Immunoglobulins in NZB-BL mice. I. Serum immunoglobulin levels and immunoglobulin class of erythrocyte autoantibody.

Determination of serum immunoglobulin levels in NZB and normal mice has indicated that an elevation of the gammaM-globulin level occurs in NZB mice. This can be demonstrated in young NZB mice well before other autoimmune manifestations are observed. A slight elevation of gammaM-globulin was also observed in NZB hybrid mice. Erythrocyte autoantibodies were analyzed by the direct Coombs' test with specific antiimmunoglobulin reagents. Autoantibodies in NZB mice can be of all immunoglobulin classes, although predominantly of gammaG(1)-class. In (NZB x NZC)F(1) hybrid mice, although Coombs' positivity develops around the same age as in NZB mice, there is a greater restriction of the autoantibodies into gammaM- and gammaG(1)-classes. Preliminary attempts to quantitate immunoglobulins coating NZB red cells have shown that there are approximately 400 molecules of gammaM and 5000 molecules of gammaG-globulin per NZB mouse red cell.

B-lymphocyte heterogeneity: ontogenetic development and organ distribution of B-lymphocyte populations defined by their density of surface immunoglobulin.

The density of total Ig and of IgM, IgG1, IgG2, and IgA on the surface of adult murine splenic B lymphocytes was measured using the technique of rapid flow microfluorometry. In addition, the density of total surface Ig and of IgM on B lymphocytes derived from adult bone marrow, lymph nodes, and Peyer's patches, and from neonatal spleen was determined. Adult spleen and lymph node B lymphocytes are characterized by the presence of a large population of cells with a low-to-intermediate density of total surface Ig, which is seen as a peak in the fluorescence profiles when these cells are labeled with fluorescein-conjugated (F1) anti-Ig. This peak is not detected when neonatal spleen or adult bone marrow are examined; the development of this peak among spleen cells occurs during the first 4 wk of life. Although the characteristic fluorescence intensity peak is not seen when adult spleen cells are labeled with Fl anti-mu, changes in the density of surface IgM do occur during the first few weeks of life and are detected as a decrease in the frequency of cells which have relatively large amounts of surface IgM. The differences seen in the fluorescence patterns of adult spleen cells labeled with Fl anti-Ig and Fl anti-mu may be due to the appearance of IgD on the surface of mature splenic B lymphocytes. This is supported by the similarity of the fluorescence profiles of adult bone marrow cells stained with Fl anti-Ig and Fl anti-mu, as the latter population of cells is reported to lack surface IgD.

Demonstration of a blocking factor in the plasma and spinal fluid of patients with subacute sclerosing panencephalitis. I. Partial characterization.

Conflicting reports on the immune responsiveness of patients with subacute sclerosing panencephalitis (SSPE) have been reported. This report shows that the leucocytes from four SSPE patients exhibited strong sensitivity to both measles and SSPE virus preparations as measured by the macrophage migration inhibition test, mixed lymphocyte virus infected cell culture test, and the lymphotoxin assay. Earlier suggestions that a factor may be operating to suppress cellular reactivity are confirmed by the demonstration that the response of lymphocytes from SSPE patients could be blocked by the addition of SSPE spinal fluid or plasma. It was determined that the blocking factor was stable at -20 degrees C, heat labile at 56 degrees C for 30 minutes, trypsin and neuraminadase sensitive, and had a mol wt greater than 150,000 as determined by Sephadex G-200 gel chromatography. The blocking factor appeared to be specific for SSPE virus and did not block the response of lymphocytes to nonspecific mitogenic agents and other viral and bacterial agents.

Time-varying magnetic fields: effect on DNA synthesis.

Human fibroblasts have exhibited enhanced DNA synthesis when exposed to sinusoidally varying magnetic fields for a wide range of frequencies (15 hertz to 4 kilohertz) and amplitudes (2.3 X 10(-6) to 5.6 X 10(-4) tesla). This effect, which is at maximum during the middle of the S phase of the cell cycle, appears to be independent of the time derivative of the magnetic field, suggesting an underlying mechanism other than Faraday's law. The threshold is estimated to be between 0.5 X 10(-5) and 2.5 X 10(-5) tesla per second. These results bring into question the allegedly specific magnetic wave shapes now used in therapeutic devices for bone nonunion. The range of magnetic field amplitudes tested encompass the geomagnetic field, suggesting the possibility of mutagenic interactions directly arising from short-term changes in the earth's field.

Sequence of the immunodominant epitope for the surface protein on sporozoites of Plasmodium vivax.

