Serological detection of variable region (Vh) subgroups of Ig heavy chains.

Serological test systems were established for determining the heavy-chain variable region (Vh) subgroups of immunoglobulin heavy chains. Myeloma proteins with known Vh subgroups based on amino acid sequence were utilized as the primary basis of reference for analysis by hemagglutination and hemagglutination inhibition. Good agreement between the chemical and serological typing was obtained and nonoverlapping systems established for the three major Vh subgroups. In a survey of 167 myeloma proteins, all except two were exclusively positive in one of the three systems. The two exceptions may represent a fourth subgroup. There was an overall incidence ratio of 1:2:3 for VhI:VhII:VhIII subgroups. Some differences in the overall ratios were encountered within the immunoglobulin classes. Certain advantages of the serological typing antisera were discussed with special emphasis on their use for studies of Vh antigens on the membranes of lymphocytes.

Characterization of amyloid fibril proteins from medullary carcinoma of the thyroid.

Amyloid fibrils were studied from two different tissues of medullary carcinoma of the thyroid (MCT). The fibrils mainly consisted of a low molecular weight protein, AMCT, which was immunologically distinct and did not react with various antisera against known amyloid fibril proteins. A specific antiserum raised against the MCT amyloid proteins gave a reaction of identity with the degraded MCT amyloid fibrils from two patients, as well as with the isolated AMCT protein, but showed no reaction with other known amyloid proteins. The AMCT protein had a blocked N terminus, but the sequence analysis of a cyanogen bromide fragment revealed identity with human calcitonin in the 11 positions studied. Although the amino acid composition was similar, there were also distinct differences, and the mol wt of 5,700 daltons was considerably larger than that of calcitonin. For these reasons the AMCT protein may represent a prohormone of calcitonin.

Connective tissue origin of the amyloid-related protein SAA.

Protein SAA is a serum protein related to the major constituent of secondary amyloid fibrils, protein AA, and has been suggested to be a precursor of the amyloid protein AA. In the present study, the origin of SAA was investigated by studying human fetal tissues and cultured human fetal fibroblasts with the indirect immunofluorescence technique. Anti-SAA antibodies reacted strongly with cultured fibroblasts producing a fine fibrillary cytoplasmic staining and with extracellular fibrils in loose connective tissues and vessel walls. The reactions were specifically inhibited by absorption with degraded amyloid material, isolated protein AA, isolated protein SAA, and sera from patients with elevated levels of SAA. In contrast, no inhibition was seen with amyloid fibril material devoid of AA protein or by human sera in which SAA was not detectable by double-diffusion tests. These observations showed that SAA-like material is produced by fibroblasts and indicate that it is a normal constituent of developing extracellular connective tissue fibers.

Class, subclass, and allelic exclusion of membrane-bound Ig of human B lymphocytes.

Membrane-bound immunoglobulins of peripheral blood B lymphocytes and of lymphocytes from an IgG chronic lymphocytic leukemia showed restriction to one immunoglobulin class, one IgG subclass, and one Gm allotype. Certain antigens in the C-terminal part of IgG Fc were not exposed on the cell surface.

New, third class of amyloid fibril protein.

An unusual protein AR was isolated from the amyloid fibril preparation derived from a patient with primary amyloidosis. Protein AR was unique in its antigenicity, and revealed no structural identity with any known amyloid proteins or with immunoglobulin chains or fragments. Thus a new third class of amyloid fibril proteins besides the immunoglobulin light-chain variable region fragments and the nonimmunoglobulin protein AS, has been characterized. A component antigenically related to protein AR was found in the serum of the patient.

Characterization of an amyloid fibril protein from senile cardiac amyloid.

A protein, ASCA, is isolated from amyloid fibrils extracted from heart tissue of five different patients with senile cardiac amyloidosis (SCA). The proteins of all five patients showed immunological identity when reacted with an antiserum raised against one of the proteins. In contrast, no reaction was obtained with antisera against a variety of other amyloid proteins. The antiserum against the subunit protein of senile cardiac amyloid did not react with any other amyloid preparations tested, nor with extracts of normal heart tissue. Thus, the subunit protein appeared to be unique to senile heart amyloid. The protein could form fibrils in vitro, had a mol wt of about 6,000 daltons and the amino acid compositions investigated in two cases showed extensive similarities but were clearly different from that of protein AA of secondary amyloid fibrils.

Evidence for recent duplications among certain gamma globulin heavy chain genes.

