Serological specificity of IgG and IgM antiglobulin antibodies in anti-Gm(a) antisera

Serological study of a chromatographed Ragg anti-Gm(a) serum has shown differences in specificity of the IgG serum agglutinators and the IgM rheumatoid factor monospecific for Gm(a). The specificity of the IgG serum agglutinators is determined by the enzymatic modification of the globulin molecules and is unrelated to the Gm type.

Both serum agglutinators and anti-Gm(a) antibodies were demonstrable in a (heterologous) baboon anti-Gm(a) serum; however, both types of antibodies in this animal were IgG.

     

The relationship between rheumatoid factors and serum agglutinators

A detailed study of the DEAE fractionated serum R. W. [anti-Gm(x)] has revealed differences in specificity between the 7S serum agglutinators of erythrocytes sensitized with anti-Rh antibodies digested with proteolytic enzymes and 19S rheumatoid factor monospecific for Gm(x). The serological reactivity and specificity of the serum agglutinators is determined by the enzymatic modification of the globulin molecules and is unrelated to the Gm type. On the other hand, the 19S rheumatoid factor [anti-Gm(x)] is reactive with undigested molecules or with those antibody molecules digested with proteolytic enzymes which leave the Fc piece intact. The 19S factor specificity is otherwise unrelated to digestion of the globulin molecule and rests solely on the intactness of the Gm site for which it is specific.     

Cross-reactivity studies of horse, goat and rabbit anti-lymphocyte globulin

In the sera of ten normal humans and twenty-eight candidates for organ transplantation, the passive haemagglutination test detected a 50% incidence of preformed antibodies of low titre directed against horse serum. Such antibodies were also found to cross react with goat or rabbit sera in most instances. Seventeen of the organ recipients were later studied after the institution of treatment with horse antihuman-lymphocyte globulin (ALG). The incidence of anti-horse-serum antibodies rose to 100%. At the same time, an increased activity against goat serum developed; cross-reactions against rabbit serum were also demonstrated but to a less pronounced degree. With immunoelectrophoresis and Ouchterlony diffusion tests, it was shown that the cross-reactivity was to similar although not necessarily identical protein components of the different heterologous sera.

These data suggest that there is an inherent risk in switching from one ALG to another, particularly if horse and goat derivatives are used sequentially. Since rabbit ALG does not cross react so extensively with horse or goat serum it would be predicted to be a relatively safer second-line agent.

These expectations seemed to have been realized in six patients who were given more than one kind of ALG, always beginning with horse globulin. When goat ALG was administered second, anaphylactic reactions tended to appear early, but when the rabbit product was given second or third, it was relatively well tolerated.

     

Naturally occurring pepsin agglutinators in the serum of subhuman primates

Antibodies directed against both human and infrahuman pepsin digested γ-globulin were present in a majority of the primate sera tested. The subhuman pepsin agglutinators paralleled previously described human pepsin agglutinators in respect to their wide distribution in normal sera, their specificity and cross-reactivity, and their immunochemical features. The pepsin agglutinators† at different primate levels appeared closely related.

Among the subhuman pepsin agglutinators a subspecificity was described for a subhuman primate antigen. This finding suggested some limited differences between the subhuman pepsin agglutinators and the human pepsin agglutinators.

Experimental immunization of four cynomologous monkeys failed to elicit or alter these serum reactants.

     

Classification of Blood-Group Antibodies as β2M or γ Globulin

Thirty selected blood-group antibodies (excluding anti-A and anti-B) have been classified as β₂M (19S γ) globulin, γ (7S γ) globulin or mixtures, using the following three methods: fractionation on a DEAE-cellulose column; indirect anti-globulin tests, using specific anti-β₂M-globulin and anti-γ-globulin sera; and treatment with 2-mercapto-ethanol. With only minor exceptions, results obtained with the three methods were in agreement.Most blood-group antibodies within the Le, MNSs and P systems appear to be `naturally occurring'' and these were found to be β₂M globulin. Blood-group antibodies within the Rh, K and Jk systems, which had arisen after an antigenic stimulus, were usually γ globulin but were occasionally β₂M globulin.Antibodies composed of β₂M globulin usually behave as agglutinins but may behave as incomplete antibodies (e.g. some examples of anti-Jk^a); conversely, antibodies composed of γ globulin usually behave as incomplete antibodies but may behave as agglutinins (e.g. an example of anti-M).The ability to bind complement seems to be related more to the blood-group specificity of the particular antibody than to its molecular size. For example, anti-Jk^a, when composed either of γ or β₂M globulin, seems invariably to bind complement, whereas potent anti-M or anti-Rh, whether composed of γ or β₂M globulin, do not bind complement.     

