Antigenic specificity of chlorpromazine-induced antinuclear antibodies

A few patients have been reported who developed systemic lupus erythematosus (SLE) in the course of prolonged treatment with chlorpromazine. Patients on this drug have also been found to have antinuclear antibodies (ANAs) although they do not develop lupus.

We have studied the antigenic specificity of ANAs in fifty-four patients on longterm chlorpromazine treatment and compared our findings with those on 175 patients on anticonvulsants, 215 patients on isoniazid, 109 SLE patients and fifty-four healthy subjects, sex and age matched to the chlorpromazine patients.

Thirty-nine per cent of patients on chlorpromazine had ANAs which were most frequently directed to single stranded (s)DNA. In contrast, patients on anticonvulsants as well as those on isoniazid had ANA directed to soluble nucleoprotein (sNP) most frequently and none of the patients on isoniazid had ANA to sDNA.

The mechanisms by which chlorpromazine, isoniazid or anticonvulsant intake results in ANAs probably differ. Our findings suggest that development of ANAs in patients on chlorpromazine may be initiated by interaction of the drug with denatured DNA.

     

The range and specificity of antinuclear antibodies in systematic lupus erythematosus

Antibodies to nine calf thymus nuclear antigens were sought by complement fixation methods in twenty-four sera from sixteen patients with systemic lupus erythematosus. These antigens included whole nuclei, native and heat denatured DNA, particulate and soluble nucleoprotein and Sm antigen. Soluble antigens were also tested by tanned red-cell agglutination tests. A wide variation in the presence and titres of antibodies to these various antigens was found in the sera studied even when from the same patient but at different times. To further test the range and specificity of antinuclear antibodies in SLE, nineteen ribonucleosides, nucleotides and monophosphoric dinucleotides were coupled to human serum albumin and used as antigens in precipitin studies. A wide variation of reactivity was also found in each serum. Exquisite specificity became apparent, capable of reacting with a nucleoside but not with the corresponding nucleotide or vice versa, with a dinucleotide but not with the nucleotides or nucleosides which it contained, with a given dinucleotide but not with the opposite sequence.

Antinuclear antibodies in systemic lupus are, therefore, markedly heterogeneous. Those to a `single' antigen such as DNA may be directed to antigenic sites which may variously be at the bases, single or in sequence, at the site of union of base and sugar–phosphate moiety, at the backbone of deoxyribophosphate or actually dependent on the secondary structure.

     

Anti-ssDNA and antinuclear antibodies in human malaria.

The incidence of serum antinuclear antibodies and serum antibodies to single stranded (ss) and double stranded (ds) DNA was investigated following acute malaria in 58 Caucasians visiting tropical countries but resident in Britain and in 24 Ghanaians resident in Ghana. In Caucasians this infection was associated with a significant increase in the incidence of speckled antinuclear antibodies (38% compared to 3% in controls; P less than 0.001) and a significant rise in antibody levels against ssDNA (14% compared to 5%; P less than 0.05), but no rise in antibodies against dsDNA. Acute malaria in Ghanaians was associated with an incidence of 25% of antinuclear antibodies and 4% of antibodies to ssDNA; these were similar to those found in healthy Ghanaians who are chronically exposed to malaria. Antibodies against dsDNA were not detected. The incidence of antinuclear antibodies and levels of anti-ssDNA antibodies was higher in the Ghanaian healthy population than in normal Caucasians. These observations indicate that malaria is associated with the development of antinuclear and anti-ssDNA antibodies. Ghanaian patients with a tropical splenomegaly syndrome or with a nephrotic syndrome, both of which conditions are suspected of having a malarial aetiology, had serum levels of anti-ssDNA higher than healthy controls. This observation adds further circumstantial evidence to the role of malaria in causing anti-DNA antibodies.     