A survey of a large number of human sera with the heavy chain genetic markers of the gamma-globulin system has revealed an unusual gene complex which is inherited as a unit through two different families. The gene complex involves two pairs of gammaG1 genetic markers which ordinarily behave as homoalleles, Gm(z) and Gm(f) for the Fd part of gammaG1 molecules, and Gm(a) and non-a for the Fc part. Isolation of the gammaG1 fraction from the unusual sera demonstrated the presence of the important non-a antigen in the gammaG1 fraction. Through the use of immunoadsorbents it was shown that these antigens were not part of a single molecule but that separate molecules were involved. The accumulated evidence indicated that the appearance of such homoalleles on the same chromosome probably resulted from a recent gene duplication, giving rise to two gammaG1 cistrons on one chromosome.

Amyloid-related serum protein SAA in endotoxin-induced amyloidosis of the mink.

The concentration of the amyloid AA-related serum protein (SAA) was markedly increased after endotoxin injections in mink, mouse, rabbit, and man. It was particularly studied during the development of endotoxin-induced amyloidosis of the mink. Protein SAA was markedly elevated in all mink 24 h after the first endotoxin injection but had fallen to relatively low levels before the onset of amyloidosis at about 4 wk. These results are consistent with SAA being a circulating precursor of the amyloid fibril protein, AA. However, both proteins may be derived by proteolysis from a common precursor.

FURTHER STUDIES ON THE gammaG-HEAVY CHAIN GENE COMPLEXES, WITH PARTICULAR REFERENCE TO THE GENETIC MARKERS Gm(g) AND Gm(n).

The recently described Gm (g) and Gm (n) genetic markers of the gammaG3- and gammaG2-subgroups of gamma-globulin were characterized in detail primarily through studies of myeloma proteins, their polypeptide chains and fragments. Antisera derived from rabbits, non-human primates and rheumatoid arthritis patients gave identical results. This contrasted with the Gm (b) system where the rabbit antisera react with a different genetic determinant (b(0)) than the sera from rheumatoid arthritis patients (b). The Gm (g) and Gm (n) antigens were detected both by precipitin analysis and by hemagglutination inhibition. The Gm (g) antigen was not associated with any of the other genetic antigens of the gammaG3-proteins which all belonged in the Gm (b) class. The genes for the latter were always allelic to the gene coding for Gm (g), with that for Gm (b(0)) constantly present when that for Gm (g) was absent. The Gm (g) and Gm (n) markers were of particular value in tracing the various gene complexes made up of the closely linked subgroup genes. Further support was gained for the concept that the different gene complexes of various population groups arose primarily through crossing-over. The Gm(g) and Gm(b) genes for the gammaG3-subgroup were extremely closely linked to those for the gammaG1-subgroup. However the Gm (n) marker indicated that the gammaG2-subgroup genes were probably further separated on the chromosome. Additional evidence was obtained for the gammaG2-gammaG3-gammaG1-order of the subgroup cistrons. Among the wide range of gene complexes a new type (gammaG2,-,gamma/G1) was described. This complex appeared to have a deletion of the gammaG3-cistron. Lower levels of gammaG3-globulin were found in the sera of the individuals with this gene in the heterozygous state. The possibility that this unusual complex arose through an unequal nonhomologous crossing-over is discussed.

Genetic variants of G4 globulin. A unique relationship to other classes of G globulin.

Two types of gammaG4 proteins, termed 4a and 4b, were characterized through antigenic studies of myeloma proteins. Both were recognized by specific antigens on the Fc fragment which were shared with other gammaG classes. The distinctive antigen of the common 4a type was shared with all gammaG1 and gammaG3 proteins but missing in those of the gammaG2 class; that for the rarer 4b type was selectively found in proteins of the gammaG2 class. Analyses on gammaG4 fractions isolated from normal sera showed that either the 4a or the 4b or a mixture of the two types was present in each serum. Evidence was obtained that these differences were on a genetic basis and that allelic genes linked to those of the Gm system were involved. Such a reciprocal occurrence in other classes of gammaG globulin of the antigenic markers distinguishing genetic variants has not been observed previously. A number of questions regarding the evolutionary development of the genes responsible are discussed. The possibility is raised that those for the gammaG4 class arose relatively early and preceded the development of those for the other gammaG classes.

Localization of amyloid-related serum protein SAA-like material to intermediate (10 nm) filaments of cultured human embryonal fibroblasts.

Further studies are presented on the intracellular localization of the amyloid-related serum protein SAA previously shown to be produced by embryonal fibroblasts. In cultured embryonal fibroblasts, the fine fibrillar cytoplasmic immunofluorescence obtained by anti-SAA was distinguished from that of microfilaments and microtubules. By using electron microscopy and cells treated with drugs known to specifically alter intracellular fibrils, SAA was localized to 10-nm intermediate size filaments. These filaments form characteristic perinuclear bundles upon treatment with drugs such as demecolcine or vinblastine which disrupt micotubules. The results indicate that SAA is a constituent of the intracellular cytoskeleton.

A serum component related to nonimmunoglobulin amyloid protein AS, a possible precursor of the fibrils.

