Experimental delayed type allergy without demonstrable antibodies: I. Absence of cytophilic antibodies

In guinea-pigs a delayed type allergy against sheep erythrocytes (SE) without detectable circulating antibodies (haemolysins) could be produced by immunization with erythrocyte—antibody complexes. Cytophilic antibodies against SE as measured by adherence of SE to macrophages could not be detected on the macrophages or in the serum of these animals as long as they had no haemolysins. Conversely skin tests were often negative in animals with cytophilic antibodies in serum. These antibodies do not seem to be involved in the allergic reaction.

Though cytophilic antibodies only occur in sera with haemolysin titres above 50–200 no constant relation between the titres of both antibodies was found in individual sera. Complete Freund's adjuvant stimulates the production of cytophilic antibodies but they are also produced by immunization without adjuvant.

     

Maternal immunity against avian influenza H5N1 in chickens: limited protection and interference with vaccine efficacy

After avian influenza (AI) vaccination, hens will produce progeny chickens with maternally derived AI-specific antibodies. In the present study we examined the effect of maternal immunity in young chickens on the protection against highly pathogenic AI H5N1 virus infection and on the effectiveness of AI vaccination. The mean haemagglutination inhibition antibody titre in sera of 14-day-old progeny chickens was approximately eight-fold lower than the mean titre in sera of vaccinated hens. After H5N1 infection at the age of 14 days, chickens with maternal antibody titres lived a few days longer than control chickens. However, only a low proportion of chickens with maternal immunity survived challenge with H5N1. In most progeny chickens with maternal immunity, high virus titres (>10⁴ median embryo infective dose) were present in the trachea during the first 4 days after H5N1 infection. In the cloaca, only low virus titres were present in most chickens. In 14-day-old progeny chickens with maternal immunity, the induction of antibody titres by vaccination was severely inhibited, with only a few chickens showing responses similar to the control chickens. It is concluded that high maternal antibody titres are required for clinical protection and reduction of virus titres after infection of chickens, whereas low antibody titres already interfere with vaccine efficacy.     

Expression of human–Torpedo hybrid acetylcholine receptor (AChR) for analysing the subunit specificity of antibodies in sera from patients with myasthenia gravis (MG)

The nicotinic AChR, a pentamer composed of α₂βγ(or ε)δ subunits, is the autoantigen in the human autoimmune disease MG. Anti-AChR antibodies in MG sera bind mainly to conformational epitopes, therefore determination of their specificities requires the use of intact AChR. Indirect antibody competition studies have suggested that most MG antibodies are inhibited from binding to AChR by MoAb to the main immunogenic region (MIR) on the α-subunits. More recently, based on the knowledge that MG antibodies show little detectable cross-reaction with Torpedo AChR, we have shown, using mouse–Torpedo hybrid AChR, that most MG antibodies that detectably cross-react with the mouse AChR bind to the α-subunit. To analyse the whole anti-AChR antibody repertoire in MG sera, we expressed on stably transfected fibroblasts a novel human α+Torpedoβγδ AChR and compared the antibody titres against human, Torpedo, and the hybrid AChR. Direct information was provided for the subunit specificity of several MoAbs and sera from 50 MG patients. On average, at least 48% of the anti-AChR antibodies in the sera were directed against the α-subunit. Interestingly, the anti-α-subunit antibodies predominated in low titre (0.6–7.4 nm) but not in high titre (10–386 nm) sera, where they comprised on average 68% versus 23% of the antibodies, respectively. Finally, the directly determined anti-α-subunit antibodies and the anti-MIR antibodies defined by antibody competition were significantly correlated, thus suggesting that at least a significant fraction of the anti-MIR antibodies in MG sera bind to the α-subunit.     