Factors influencing bonding of bromelain agglutinators and their Fab fragments

The complex of bromelain agglutinators and their homologous Fab fragments is dissociated by gel chromatography under certain conditions. When albumin is present as a source of thiol groups, Fab fragments previously treated with N-ethylmaleimide (NEM) will dissociate from the agglutinators (fluid phase). If anti-Rh Fab fragments are bound to Rh-positive erythrocytes, the agglutinates are not dissociated by thiols (cellular phase). Prior to erythrocyte sensitization, the agglutinator site on Fab fragments can be blocked by thiol-disulfide exchange. Once the Fab fragments are coated on erythrocytes, the agglutinator site is more readily available than it was prior to sensitization, as evidenced by inhibition with 0.01 M NEM. The differences between the bonding characteristics of the fluid phase and the cellular phase and the influence of mercaptoalbumin on the agglutinator-Fab complex suggest that the agglutinators are not antibodies.     

Precipitation test for infectious mononucleosis and enterovirus diseases

An extract of bovine kidney obtained by trypsin digestion was used in double diffusion gel precipitation tests. Positive results were obtained with seven of twenty-seven infectious mononucleosis sera, five of twenty-five sera of patients with enterovirus diseases, and with only two of sixty normal human sera.

The precipitating factor was unrelated to heterophile antibodies detectable by sheep cell haemagglutination and ox cell haemolysis. On the other hand, the precipitating factor appeared to be closely related to the serum factor previously demonstrated by mixed agglutination reactions with bovine kidney cell cultures.

     

Heterophile antibodies indicate progression of autoimmunity in human type 1 diabetes mellitus before clinical onset

We previously reported serum cytokines in a group of long term non-progressors to Type 1 diabetes; this reactivity detected in ELISA is now identified as heterophile antibody in some sera. Here, we characterize heterophile antibody activity. A 14 kDa-polypeptide from heterophile antibody containing serum bound to an anti-IL-4 column, but IL-4 was not detected by Western blot or by MS/MS sequencing. However, in 2/13 heterophile antibody positive sera, T-cell growth was potentiated and was blocked by an anti-human immunoglobulin. To examine the relationship between low affinity heterophile antibody presence and disease progression, 1100 archived serum samples were analyzed with two pairs of antibodies from 443 diabetes-free first degree relatives of Type 1 diabetes mellitus patients for heterophile antibody; 95 individuals developed diabetes on follow-up. Twenty-two individuals, whose serum was heterophile antibody positive with the second pair of antibodies (but negative with the first pair of antibodies), had a significantly higher incidence of developing diabetes after five years. Thirty-seven individuals with heterophile antibody reactivity with the first pair of antibodies, regardless of reactivity with the second pair of antibodies, had a significantly lower incidence of developing diabetes. While we cannot exclude the presence of genuine cytokine in all sera, these data indicate the presence of distinct groups of heterophile antibodies in patients at high risk to develop diabetes. Thus, anti-Ig heterophilic antibodies with different immunochemical reactivities are linked to the progression of or protection from Type 1 diabetes autoimmunity.     

The influence of infection on titres of antiglobulin antibodies

The influence of acute and chronic infection and hypergammaglobulinaemia on the titres of anti-globulin antibodies was studied in 128 patients. Very high titres of serum agglutinators were found to be associated with gram positive septicaemia. This infection had no influence on the titres of rheumatoid factors. Elevated titres of serum agglutinators may be helpful in differentiating `benign' from `malignant' gammopathy.     