Occurrence, immunoglobulin pattern and specificity of antinuclear antibodies in sera of procaine amide treated patients

Thirty-one out of fifty patients receiving procaine amide were found to develop antinuclear antibodies belonging in most cases to the IgM and IgG classes and to a lesser extent to the IgA class. Specific interaction between these antibodies and a nucleoprotein component could be demonstrated in a large proportion of cases.No relation was found between occurrence of antinuclear antibodies and development of an immune response to procaine amide itself.     

Immunomorphological characterisation of antinuclear antibodies in chronic liver disease.

Two immunofluorescence procedures to evaluate antinuclear antibodies were compared in a series of 221 patients with chronic liver disorders of various aetiologies. The use of HEp-2 cells allowed us to discriminate with more confidence between the homogeneous and speckled patterns, to show the presence of associated patterns in the same serum, and, above all, to identify two specificities, unrecognizable on tissue sections. The anticentromere antibody was found in 10% of cases of primary biliary cirrhosis and occasionally in other conditions; the antibody staining multiple nuclear dots was strictly confined to primary biliary cirrhosis (17%). With the exception of autoimmune chronic active hepatitis the prevalence of antinuclear antibodies increased in all groups, particularly in primary biliary cirrhosis. Homogeneous antinuclear antibody was associated by both immunofluorescence procedures with autoimmune chronic active hepatitis. The multiple nuclear dot antinuclear antibody turned out to be an additional marker of primary biliary cirrhosis, helpful for the positive diagnosis of primary biliary cirrhosis in a proportion of cases negative for antimitochondrial antibody. Absorption experiments showed that multiple nuclear dot and antimitochondrial antibody are antigenically distinct. Moreover, multiple nuclear dot antinuclear antibody was associated with the finding of a dry Schirmer's test.     

Association of antinuclear and antinucleolar antibodies in progressive systemic sclerosis.

Antinuclear and/or antinucleolar antibodies were demonstrated in the sera of 74 of 76 patients (97%) with progressive systemic sclerosis, using tissue culture cells (HEp-2) as substrate in the indirect immunofluorescent method. Six patterns of nuclear staining and three nucleolar patterns were recognized. The nuclear patterns were centromere, fine speckles, coarse speckles, diffusely grainy, homogeneous and nuclear dots. The nucleolar patterns were speckled, homogeneous and clumpy. The results of digestion studies with ribonuclease, deoxyribonuclease and trypsin suggested that the nuclear antigens are proteins, some of which may be associated with chromatin. The nucleolar antigens appeared to be nucleic acid in nature. Certain characteristic serologic and clinical features associated with staining patterns were observed. The diffusely grainy pattern was seen only in sera containing antibody to Scl-70 antigen. Centromere staining was confirmed to be highly selective for the CREST (Calcinosis, Raynaud's phenomenon, esophageal involvement, sclerodactyly and telangiectasis) variant of progressive systemic sclerosis with rheumatoid factor titres higher in these patients with anti-centromere antibodies.     

Tissue specificity of anti melanoma monoclonal antibodies analyzed on cell lines

Twenty-eight monoclonal antibodies (MoAbs) from the NIH melanoma exchange program were analyzed in a binding assay for reactivity with a panel of 22 cell lines which included melanomas, carcinomas, fibroblasts, and lymphomyeloid cells. In this test, most MoAbs showed reactivity with a wide range of cell lines. The MoAbs were also tested for binding with freshly removed tumor cells and normal peripheral lymphocytes.     

Differential induction of lupus associated antinuclear antibodies in MRL mice by monoclonal anti-Sm antibodies.

The effect of monoclonal autoantibodies on immunoregulation was investigated in MRL/MpJ-lpr/lpr mice. Passive transfer of KSm2 (a monoclonal IgG2a antibody directed against the 16 kD polypeptide of Sm) induced IgG antibodies to the other major immunoreactive polypeptides of Sm (28 and 29 kD) in all mice studied, and to polypeptides of the closely related antigen nRNP/Sm in 63% of the mice. In addition an increment in IgG anti-dsDNA antibodies, and in IgA and IgM anti-Sm antibodies, over control levels was observed. These effects were not due to polyclonal activation since anti-histone antibody levels were unaffected. Two other IgG2a monoclonal antibodies: KSm5 (directed against the 28 and 29 kD Sm polypeptides) and OX 12 (directed against an irrelevant antigen) failed to modulate the autoimmune responses of the mice in any way. These results demonstrate specific antibody-mediated connectivity between B cell clones producing autoantibodies against three distinct antigens.     