Antibody production by lymph-borne immunoblasts following subcutaneous injection into xenogeneic recipients

Efferent lymph was collected from individual lymph nodes of unanaesthetized sheep following stimulation of the nodes with a variety of antigens. At the height of the immune responses (i.e. 80–120 hours after stimulation) 15–30 per cent of the lymph cells were large, basophilic immunoblasts. Lymph cells collected during these times were washed and injected subcutaneously into small laboratory rodents (usually C57 black mice); each recipient received a single injection of lymph cells containing 1–2 × 10⁸ immunoblasts. Between 24 and 100 hours later the recipients were killed and exsanguinated. By the latter time substantial titres of specific agglutinating or lytic antibodies were demonstrable in the sera of the recipients and immunodiffusion and immunoelectrophoresis studies confirmed the presence in these sera of sheep immunoglobulins. Only trivial titres of antibody appeared in the recipients' sera when lysed lymph cells had been injected and no antibody or immunoglobulin was detectable following the injection of normal small lymphocytes from unstimulated or hyperimmune sheep.

An autoradiographic, electron microscope study showed that although many of the injected immunoblasts degenerated rapidly a significant proportion survived long enough to develop lamellar endoplasmic reticulum.

It was concluded that some of the injected cells survived long enough to synthesize and secrete specific antibody; on the basis of mercaptoethanol sensitivity it was concluded that both 19 and 7S antibodies were produced.

When sheep immunoblasts were injected subcutaneously into isogeneic or allogeneic sheep specific antibody appeared in the local tissue fluid and reached a relatively high titre some 50 hours after the injection.

     

The response of normal, thymectomized and reconstituted mice in contact sensitivity

An indirect immunofluorescence test for titration of IgM cytomegalovirus (CMV)-antibodies is presented as a sensitive and rapid method for the diagnosis of CMV-infection in adults. The development of these antibodies was followed in thirty-two serum samples from nine patients with a CMV-infection. No IgM antibodies were detected in pre-illness sera or in four sera, obtained as early as 1–7 days after onset of symptoms. Positive specimens were collected 11 days or more after the development of symptoms. After reaching a maximum level, antibody titres decreased gradually. In thirty-one control sera, obtained from a group of twenty-four individuals, either healthy, or suffering from other herpes-group virus infections, IgM CMV-antibody titres did not exceed 8. In this investigation, titres of 16 or greater were concluded to be evidence of recent infection. It is suggested that the testing of only one serum specimen can be sufficient to support the clinical diagnosis.     

Plasmodium falciparum-specific immunoglobulin G (IgG), IgM, and IgE antibodies in paired maternal-cord sera from east Sepik Province, Papua New Guinea.

Enzyme-linked immunosorbent assay (ELISA) and Western blot (immunoblot) serological analyses for immunoglobulin G (IgG), IgM, and IgE antibodies to Plasmodium falciparum were made from 46 maternal-cord serum pairs obtained from parturient East Sepik (Papua New Guinea) women and their newborn. Concurrent study of these women had shown that placental parasitemia rates were related to parity with the highest rate (41%) in the primiparous group and the lowest rate (3%) in the women who had given birth more than three times (> 3 parity group). Overall ELISA positivity rates for antimalarial IgG, IgM, and IgE antibodies in the maternal sera were 54.3, 28.2, and 8.3%, respectively, while those for the cord sera were 36.9, 0, and 16.6% respectively. Seropositivity rates were not related to maternal parity group, except for maternal IgE, in which there was a higher rate, of borderline significance, in the > 3 parity group than in the primiparous group. Cord IgE positivity was largely independent of maternal positivity and vice versa. Cord and maternal IgG immunoblot pairs showed near homology. IgG antibodies to the P. falciparum antigens of sizes < 36 kDa were either weak or absent in parity group 1 and 2 maternal-cord serum pairs. Neither ELISA or immunoblot revealed IgM antibody in the cord serum samples. Maternal IgM antibodies showed a heterogeneity of responses both between paired IgG immunoblots and between different serum samples. The IgE immunoblots exhibited a similar diversity, albeit of less complexity. The presence of P. falciparum-specific IgE in the cord sera would indicate that prenatal immune hypersensitization of the fetus to malaria had occurred.     