A nonimmunoglobulin protein with the molecular weight of 9,145 (protein AS) has been shown to be a principal component of the amyloid fibrils in different clinical types of amyloidosis. A protein component, antigenically closely related to protein AS, was detected in human sera. The protein AS-related component (protein ASC) was found in the sera of many groups of patients, including 48 out of 55 patients with various clinical types of amyloidosis. No structural relationship of protein ASC to the plasma component of amyloid was found. Protein ASC was also present with high frequency in the serum of diseases known to be frequently complicated by amyloidosis. In some cases, ASC was found in the sera of patients 2-3 yr before the diagnosis of amyloidosis was established. Protein ASC was also frequently found in hypogammaglobulinemia. Among normal individuals, protein ASC was seldom detected in the serum by our techniques, but there was a noticeable increase with age and during pregnancy. Moreover, a more sensitive technique, immunoelectro-osmophoresis, revealed protein ASC in a higher number of sera from both patients and normal controls. Thus protein ASC was suggested to be a normal serum constituent, usually present only in minor quantities. Under certain conditions, protein ASC increases considerably in serum, and may in such instances act as a precursor for the deposition of amyloid fibrils in the tissues.

Rheumatoid factors isolated from patients with autoimmune disorders are derived from germline genes distinct from those encoding the Wa, Po, and Bla cross-reactive idiotypes.

To better understand the structural basis for rheumatoid factor activity, the nucleotide sequence of the light chain variable regions of nine human monospecific IgM rheumatoid factors were analyzed. Rheumatoid factors were isolated from three patients with rheumatoid arthritis, a patient with systemic lupus erythematosus, and a normal individual. The VL gene segments used by these rheumatoid factors are not as restricted as previous work on mixed cryoglobulin rheumatoid factors had suggested. Each of the different VK families is represented and there are two examples where a V lambda gene segment is used. Molecules with structures similar to those of the Wa and Po CRI, characteristic of mixed cryoglobulin rheumatoid factors, are not common among these rheumatoid factors isolated from patients with rheumatoid arthritis. While there are clear examples of rheumatoid factors that are direct copies of germline genes, most of the sequence data suggest that the processes of antigenic selection and somatic mutation contribute significantly to the generation of monospecific rheumatoid factors in patients with autoimmune disease.

Preformed ribozyme destroys tumour necrosis factor mRNA in human cells.

Maintaining RNA stability is a major problem in the delivery of preformed inhibitory RNA to target cells. In this study, we delivered a hammerhead ribozyme directed against tumour necrosis factor alpha into human promyelocytic leukaemia cells by cationic liposome-mediated transfection. Delivering a ribozyme in this manner reduced by 90% and 85% tumour necrosis factor alpha mRNA and protein, respectively. A modified ribozyme with a bacteriophage T7 transcription terminator at its 3' end was more stable than one lacking this sequence. This indicates that ribozyme stability can be improved by the addition of terminal sequences expected to protect against cellular nucleases.

Heterogeneous RF structures between and within healthy individuals are not related to HLA DRB1*0401.

To study variations in Rheumatoid Factor (RF) autoantibodies between and within healthy individuals, we have produced and analysed the variable heavy chain (VH) regions of 18 new monoclonal IgM RFs from the peripheral blood of two healthy subjects before and after immunization with tetanus toxoid (TT). The majority of these RFs used germline genes of the VH3 family, but RFs of the VH1, VH2 and VH7 families were also found. No RF of the VH4 RF is found. Fourteen different VH germline (GL) genes encoded the RFs, suggesting an extraordinary heterogeneity in structure. Consequently, changes in RF V region structures following immunization were difficult to identify. There were, however, structural differences between RFs from the two donors. RFs from one donor (IP) used more lambda light chains than RFs from the other donor and previously described RFs. In addition, 50% of the RFs from donor IP were encoded by GL genes frequently found to encode RFs from patients with Rheumatoid Arthritis (RA) but not described in RFs from other healthy subjects. The predominant use of VH3 RFs in the two healthy donors contrasts with the over-expression and expansion of VH1 RFs in one previously described healthy immunized donor. There are thus large individual differences in RF structures, which might be related to the immunological status, environment or genetic background of the donors. However, since these three donors are all HLA DRB1*0401, it is unlikely that this HLA type, associated with seropositive RA, accounts for the individual differences.

Localized laryngeal amyloidosis: partial characterization of an amyloid fibril protein AL.

Amyloid fibrils were extracted from a patient Wr with more than 10 yr history of localized laryngeal amyloidosis. Degraded amyloid fibrils reacted in immunodiffusion with an antiserum against an amyloid protein of immunoglobulin kappa light chain origin, showing a line of identity with a kappa I amyloid protein. The protein Wr had a blocked aminoterminal, previously only reported in lambda chains. Amino acid sequence analysis of a fragment of the protein showed it to be an immunoglobulin light chain protein of V kappa I or V kappa III subgroup. The protein had a few unusual amino acid residues as compared to other kappa light chains. The findings support the view that the fibrils in localized, tumour-like amyloidosis are composed by homogeneous immunoglobulin light chain proteins in the same way as is seen in primary and myeloma associated systemic amyloidosis. It is possible that unusual light chains are over-represented in amyloid fibrils.