Occurrence and cross-reactivity of heterophile antibodies and anti-kidney antibodies in kidney transplanted patients and patients with renal disease

Increased titres of heterophile antibodies to rat erythrocytes occurred in twelve of twenty-seven patients after renal transplantation. In seven of these twelve patients the titre rise appeared to be associated with rejection. Heterophile antibody formation showed no consistent kinetic pattern after transplantation and no definite relationship between rise in antibody titre and rejection can be claimed. Patients with very high heterophile antibody titres were however prone to rejection.Heterophile antibodies to rat erythrocytes cross-reacted with human and monkey kidney cells and a subpopulation of these antibodies also with human B erythrocytes. The antibodies were not of the Forssman or Paul-Bunnel-type and their appearance could not be related to ABO or HL-A incompatibility. The heterophile antibodies, primarily of IgM class, are suggested to be produced in response to B-substance related antigens in Gram-negative bacteria and non-HL-A isoantigens.Approximately 35% of transplantation sera or sera from patients with kidney disease had IgG antibodies reacting with human and monkey kidney cells, human thyroid cells and A and B erythrocytes. Anti-kidney IgG antibodies in certain sera cross-reacted with rat erythrocytes. One-third of the patients with renal disorders had increased heterophile antibody titres.     

Studies on inherited antigenic variation of human serum β-lipoprotein by passive hemagglutination

Sera from 39 unselected children with thalassaemia who had received multiple blood transfusions were examined for the presence of isoantibodies to β-lipoprotein antigens. Precipitating antibodies were found in 8 sera. In 5 of these, additional non-precipitating antibodies were detected by the passive hemagglutination method. In another 7 serum samples, only non-precipitating antibodies were present. Thus, 15 out of 39 sera (38.5 %) contained isoantibodies to β-lipoprotein antigens. This frequency of antibodies to β-lipoprotein antigens in patients with thalassaemia is the highest ever reported for any group of multiply-transfused patients.
A total of 30 antibodies were found in these 15 sera.     

Physico-Chemical Characterization of the Cold Auto-Antibodies of Acquired Haemolytic Anaemia

Cold agglutinins were separated from the sera of eleven patients suffering from the cold-antibody type of acquired haemolytic anaemia by the dissociation of the specific antigen-antibody complexes. The electrophoretic mobility of the cold antibody was found to correspond to that of γ₁ globulin in each case.

Ultracentrifugal studies carried out on eluted cold antibodies prepared from the sera of seven patients demonstrated that the antibodies were macromolecules (S_20.w~18.1). In one instance the sedimentation coefficient of the cold antibody was demonstrated to be similar to that of electrophoretically separated `γ₁ globulin' prepared from the same serum, indicating their identical physical state.

The results of treating the sera of nineteen patients with the cold-antibody type of acquired haemolytic anaemia with mercapto-ethanol further confirmed that the cold antibodies were all macromolecules, whether derived from idiopathic cases or cases secondary to some other known disorder.

     

Management of cytomegalovirus antibody negative patients undergoing heart transplantation.

In a series of 61 consecutive patients undergoing heart, heart and lung, and lung transplantation, 24 patients were known to be cytomegalovirus (CMV) antibody negative on the day of transplantation. Enzyme linked immunosorbent assays (ELISA) for CMV IgG were performed on donor samples on the day of operation. In 16 of the 24 susceptible patients the test was negative and the only preventive measure taken was the use of blood and blood products from CMV-antibody negative blood donors. None of these patients acquired primary infection with CMV. In another six patients the donor serum was found to contain CMV specific IgG, and in these patients, including one heart and lung transplant recipient, prophylaxis with CMV specific hyperimmune globulin was given. All six patients developed CMV IgM antibodies and in five there was an associated but clinically mild illness. None of these patients required treatment. In the remaining two patients ELISA tests on the donor sera gave equivocal results and hyperimmune globulin was withheld. Both patients developed primary CMV infection of greater severity than those given hyperimmune globulin and one required treatment. Reference tests confirmed that the donor sera contained CMV antibodies. Primary CMV infection in susceptible patients after heart transplantation can be avoided by the use of screened blood and blood products where the organ donor is seronegative to CMV and it can be improved by the use of prophylactic hyperimmune globulin where the donor is CMV antibody positive.     

Detection of a Third Lymphocyte-Like Cell Type by Rosette Formation with Erythrocytes Sensitized by Various Anti-Rh Antibodies

Abstract. Assay systems with erythrocytes sensitized by three different human anti-Rh isoantibodies, anti-Rh Ri, Lor and 3360 have been compared for their sensitivity and specificity in detecting Fc receptor-bearing lymphocyte-like cells. When carefully standardized, all these test systems detected primarily the 'third lymphocyte-like cell type', different from B and T lymphocytes as detectable by conventional membrane markers.     