Passive Acquisition of Protective Antibodies Reactive with Bordetella pertussis in Newborns via Placental Transfer and Breast‐feeding

Although acquisition of anti‐pertussis antibodies by the newborn via placental transfer has been demonstrated, a subsequent recrudescence of pertussis infection is often observed, particularly in infants. The present study investigated the passive transfer of anti‐pertussis IgG and IgA antibodies to term newborns and their ability to neutralize bacterial pathogenicity in an in vivo experimental model using mice intracerebrally challenged with viable Bordetella pertussis. Forty paired samples of maternal/umbilical cord sera and colostrum were obtained. Anti‐pertussis antibodies were analysed by immunoenzymatic assay and by Immunoblotting. Antibody neutralizing ability was assessed through intracerebral B. pertussis challenges in mice. Anti‐pertussis IgG titres were equivalent in both maternal and newborn sera (medians = 1:225 and 1:265), with a transfer rate of 118%. The colostrum samples had variable specific IgA titres (median = 1:74). The immunoblotting assays demonstrated identical recognition profiles of paired maternal and newborn serum pools but different bacterial recognition intensities by colostrum pools. In the animal model, significant differences were always observed when the serum and colostrum samples and pools were compared with the positive control (P < 0.05). Unlike samples with lower anti‐pertussis titres, samples with high titres showed protective capacities above 50%. Pertussis‐absorbed serum and colostrum pools protected 30% of mice and purified IgG antibodies protected 65%. Both pooled and single‐sample protective abilities were correlated with antibody titres (P < 0.01). Our data demonstrated the effectiveness of anti‐pertussis antibodies in bacterial pathogenesis neutralization, emphasizing the importance of placental transfer and breast‐feeding in protecting infants against respiratory infections caused by Bordetella pertussis.     

Rheumatoid factor as a cause of positive reactions in tests for Epstein–Barr virus-specific IgM antibodies

Sera from twenty-eight patients with rheumatoid arthritis (RA) were titrated in indirect immunofluorescence tests for Epstein–Barr virus (EBV) specific antibodies. All had IgG antibodies to viral capsid antigen (VCA), 64% at titres [unk] 320, and 71% reacted also in tests for VCA-specific IgM antibodies at titres ranging from 20 to 640. The reactions observed in the IgM test were not due to VCA-specific IgM antibodies, however, but rather to rheumatoid factor (RF) usually an IgM antibody to the Fc regions of IgG. The titres recorded in the anti-VCA IgM test correlated significantly with the RF titres and both reactivities were abolished by adsorption onto IgG coated latex particles. In addition, they clearly depended upon the height of the IgG antibody titre to VCA, indicating that the more VCA-specific IgG molecules are present the more likely it is that RF will combine with them in sufficient quantity before or after their attachment to VCA-positive test cells so as to become detectable by the fluorescent antibodies to human IgM. Results comparable in every aspect were obtained with those sera from patients with Hodgkin's disease, nasopharyngeal or cervical carcinomas which reacted in the anti-VCA IgM test. Sera from patients with infectious mononucleosis may also contain RF, but in such cases its removal by adsorption onto IgG-coated latex particles did not generally reduce the VCA-specific IgM antibody titre. Removal of RF from any of the sera studied did not affect the titres of VCA-specific IgG and, where applicable, IgA or heterophil antibody titres. These results re-emphasize the pitfall created by RF noted previously in tests for virus-specific IgM antibodies.     

Placental Transfer of IgG and IgG Subclass Antibodies Anti-Purified Escherichia coli LPS O16, O6 and O111

We evaluated 22 paired maternal and cord sera regarding the presence of IgG and IgG subclasses against purified Escherichia coli LPS O6, O16 and O111 employing ELISA for titre and avidity analysis, isoelectric focusing associated with affinity-blotting for spectrotypic analysis, and the Western-blotting technique for recognition of the various bands in lipopolysaccharide (LPS). Levels of anti-LPS IgG antibodies in cord sera were equivalent to their respective maternal sera, showing a significant correlation ( P  < 0.0001). IgG1 antibody levels were higher in cord sera than in maternal sera ( P  < 0.005 for anti-O111, P  < 0.05 for anti-O16 and P  < 0.02 for anti-O6). Cord IgG2 antibody levels were not different from the maternal levels ( P  > 0.1). The levels of IgG3 and IgG4 were undetectable. The avidity of anti-O6 and anti-O111 IgG in 10 cord sera showed an extremely significant correlation with maternal antibody avidity ( P  < 0.0001). Identical patterns of recognition were found in the paired samples analysed by Western blotting. Most of the serum samples recognized the O-repetitive chains and also the region corresponding to core and lipid A. Although the antibody spectrotypes varied among individuals, paired cord and maternal serum samples showed identical patterns. Our findings suggest the occurrence of placental transfer of IgG antibodies against LPS O6, O16 and O111, mainly involving the IgG1 or IgG2 subclasses.     

