysed but not intact platelets neutralized the rabbit SMA sera activity.     

High titres of antibody to murine leukaemia virus in lymphocyte (Ly) alloantisera

The radioimmune precipitation (RIP) assay was used to examine the antibody titres against endogenous AKR murine leukaemia virus (MuLV) in a number of antisera to lymphocyte (Ly) alloantigens. The sera from normal donor and unimmunized recipient mice used in raising the alloantisera were also examined for anti-MuLV activity. It was found that all the antisera had high anti-MuLV titres and that in all but one case alloantigen immunization augmented the anti-viral titres. The degree of augmentation did not appear to be related to the anti-MuLV titre in the donor strain sera. Three I-region antisera were also examined for anti-MuLV antibodies and were found to have lower anti-viral titres than the Ly antisera even though immunization to I-region products greatly augmented the anti-viral titre. These results caution against the use of Ly antisera in characterizing the phenotype of lymphoid tumour cells without prior virus absorption.