Presence of hepatitis-associated antigen in systemic lupus erythematosus

Presence of hepatitis-associated antigen (HAA) was investigated in 504 sera from 116 patients with SLE and was found in 41% of them.

HAA was present in at least one serum in 75% of the patients but there were variations in presence and titres in the same patient at different times.

Except for a tendency of HAA to appear or rise in titre with lupusi nactivation following corticosteroid or immunosuppresive therapy, there was no correlation between its presence and disease activity, specific organ involvement, antinuclear antibodies or immunoglobulin levels.

All but one of twelve lupus patients with recurrent bacterial infections had HAA at high titres. HAA appeared in the serum of a patient upon development of IgA deficiency.

HAA antigenaemia in systemic lupus erythematosus seems a consequence rather than a cause of the immunological derangement in this disease.

     

Biophysical and immunological studies on bovine immune globulins with evidence for selective transport within the mammary gland from maternal plasma to colostrum

Three immune globulins in maternal serum and colostrum and newly born calf serum, have been characterized and compared. An examination was made to determine first, which of the maternal serum immune globulins accumulate in the circulation of the calf and secondly, the selectivity of the mammary gland for these proteins compared with the intestinal mucosa of the newly born calf.

By difference in their electrophoretic mobilities three antigenically related immune globulins were isolated from bovine serum.

The immune lactoglobulins in bovine colostrum were qualitatively similar to those in serum. However, marked differences were observed between the relative concentrations in serum and colostrum of the three immune globulins.

An electrophoretically fast immune globulin (C1), present in colostrum at high concentration, was shown to be antigenically similar to an immune globulin (S1) present in the maternal serum at low concentration.

These findings indicate that the mammary gland showed a highly selective preference for, and hence ability to concentrate in, colostrum, the electrophoretically fastest serum immune globulin.

The slowest serum immune globulin and the component with intermediate electrophoretic mobility (S3 and S2 respectively) were both present at high concentration in bovine maternal serum, but were transmitted at different rates into the colostrum, so that the slowest serum immune globulin (S3) was present in the colostrum as a comparatively minor component (C3).

In contrast to the mammary gland, the intestine of the newly born calf (permeable to undegraded protein during the first 24 hours of life) showed no selectivity. Immune globulins showing the three electrophoretic mobilities were absorbed equally readily.

Thus, while the bovine mammary gland showed a highly selective preference for certain electrophoretically different serum proteins, no comparable selectivity was shown by the intestinal mucosa of the newly born calf.

The results emphasize the heterogeneity of bovine immune globulins and show that the calf receives into its circulation from ingested colostrum selected maternal serum immune globulins. This selection of proteins from maternal plasma, for admission to the calf's circulation, occurs within the mammary gland during the formation of colostrum but not during absorption across the calf's intestinal mucosa.

     

Naturally occurring anti-tissue antibodies in rat sera

Seventy per cent of normal rat sera have been shown to contain heat labile serum component(s) active against various rat organ homogenates as demonstrated by haemolytic complement fixation and passive haemagglutination tests. The main antigenic activity in rat liver has been found in the mitochondrial fractions.

It was also demonstrated by the indirect fluorescent antibody technique that both guinea-pig complement and high molecular weight rat globulins were fixed to rat organ sections.

Chemotactic activity has also been observed with rat serum and rat liver mitochondria and it is suggested that these naturally occurring antibodies may be implicated in the removal of tissue breakdown products.

     

Studies of the antibody nature of the rheumatoid factor: Reaction of the rheumatoid factor with sheep erythrocytes sensitized with human anti-sheep cell antibodies and with O Rh positive cells sensitized with incomplete anti-Rh antibodies

The reaction of the rheumatoid factor (RF) with 7S γ-globulin antibodies of nine persons immunized with sheep erythrocytes was studied. All of a panel of rheumatoid sera with high Waaler-Rose titres agglutinated sheep cells sensitized with the human anti-sheep cell antibodies and O Rh positive cells sensitized with the `diagnostic' anti-CD serum Ripley. The RF measurable with these systems could be absorbed to diphtheria toxoid—human antitoxin precipitate, whereas the absorption with egg albumin—rabbit anti-egg albumin precipitate did not result in any appreciable titre reduction. However, the eluate from the rabbit precipitate was highly active in these systems, whereas Rh positive cells sensitized with anti-Rh sera suitable for Gm(a) typing were not regularly agglutinated.