Development of anti-tissue antibodies in rats

The sera of `normal' rats have been found to contain anti-tissue antibodies from shortly after birth. The antibody titres rose rapidly over the first 3 weeks of life reaching adult levels by the time the rats were 3–6 weeks old. Neonatal rats were rendered tolerant by exposure to bovine serum albumin (BSA). In contrast, higher levels of anti-tissue antibody were found in the sera of neonatal rats exposed to tissue antigens released by experimental liver damage than in the sera of littermate controls. It is suggested that the particulate nature of the tissue antigens may be associated with their ability to provoke an immune response in neonatal rats and that this in turn may be a factor hindering the induction of tolerance to these antigens.     

Immunological studies in ulcerative colitis. III. Incidence of antibodies to colon-antigen in ulcerative colitis and other gastro-intestinal diseases

The presence of anti-colon antibodies in sera from patients with ulcerative colitis was investigated with indirect haemagglutination and the fluorescent antibody procedure. The antigen was obtained from germ-free rats. There was a good correlation between the results obtained with both procedures.

Of a group of 101 ulcerative colitis patients, 56% were found to have a haemagglutination titre of ≥ 1:16. In a group of forty-five age- and sex-matched healthy controls or surgical cases 13% had such titres. This difference was statistically significant. In an additional control group of patients with bronchial asthma, the incidence of elevated titres was higher than in the healthy controls but lower than in ulcerative colitis. Of 109 patients with other gastro-intestinal disorders (chronic diarrhoeas of unknown aetiology, bacillary dysentery, Salmonella infections, cancer of colon and rectum, coeliac disease) only eight had a titre of ≥ 1:16. In fifteen South African patients with amoebic dysentery but of unknown clinical status, the incidence of elevated haemagglutination titres was also higher than in the healthy controls (33%). Of eighteen patients with regional enteritis, twelve (67%) had titres of 1:16 or higher. However, eleven out of these eighteen had lesions in both large and small intestine. Fluorescent antibody staining of rat colon sections confirmed these results.

In ulcerative colitis, the incidence of elevated titres (≥ 1:16) was independent of the patients' age but was significantly higher in females older than 25 years than in male patients of the same age. In patients younger than 25 years, this difference between the sexes was less marked and statistically not significant. Colectomy (including pancoloproctectomy) was without influence on antibody titres, which sometimes were found to be elevated as long as 2–10 years after surgery. There was no significant correlation between antibody titres and duration or severity of the disease, the extent of colonic involvement and the occurrence of extra-colonic manifestations.

     

Immunoglobulin class of antibodies to cow's milk casein in infant sera and evidence of low molecular weight IgM antibodies

Use of the red cell linked antigen—antiglobulin reaction with specific antiglobulin sera allows the recognition of the immunoglobulin class of the reacting antibody.In the youngest infants IgG was the only immunoglobulin reacting with casein. With the particular sera studied the antibody measured was probably maternal. In older infants IgA and IgM antibodies were also detected but they were usually present at lower levels than IgG antibodies.In serum from a boy with Aldrich''s syndrome the IgA level was very high and IgA antibody to casein (titre 128,000) was higher than the IgG antibody.In selected sera containing IgM antibody to casein, evidence is produced of a low molecular weight form of μ-specific antibody. In Sephadex G-200 filtration it was located in the `7S'' fraction containing IgG antibody.     

The use of cord serum and indirect viral immunofluorescence as a method of evaluating the immunological specificity of fluorescein-labelled anti-human IgA conjugate

Maternal and cord sera, obtained simultaneously, were compared by indirect immunofluorescence for the presence of IgG and IgA antibody specific for poliovirus, measles virus and herpes simplex virus. In paired maternal and cord sera virus-specific IgG is present in equal amounts; virus-specific staining by an anti-IgA conjugate was produced by maternal sera and not by cord sera. We believe that a comparison of virus-specific staining by a maternal serum with that produced by the corresponding cord serum using fluorescein-labelled anti-human IgG and IgA conjugates provides an excellent method of excluding cross-reactivity between an anti-human IgA conjugate and IgG.     