Demonstration of antinuclear antibodies in mercuric chloride-induced glomerulopathy in the rat.

Serial administration of mercuric chloride to PVG/C rats induced a glomerulopathy associated with immune complex deposition along the glomerular basement membrane and in the mesangial area. These deposits could be demonstrated by immunofluorescence and electron microscopy 5 to 8 weeks after the first injection of mercuric chloride. At this time antinuclear antibodies could be demonstrated in the sera and in the eluates from the renal cortices from diseased animals. These findings suggest a possible role for antibodies directed against nuclear antigen in the pathogenesis of this type of experimental glomerular disease.     

Prevalence of positive antinuclear antibodies in healthy children

Antinuclear antibodies (ANA) frequently arise in the sera of children with connective tissue disease and is used in the diagnosis of these diseases. Therefore it is also important to know the prevalence of ANA in normal children. The main objective of the present study was to determine the prevalence of antinuclear antibody (ANA) in healthy children. Ninety-nine serum samples from a serum bank and 108 samples from patients who had attended elective surgery and whose blood had been withdrawn for other investigations, were tested for ANA by indirect immunofluorescence method using HEp-2 cells as substrate. Sera from 52 children with SLE were also tested during the same period. It was found that antinuclear antibodies were present in 32 (15%) of the 207 sera of healthy children at a dilution of 1:40 or higher. ANA were positive in 9% at a serum dilution of 1:40, in 3% at 1:80 and in 3% at 1:160. The patterns of immunofluorescence staining were as follows: homogeneous in 46.7%, speckled in 20%, and nucleolar in 10%. In SLE patients, ANA were positive in 91%; 13% at a serum dilution of 1:40, 7% at 1:80, 20% at 1:160, 15% at 1:320, 9% at 1:640, 20% at 1:1,280 and 9% at > or = 1:2,560. It was concluded that the prevalence of positive ANA using the HEp-2 cells as substrate was 15% in healthy children at dilutions of 1:40 or higher. Using the cutoff serum dilution of 1:40, the sensitivity of this test was 91%, the specificity was 85%, the positive predictive value was 57% and the negative predictive value was 97%.     

Tubuloreticular structures and antinuclear antibodies in autoimmune and non-autoimmune diseases.

Tissues obtained at random from patients suffering from autoimmune and non-autoimmune diseases were studied for the presence of tubuloreticular structures (TRS). It could be demonstrated that the occurrence of TRS in renal tissue is not a specific characteristic of systemic lupus erythematosus whereas the presence of such structures in skin tissue might be suggestive for this disease. The serum of some of the patients could be studied for the presence of antinuclear antibodies (ANA). A statistically significant correlation was found between TRS and ANA in the group of patients with autoimmune diseases. The possibility is discussed that this correlation might favour the theory that viruses may be involved in the aetiology of autoimmune diseases, particularly of systemic lupus erythematosus.     

Incidence of circulating antinuclear antibodies in cancer patients

The following study was conducted to know the incidence of antinuclear antibody [ANA] in various types of cancers in different age groups of both sexes. Results revealed that an overall level of ANA in female is higher than males. This study also showed that out of 50 only 20 cancer patients had raised level of ANA. Whereas in control group only one was positive for antinuclear antibody. Of these 20 positive cases, 4 were having very high titer of ANA, 13 showed high titer and 3 samples were moderately high titer of ANA. The high prevalence of autoantibodies found in aged cancer subjects could be attributed to several cellular and humoral immunological aberrations, which occur with the aging process. The results of this study confirm the earlier observation of necrosis of tumour tissue could be an important contributing factor for production of autoantibodies.     