Plasmodium vivax is one of the four malaria parasites that cause disease in humans. The structure of the immunodominant repeating peptide of the circumsporozoite (CS) protein of P. vivax was determined. A fragment of P. vivax DNA that encodes this tandemly repeating epitope was isolated by use of an oligonucleotide probe whose sequence is thought to be conserved in CS protein genes. DNA sequence analysis of the P. vivax clone indicates that the CS repeat is nine amino acids in length (Gly-Asp-Arg-Ala-Asp-Gly-Gln-Pro-Ala). The structure of the repeating region was confirmed with synthetic peptides and monoclonal antibodies directed against P. vivax sporozoites. This information should allow synthesis of a vaccine for P. vivax that is similar to the one being tested for P. falciparum.

Differential susceptibility of human IgA immunoglobulins to streptococcal IgA protease.

IgA protease, a proteolytic enzyme found in human saliva and colonic fluid, hydrolyzes human serum IgA immunoglobulins to yield Fab(alpha) and Fc(alpha) fragments. The enzyme is produced by organisms in the normal human microflora and can be purified from culture filtrates of the common human oral organism Streptococcus sanguis (American Type Culture Collection no. 10556). IgA protease is inactive against all other protein substrates examined including the other classes of human immunoglobulins. The role of this enzyme in affecting the function of the secretory IgA immune system is unknown. To further characterize and explain this unusual substrate specificity, the susceptibility of 31 human IgA myeloma proteins of both subclasses was investigated. 16 IgA1 and 15 IgA2 myeloma paraproteins were treated with enzyme and the extent of proteolysis was determined by cellulose actate electrophoresis, immunoelectrophoresis, polyacrylamide gel electrophoresis, and column chromatography. All IgA1 proteins were enzymatically cleaved to Fab(alpha) and Fc(alpha) fragments, but all IgA2 proteins were resistant, yielding no fragments after prolonged enzymatic treatment. N-terminal amino acid sequence analysis of the purified Fc(alpha) fragment of a single IgA1 paraprotein was as follows: Thr-Pro-Ser-Pro-?-Thr-Pro-Pro-Thr-Pro-Ser-Pro-Ser. Comparison of this sequence to that reported for the IgA1 heavy chain shows that the enzyme-susceptible peptide bond is a Pro-Thr in the IgA1 hinge region. The most likely explanation of the resistance of the IgA2 subclass to IgA protease is a deletion in the heavy chain which commences with the critical threonine of the susceptible Pro-Thr bond.

The identification of the subclasses of human IgG by analytical isotachophoresis.

A new method is described for demonstrating the individual subclasses of human IgG immunoglobulins by means of analytical isotachophoresis. Individual subclasses of IgG purified from sera of multiple myeloma patients have distinct mobility patterns. When a mixture with purified IgG for each of the 4 subclasses is analyzed, a characteristic mobility pattern with 4 unique peaks is obtained. An IgG of unknown subclass can be identified by the superimposition of the peak for the unknown upon one of the 4 well characterized peaks. The method using microgram quantities protein in a 1.0 microliter volume can be performed easily in 15 min.

Use of monoclonal antibodies to quantify subclasses of human IgG. I. Development of two-site immunoenzymometric assays for total IgG subclass determinations.

Dissection of the IgG antibody response into its subclass components has been difficult largely because of the lack of adequate supplies of specific reagents. The development of monoclonal antibodies (Mcab) promises to overcome this problem, but the use of such antibodies has certain inherent problems. It has been shown recently that Mcabs which were avid, potent and specific for well defined epitopes may partially or completely lose their activity depending on the assay system in which they were used. In order to identify Mcabs that would be specific and useful as capture antibodies in a simple two-site enzymometric assay, a panel of 18 Mcabs was screened and one Mcab to each of the four IgG subclasses was identified for quantitation of subclass levels in human serum.

Gastric lavage: a simple method to obtain IgA-rich intestinal secretions from the rabbit.

A lavage procedure was developed to obtain intestinal secretions from rabbits. The procedure facilitated the repeated monitoring of the intestinal IgA immune response of these animals to enteric infection with Campylobacter jejuni. This non-invasive technique was easily performed, reproducible and yielded consistent levels of IgA from rabbit intestinal secretions. It is anticipated that this procedure will aid in the study of the intestinal immune response of rabbits to other enteric pathogens.

Immune response of guinea pigs to Schistosoma mansoni. I. In vitro effects of antibody and neutrophils, eosinophils and macrophages on schistosomula.

Schistosomula of Schistosoma mansoni were rapidly killed in vitro by IgG2 antibodies from serum of schistosome-infected guinea pigs and heat-labile factors present in normal serum. Addition of polymorphonuclear neutrophilic leukocytes greatly increased the rate and degree of killing. Eosinophils and macrophages did not increase the level of killing, though they did react with schistosomula already damaged or killed by antiserum. Neutrophils and eosinophils reacted with schistosomula only in the presence of specific antibody, while macrophages nonspecifically attacked dead schistosomula. Serum antibody levels reached a plateau at approximately 6 weeks after a single infection. Attempts to precoat schistosomula with antibody prior to exposure to complement were largely unsuccessful.