Analysis of 'natural' and vaccine-induced haemophilus influenzae type B capsular polysaccharide serum antibodies for 3H1, a V3-23-associated idiotope.

The variable (V-) region repertoire of antibodies (Abs) to Haemophilus influenzae capsular polysaccharide (Hib PS) has been extensively studied in individuals vaccinated against the microbe, but to a lesser extent in subjects who generated such Abs in response to a 'natural' encounter with this microbe or its antigenic mimics. To gain an insight into the repertoire of Hib PS-reactive Abs in vaccinated and non-vaccinated individuals, we used a monoclonal Ab, 3H1, which detects an idiotypic marker associated with an Ab V-region gene, V3-23. We show here that Hib PS-reactive Abs with detectable 3H1 idiotope can be quantified by an indirect inimunoezymatic assay in serum samples of non-vaccinated healthy adults as well as of recently vaccinated healthy infants. The percentage of Abs that was simultaneously Hib PS-reactive and 3H1-positive ranged widely (from 0 to 68%) among individual serum samples from both groups of subjects. No dramatic differences in the expression of 3H1 idiotope on Hib PS-reactive Abs were found between vaccinated and non-vaccinated individuals. Our results are consistent with the hypothesis that the utilization of V-region genes in Hib PS-reactive Abs that individuals generate after a 'natural' encounter with Hib PS or its mimics is similar to that in these Abs elicited by Hib PS conjugate vaccines.

Effects of nonsteroidal antiinflammatory drugs on the immune network.

The immune system is extremely complex. It comprises many different types of cells and their products. In patients with rheumatic diseases the immune system is activated and has disturbed regulation. It is also believed that immune reactions are involved in the pathogenesis of these disorders. Nonsteroidal antiinflammatory drugs (NSAID) have therapeutic effects on rheumatic diseases. These effects can all be explained by inhibition of prostaglandin production locally in the diseased joints, leading to reduced inflammation. Little or no effects on the number of circulating lymphocyte subpopulations or on peripheral blood mononuclear cell immune reactions can be seen after treatment of patients with rheumatoid arthritis (RA) with NSAID. The possibility, however, exists that immune reactions locally in the diseased joints are modulated by NSAID secondary to reduced prostaglandin production.

Rheumatoid factor autoantibodies in health and disease.

Recent advances in molecular biological and human cell hybridization technology have significantly advanced the knowledge of mechanisms that underlie human rheumatoid factor (RF) production. These advances have provided insight into the etiopathogenesis of synovial inflammation and lymphocyte recruitment in rheumatoid arthritis (RA) joints. We have examined the mechanisms that lead to RF production in RA patients and those that regulate RF production in normals. The studies revealed structural features that distinguish RF produced in normals from those produced in RA synovial tissue. There are significant differences in the use of VL and VH genes between the two RF populations. Furthermore, IgV genes encoding synovial RF in RA have extensive evidence for nucleotide changes, leading to amino acid replacement in the complementarity determining regions (CDRs). In addition, RF produced in RA synovia show evidence for affinity maturation, isotype switch to IgG RF, and repertoire shift indicative of a continued recruitment of B cells. Together with computer modeling and crystallographic studies, our data suggest that the mechanisms that operate on RF selection in RA synovia are similar to immune responses to exogenous antigens. In contrast, RF established from human immunized donors (HID) are characterized by a very low ratio of replacement to silent (R:S) nucleotide changes in the CDR1+2. In addition, there is little increase in affinity with increasing numbers of mutations. There is thus evidence for regulatory mechanisms that limit affinity maturation of RF in normals.

V-gene repertoire and hypermutation of rheumatoid factors produced in rheumatoid synovial inflammation and immunized healthy donors.

We have compared RF from normal, immunized donors and RF derived from the synovial tissues of RA patients. We have found a difference in the preferential use of VL and VH gene families. In both conditions, RFs were found to have accumulated somatic mutations. However, there was a striking difference in the patterns of mutation. RFs from normals were characterized by a very low R:S ratio in the CDR1+2, considerably lower than seen among the RARFs. In addition, there was little increase in affinity with increasing numbers of mutations in a group of clonally related RFs from an immunized normal. This contrasts with RF from RA, where there is evidence of both affinity maturation and class switching. Together these data suggest that in healthy persons there is a controlling mechanism to limit the affinity of RF autoantibodies, and that this is lost in RA. The higher affinity of the RA-derived RF may be of significance in the pathology of the disease.