Immunoglobulin G subclass of human antinuclear antibodies

Fourteen sera containing antinuclear antibodies were studied to determine whether the immunoglobulin G subclass of these antibodies correlated with the relative complement fixing activity of the antibodies or the presence or absence of lupus nephritis. Sera studied included eight with high complement fixing activity, all from patients with active lupus nephritis and six with low complement fixing activity, three from patients with lupus without nephritis and three from patients with antinuclear antibodies induced by procainamide. Purified antinuclear antibodies reactive with desoxyribonucleoprotein and desoxyribonuclease were prepared by elution, and subclass typing was carried out by radial immunoassay with specific antisera.In general, subclasses represented were similar in frequency to the occurrence of the subclasses in normal immunoglobulin G. There was no striking predominance or absence of any single subclass which might correlate with presence of nephritis, or differing complement fixing activity of sera. There was a trend to a predominance of subclasses high in complement fixing activity in antinuclear antibodies from lupus patients with active nephritis and subclasses low in complement fixing activity from those without nephritis.     

`Nuclear' antigens and antinuclear antibodies in mink sera

Aleutian disease of mink is transferrable by cell-free extracts and is characterized by hepatitis, vasculitis, nephritis, and hypergammaglobulinaemia. Because of increasing evidence incriminating antigen—antibody complexes in vasculitis disorders, the presence of nuclear antigens and antinuclear antibodies in mink sera was investigated.

Serum pools as well as individual serum specimens were obtained from uninfected mink, mink with experimentally induced Aleutian disease, mink with spontaneous Aleutian Disease, all of genotype Aa as well as from `normal' mink of similar age from colonies without Aleutian disease.

The serum pool from mink before and after experimentally induced Aleutian disease appeared to contain `nuclear' antigens detectable by rabbit anti-DNA antibodies in complement fixation and precipitin tests. The protein-free extracts of these serum pools gave strong reactions for deoxypentose in the diphenylamine tests. These serum pools also were shown to contain antinuclear antibodies by the immunofluorescence tests on human leucocyte nuclei and in precipitin tests against single strand calf thymus DNA. Sera from individual mink were similarly shown to contain `nuclear' antigens and antinuclear antibodies. The incidence and quantity of antigens and antibody detected were much greater in sera from mink after experimentally induced disease than in sera taken from mink before inoculation. The presence of `nuclear' antigens and antinuclear antibodies did not correlate with the degree of hypergammaglobulinaemia. Sera from `normal' mink in colonies without overt disease had neither antigens nor antibodies detectable in precipitin tests. Sera from mink with spontaneously acquired Aleutian disease had a high incidence of `nuclear' antigens and anti-DNA antibodies detectable in precipitin tests.

The `nuclear' antigens were detectable in Ouchterlony precipitin tests by specific rabbit anti-DNA antibodies. The precipitins formed lines of partial identity with those between the rabbit anti-DNA antibodies and single strand calf thymus DNA. However, the antigens in mink sera were not destroyed by prior incubation with DNAse which had been the case with DNA antigens detected in some human and mouse sera.

The antinuclear antibodies were detected in immunofluorescence tests using specific antibodies to mink γ-globulins, were shown to fix complement with single strand calf thymus DNA, but not with DNA that had been digested with DNAse, and formed precipitins with single strand calf thymus DNA which showed complete identity with precipitins formed by rabbit anti-DNA antibodies. Evidence for the simultaneous presence of `nuclear' antigens with antinuclear antibodies in the serum from mink with Aleutian disease was frequently evident. This observation is consistent with the hypothesis for the pathogenetic role of antigen-antibody complexes.

Aleutian disease of mink has certain clinical pathological and serological similarities with disease in New Zealand Black mice and in man with systemic lupus erythematosus. Since Aleutian disease of mink and disease of New Zealand black mice may both be examples of `slow virus' infections, a similar aetiology should be considered for certain autoimmune diseases of man, e.g. systemic lupus erythematosus.