A markedly greater concentration of native than of heat-aggregated γ-globulin was needed for inhibition of the agglutination by the RF of cells heavily sensitized with the human anti-sheep cell antibodies. No appreciable difference in this respect was seen when using lightly sensitized cells. Only the heavily sensitized cells fixed complement. The `natural' 7S γ-globulin antibodies against sheep cells were neither suited for demonstration of RF nor did they fix complement.

Sheep cells coated with 7S γ-globulin antibodies of a Gm(a+) person were agglutinated by a non-rheumatoid anti-Gm(a) serum, and this system was well suited for Gm(a) typing, whereas cells coated with antibodies of a Gm(a-) person were not agglutinated. Rheumatoid anti-Gm(a) sera agglutinated cells sensitized with antibodies of both Gm(a+) and Gm(a-) persons. Using cells coated with Gm(a+) antibodies, some distinction between Gm(a+) and Gm(a-) sera could be obtained under carefully controlled conditions. The use of a Gm(a-) coat resulted in a slight difference in the inhibiting capacity, which had no relation to the serum's Gm(a) type.

The results suggest that the reaction of the RF with sheep cells heavily sensitized with human anti-sheep cell antibodies is essentially an interaction of the RF with immune aggregated γ-globulin, whereas when using lightly sensitized cells the individual γ-globulin molecules play a prominent role.

     

Some aspects of immunity in patients with cystic fibrosis

Various aspects of the immune status were examined in patients with cystic fibrosis (CF) and the following observations were made.

(1) Ten per cent of the CF patients had elevated or reduced serum IgG concentrations and there seems to be a transient low serum IgA concentration in the same number. Serum IgM concentrations were normal in CF patients. Normal levels of the three serum immunoglobulins (IgG, IgA and IgM) were detected in the siblings and heterozygotes.

(2) Serum IgE concentrations were elevated in 32% of patients with CF but normal concentrations were detected in heterozygotes and siblings.

(3) Precipitating antibodies to various antigens and allergens were detected in the sputum of most patients with CF, but their sera contained lower or undetectable titres of these antibodies.

(4) The precipitating antibodies in CF sputum to bacteria, to agar, to seminal fluid, to urine and to human serum may be related to a common antigenic determinant of the glycoproteins present in all these fluids.

(5) The total protein concentration in the sputum of CF patients seemed to reflect the extent of lung damage.

(6) The third component (C₃) of serum complement concentration was normal, or moderately elevated in all CF patients tested.

(7) Serum transferrin concentrations were normal in CF patients but their prealbumin levels were depressed, suggesting marginal undernutrition.

(8) Peripheral blood lymphocyte transformations with PHA and PWM appear to be normal in CF patients.

(9) Precipitins to the food antigens and the high percentage of positive sera with reticulin antibodies may be the result of malabsorption from the gut as well as a defect of the SIgA system.

(10) The presence of free J-chain in the sputum of patients with CF may be further evidence of SIgA structural abnormality.

     

Amyloidosis in neonatally thymectomized and anti-lymphocyte serum treated mice

A marked lymphocyte depletion and a striking impairment of their capacity to form antibodies against sheep erythrocytes was shown by outbred Swiss albino mice thymectomized or sham-thymectomized at birth and later treated with anti-lymphocyte serum.

Ninety-four per cent of allogeneic and 53% of heterogeneic skin grafts applied to the former, and 63% and 0% of those applied to the latter, survived up to the time of killing, i.e. 41 days after transplantation.

The remaining allogeneic and heterogeneic skin grafts were rejected by mice belonging to both experimental groups in a minimum of 18 days and a maximum of 35 days, which is much longer than is usually required by normal recipients (allogeneic grafts = about 10–11 days; heterogeneic grafts = about 7–8 days).

Despite the severe immunological depression caused by anti-lymphocyte serum treatment, either associated or not with neonaial thymectomy, all the mice chronically exposed to casein developed amyloidosis.

The present results are in accordance with previous findings indicating that mechanisms other than immunity may be involved in the pathogenesis of amyloidosis.