Discordance of Epstein–Barr Virus (EBV) specific humoral and cellular immunity in patients with malignant lymphomas: elevated antibody titres and lowered in vitro lymphocyte reactivity

The relationship between specific viral cellular and humoral immunity to the Epstein–Barr Virus (EBV) was investigated in thirty-one untreated patients with malignant lymphoma (ML) and sex- and age-matched controls. In vitro reactivity of peripheral blood lymphocytes to heatinactivated purified EBV, in an optimal stimulating concentration, was measured with ³H-thymidine uptake. In seropositive individuals EBV lymphocyte reactivity was positively related to EBV virus capsid antibody titres. In contrast, ML patients, especially those with Hodgkin's disease, showed markedly (P<0·01) impaired EBV lymphocyte reactivity in association with raised EBV virus capsid antibody titres. The disturbance of EBV lymphocyte reactivity was not related to the impairment of non-specific lymphocyte reactivity to mitogens and recall antigens in these patients. This study supports the hypothesis that depressed cellular immunity may cause raised EBV antibodies in ML, especially in Hodgkin's disease.     

The impact of timing of maternal influenza immunization on infant antibody levels at birth

Pregnant women and infants are at an increased risk of severe disease after influenza infection. Maternal immunization is a potent tool to protect both these at-risk groups. While the primary aim of maternal influenza vaccination is to protect the mother, a secondary benefit is the transfer of protective antibodies to the infant. A recent study using the tetanus, diphtheria and acellular pertussis (Tdap) vaccine indicated that children born to mothers immunized in the second trimester of pregnancy had the highest antibody titres compared to children immunized in the third trimester. The aim of the current study was to investigate how the timing of maternal influenza immunization impacts infant antibody levels at birth. Antibody titres were assessed in maternal and cord blood samples by both immunoglobulin (Ig)G-binding enzyme-linked immunosorbent assay (ELISA) and haemagglutination inhibition assay (HAI). Antibody titres to the H1N1 component were significantly higher in infants born to mothers vaccinated in either the second or third trimesters than infants born to unvaccinated mothers. HAI levels in the infant were significantly lower when maternal immunization was performed less than 4 weeks before birth. These studies confirm that immunization during pregnancy increases the antibody titre in infants. Importantly, antibody levels in cord blood were significantly higher when the mother was vaccinated in either trimesters 2 or 3, although titres were significantly lower if the mother was immunized less than 4 weeks before birth. Based on these data, seasonal influenza vaccination should continue to be given in pregnancy as soon as it becomes available.     

Serological investigations on Indian kala-azar.

Detailed serological investigations were carried out in forty-nine active kala-azar (KA) cases in North Bihar, India. Various classes of immunoglobulin (IgG, IgA, and IgM) and third component of complement (C3) levels were determined in these sera and results were compared with those obtained in normal controls. Antibody titres were determined by the indirect haemagglutination (IHA) method using soluble Leishmania antigen. Immunoglobulin G and M class-specific antibody titres were also determined separately by the enzyme-linked immunosorbent assay (ELISA) method. Polyclonal hypergammaglobulinaemia with marked increase in serum IgG (and to a lesser extent in IgM) level was a rather common feature in the majority of these sera. Much of this immunoglobulin increase, however, appeared to be non-specific in nature and no absolute correlation could be noted between serum IgG or IgM levels and corresponding IgG or IgM antibody titres. Significant decrease in serum C3 level was observed in KA and this decrease was found to be independent of immunoglobulin levels or specific antibody titres. A fairly good correlation between aldehyde test and serum IgG level was evident from this study. Aldehyde-positive KA sera usually gave higher antibody titres than aldehyde-negative ones. Anti-leishmanial antibodies belonged mostly to IgG class although some IgM antibodies were also demonstrable. The latter class of antibodies probably appeared early in KA infection although their serological specificity was poorer to IgG antibodies. Out of forty-nine KA sera examined in this study thirty-six (73.5%) gave positive IHA titres while forty-six (93.9%) were positive by IgG-ELISA which appeared to be a highly specific and sensitive serodiagnostic method particularly for the early detection of KA cases.     