     

The value of antinuclear antibodies in primary biliary cirrhosis

Objective

Although autoantibodies have been used for the diagnosis of primary biliary cirrhosis (PBC), their role has not been clarified. In this study, we try to explore the value of gp210 antibody and anti-centromere antibodies (ACA) in PBC.

Methods

Anti-gp210 and ACA were tested in 140 PBC patients by ELISA and indirect immunofluorescence respectively. Their association with clinical, pathological data and prognosis was analysed.

Results

30.5% of PBC patients had positive anti-gp210 antibody and 29.2% had ACA. The anti-gp210 antibody positive group had higher Mayo risk scores and lower serum albumin levels compared to the negative one. Patients with positive anti-gp210 antibody were more likely to develop hepatic failure (p < 0.05, OR = 9.8460, 95% CI: 1.067–90.901) than patients with negative anti-gp210 antibody. More patients with positive ACA developed portal hypertension than patients with negative ACA (p < 0.05, OR = 9.259; 95% CI: 1.027–88.410). Furthermore, concurrent Sjögren’s syndrome (SjS) and PBC was significantly more likely in the ACA positive group than in the negative ones (68.4% in ACA positive group, 20.7% in ACA negative group p < 0.001).

Conclusions

Both anti-gp210 antibody and ACA are related to severe disease course and poor prognosis. For PBC patients with positive ACA, further examinations should be made to detect underlying SjS.

     

Isoform specificity of commercially-available anti-TGF-beta antibodies.

The transforming growth factor-beta (TGF-beta) cytokine family has important and complex effects on many biologic processes. Mammals have three TGF-beta isoforms which differ in their primary amino acid sequence, receptor binding characteristics, distribution, and function. Characterization of TGF-beta production and localization is critically dependent upon appropriate reagents, including antibodies. We have analyzed the isoform specificity of eight commercially-available TGF-beta antibodies, including one monoclonal antibody and seven polyclonal antibodies. We carried out semi-quantitative Western blot analysis using recombinant TGF-beta1, beta2, and beta3 as targets. We found that sensitivity and isoform specificity are dependent in part upon the presence or absence of reducing conditions. The antibodies tested showed a broad range of sensitivity, with an ability to detect 50 pg to 20 ng. Cross-reactivity with another, incorrect isoform was seen with several antibodies, and ranged from 0.2% to 42%. Nevertheless, we identified TGF-beta antibodies directed against each isoform which provide moderate-to-high sensitivity and specificity when used in Western blot analysis. These results may have relevance for investigators who wish to detect particular TGF-beta isoforms with techniques other than Western blot analysis, particularly when these techniques involve denatured proteins.     

The specificity of nephritogenic antibodies. III. Binding of anti-Fx1A antibodies in glomeruli is dependent on dual specificity.

Rabbit antibodies against rat tubular brushborder antigens (Fx1A) give rise to in situ formation of immune aggregates along the glomerular capillary walls after intravenous injection into rats. These antibodies (anti-Fx1A), able to produce heterologous immune complex glomerulopathy (HIC) in the rat, have previously been shown to bind with brushborders (anti-BB) as well as with rat thymocytes (anti-T). In the present communication, this dual specificity was also demonstrated in antibodies eluted from kidneys of rats with HIC. It further appeared that, when the anti-thymocyte binding activity was selectively removed from these antibodies, using immunoabsorption with rat tissue extracts, these anti-Fx1A antibodies were no longer able to stain glomerular basement membranes (GBM) as demonstrated at the ultrastructural level using the peroxidase technique. Following perfusion of these antibodies in the normal rat kidney ex vivo, binding along the capillary walls was also below the detection level, in contrast to non anti-T depleted anti-Fx1A IgG. Biochemical analysis (including immunoblotting) showed that the anti-T moiety of anti-Fx1A was directed to a 90 kD component of Fx1A, since selective absorption of this specificity prevented staining of this 90 kD component. It is concluded, that this anti-T specificity within rabbit anti-Fx1A plays a crucial role in local immune complex formation in the rat kidney ex vivo. Whether this holds also for its role in the pathogenesis of HIC in vivo awaits further confirmation.