The development of anti-immunoglobulin antibodies in rhesus monkeys repeatedly exposed to Schistosoma japonicum.

The development of anti-immunoglobulin (Ig) antibodies in rhesus monkeys repeatedly exposed to Schistosoma japonicum cercariae was studied. Anti-Ig developed in all 8 monkeys exposed 5 times to cercariae of the Formosan strain, while none of 4 monkeys exposed once to the Philippine strain developed such antibodies in the same period. All monkeys developing anti-Ig had specificities for IgA, 6 of 8 for IgM and IgG, and 7 of 8 for rabbit Ig. The persistence of anti-Ig was greatly extended in the monkeys exposed initially to the Formosan strain and then challenged with the Philippine strain. A single monkey exposed once to the Philippine strain developed anti-IgA and anti-rabbit Ig 85 weeks postinfection. No relationship between host reaction to trapped eggs and the development of anti-Ig was discerned. The results suggest that immunization protocols designed for humans be carefully examined for their potential immunopathological side effects.

Immune response of humans to the circumsporozoite protein of Plasmodium falciparum: limited T cell response to the immunodominant central repeat region.

Most adults in highly malarious areas have antibodies to the repeat region of the circumsporozoite protein of Plasmodium falciparum. To determine if a T cell epitope on the repeat region stimulated T cell help for this antibody, we used R32tet32, a recombinant construct derived from the repeat region of the circumsporozoite protein of P. falciparum, to stimulate in vitro mononuclear cells from residents of an area hyperendemic for malaria. Three groups differing in the length of time they had resided in a malarious area were studied. The percentage of individuals in each group who had positive antibody responses to R32tet32 increased with increased exposure to malaria. However, antibody positivity was not correlated with in vitro lymphocyte proliferation responses to the antigen. Lymphocytes from 79% of the individuals showing serum antibodies to R32tet32 failed to respond in a lymphocyte transformation assay, suggesting that T cell helper activity in these individuals was based upon the recognition of a T cell epitope not located within this peptide.

A new protocol to digest human IgM with papain that results in homogeneous Fab preparations that can be routinely crystallized.

A method has been developed for the routine production of Fab fragments from human IgM in high yield. After the IgM is purified at physiological pH, it is digested with papain in the presence of cysteine at room temperature for 16 hours. The Fab fragments are purified initially by gel filtration and then by ion exchange chromatography. The yield of Fab has been 60-80%. Some heterogeneity in the size of the Fabs from the different monoclonal IgMs has been observed. Fab fragments from four different IgM rheumatoid factors (RF) have been crystallized after such digestion and purification, in a variety of conditions including phosphate buffer alone or with the precipitating agents ammonium sulfate, polyethylene glycol or methylpentanediol. This modified papain digestion method has also been used for another non-RF monoclonal human IgM with equally good yield. Biological activity can be detected in the purified Fab fragment indicating that this procedure does not destroy the native conformation of the molecule.

Characterization of lymphocyte inhibition by supernatants of crowded lymphocytoblasts.

Crowded lymphocytoblasts produce a factor (or factors) that inhibits the response of peripheral blood lymphocytes to mitogens. This factor is destroyed by pepsin in 1 hr but not by neuraminidase. No activity was lost when the factor was incubated with packed human red blood cells, human lymphocytes, mouse spleen cells, or tissue culture cells. An antiserum was made to the factor which was capable of partially inhibiting its activity. The factor is active at about 25 mug/ml of protein. Maximal inhibition occurs if the factor is present for the duration of the culture period. This factor may play a regulatory role in the immune response.

Comparative specificities of serum and synovial cell 19S IgM rheumatoid factors in rheumatoid arthritis.

Rheumatoid factor (RF) may play a role in sustaining the inflammatory events and tissue damage in rheumatoid arthritis (RA). However, many serum RF have greater specificity for rabbit IgG than for human IgG, thus raising questions about RF pathogenicity in RA. Serum RF also has specificity for human IgG subclasses 1, 2 and 4, but not for IgG3. The synovium is central to the pathology of RA; thus, RF made there may have greater pathogenicity than serum RF. We examined the specificity of 19S IgM RF in an RF plaque forming cell assay (RF-PFC) using RA synovial cells (RSC). We found that: (1) RSC produced greater numbers of RF-PFC/10(6) cells than did RA peripheral blood mononuclear cells (PBM); (2) RSC RF-PFC had greater specificity for human than for rabbit IgG compared to autologous serum RF; (3) RSC RF had significantly greater specificity for human IgG3 relative to autologous serum RF. In contrast, RSC RF and autologous serum RF had the same relative specificities for polyclonal human IgG, IgG1, IgG2, and IgG4. Thus, the specificity of much of the RF synthesized by RSC differed from serum RF. The potential pathogenic significance of these observations is discussed.