An analysis of gastric parietal cell antibodies and thyroid cell antibodies in patients with pernicious anaemia and thyroid disorders

An analysis of immunoglobulins responsible for gastric parietal cell antibodies and for antibodies to thyroid cell has been performed in twenty-four patients with pernicious anaemia and thirteen patients with thyroid disorders. Immunofluorescent technique with conjugated antisera specific to each of the three main immunoglobulins and to β_1c-globulin was used.

IgG-globulin was responsible for gastric parietal cell antibodies in all patients investigated; IgA was found in gastric parietal cell antibodies of four thyroid patients but in none of the pernicious anaemia patients; IgM antibodies were found in two pernicious anaemia patients and in six thyroid patients. With regard to thyroid cell antibodies, IgG and IgA were both found responsible for antibody activity in thirteen cases and IgM in ten cases. In six patients, antibodies to thyroid cell were not β_1c-fixing, whereas all sera with gastric parietal cell antibodies fixed β_1c-globulin.

An obvious relationship could not be found either between the titres of the different types of antibodies within the same category of antibody, or between the titres of gastric parietal cell antibodies and thyroid cell antibodies in the same individual. An occasional relationship was found between the titres of IgG-parietal cell or thyroid cell antibodies and the capacity to bind β_1c-globulin.

These results demonstrate that each of the three main immunoglobulins may contribute to gastric parietal cell antibodies and to antibodies to thyroid cell.

     

Functional heterogeneity of antibody to penicilloyl coated erythrocytes

Human sera with anti-BPO (benzyl penicilloyl) specificity usually fail to lyse erythrocytes coated with BPO groups in vitro, when exposed to either guinea-pig or human C (complement). Of 350 haemagglutinating sera only seven caused lysis at levels 64–128-fold less than the corresponding haemagglutinin titres. However, of 100 sera which failed to cause lysis in vitro, twenty-one appeared capable of deviating part of the complement sequence as measured by assay of residual haemolytic C activity. Both BPO coated cells and alkaline PG solutions were effective antigens, the latter possibly as a result of polymerization which occurs readily in PG (benzyl penicillin) solutions.

Rabbit anti-human IgG sera caused haemolysis of BPO coated cells with adsorbed IgG antibody molecules. In these circumstances haemolysis was most marked with 2-mercaptoethanol reduced sera. Haemagglutination by IgM antibody in non-mercaptoethanol treated sera prevented haemolysis with anti-IgG and C, and this effect appeared to be related to the degree of aggregate formation, possibly by a steric hindrance effect of compact aggregates.

     

Presence of interferon and antibodies to BK virus in amniotic fluid of normal pregnant women

Paired samples of maternal sera, umbilical cord sera and amniotic fluids were tested for antibodies to BK virus (BKV) and for interferon content. It was found that two out of 14 pregnant women were antibody negative, the latter were estimated to be susceptible to BKV primary infection. In contrast, there was no foetal case among those which were at risk of foetal infection with BKV as judged from the presence of BKV antibody in maternal sera and interferon in the placentas. It might be hypothesized that BKV may be transmitted to the foetus only in the case of BKV primary infection of antibody-negative pregnant women with undetectable amount of interferon (IFN) in the placenta.     

The validity of the ammonium sulphate precipitation technique of estimation of antibody amount and avidity

Determinations of antibody titres with the ammonium sulphate precipitation technique showed an accuracy of ± 10 per cent (P<0.05). Calculation of antibody avidities with Sips' distribution formula, using suitably diluted antisera, gave a ten times greater accuracy than either undiluted or too highly diluted antisera. The accuracy was lower with the formula of Celada, Schmidt and Strom (1969) probably due to the narrow antigen ranges employed. Comparison of avidity calculation according to Sips (1948) and Celada et al. (1969) showed a good correlation (r = 0.73–0.79; P<0.01). No correlation between antibody titre and antibody avidity values in the sera (r = 0.03–0.19) was observed.