Antibody Production In Patients Research Articles

SAT0244 Correlation between circulating TFH cells and disease activity as well as autoantibody production in chinese patients with systemic lupus erythematosus

BackgroundPreviously we have shown that interleukin-21 (IL-21) level was tightly associated with anti-nuclear antibody (ANA) production in cultured peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE)...

Donor-derived HLA antibody production in patients undergoing SCT from HLA antibody-positive donors

Pre-existing donor-specific HLA antibodies in patients undergoing HLA-mismatched SCT have increasingly been recognized as a risk factor for primary graft failure. However, the clinical implications of the presence of HLA antibodies in donors remain unknown. We prospectively examined 123 related donors for the presence of HLA antibodies by using a Luminex-based single antigen assay. Of these, 1/57 (1.8%) male, 6/27 (22%) parous female and 0/39 (0%) nonparous female donors were HLA antibody-positive. Then, we determined the presence of HLA antibodies in seven patients who received SCT from antibody-positive donors. Of these, four became HLA antibody-positive after SCT. The specificities of the antibodies that emerged in the patients closely resembled those of the antibodies found in the donors, indicating their production by donor-derived plasma cells. Moreover, the kinetics of the HLA antibody levels were similar in all four patients: levels started increasing within 1 week after SCT and peaked at days 10-21, followed by a gradual decrease. These results suggest that donor-derived HLA antibody production frequently occurs in patients undergoing SCT from antibody-positive donors. Further studies are warranted for clarifying the clinical significance of donor-derived HLA antibodies, including the role of these antibodies in post transplant platelet transfusion refractoriness.

Contribution of an adenine to guanine single nucleotide polymorphism of the matrix metalloproteinase-13 (MMP-13) −77 promoter region to the production of anticyclic citrullinated peptide antibodies in patients with HLA-DRB1*shared epitope-negative rheumatoid arthritis

We examined whether matrix metalloproteinase-13 (MMP-13) contributes to disease susceptibility or severity of rheumatoid arthritis (RA). Eighty-seven patients with RA whose disease duration was <2 years and 71 healthy controls were enrolled in the study. Adenine (A) to guanine (G) single nucleotide polymorphism (SNP) of the −77 MMP-13 promoter region in RA and healthy controls was determined by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) technique. Human leukocyte antigen (HLA)-DRB1 genotyping was also performed using the same populations. Anticyclic citrullinated peptide (anti-CCP) antibodies from RA patients at entry were studied, and their relationships were examined. The genotype and allele frequency of SNP of MMP-13 at −77 did not differ between RA patients and healthy controls. We focused on the RA patients who were negative for HLA-DRB1*shared epitope (SE) alleles and found that the seropositivity of anti-CCP antibodies with a titer >25 U/ml was high in the A/A genotype compared with the G/G genotype. The same characteristic was also found in HLA-DRB1*0405 allele-negative patients. Our data suggest that SNP of the −77 MMP-13 promoter region acts as a surrogate marker of anti-CCP antibody production in HLA-DRB1*SE allele-negative RA patients, which may reflect RA severity.

Contribution of an adenine to guanine single nucleotide polymorphism of the matrix metalloproteinase-13 (MMP-13) −77 promoter region to the production of anticyclic citrullinated peptide antibodies in patients with HLA-DRB1*shared epitope-negative rheumatoid arthritis

We examined whether matrix metalloproteinase-13 (MMP-13) contributes to disease susceptibility or severity of rheumatoid arthritis (RA). Eighty-seven patients with RA whose disease duration was <2 years and 71 healthy controls were enrolled in the study. Adenine (A) to guanine (G) single nucleotide polymorphism (SNP) of the -77 MMP-13 promoter region in RA and healthy controls was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Human leukocyte antigen (HLA)-DRB1 genotyping was also performed using the same populations. Anticyclic citrullinated peptide (anti-CCP) antibodies from RA patients at entry were studied, and their relationships were examined. The genotype and allele frequency of SNP of MMP-13 at -77 did not differ between RA patients and healthy controls. We focused on the RA patients who were negative for HLA-DRB1*shared epitope (SE) alleles and found that the seropositivity of anti-CCP antibodies with a titer >25 U/ml was high in the A/A genotype compared with the G/G genotype. The same characteristic was also found in HLA-DRB1*0405 allele-negative patients. Our data suggest that SNP of the -77 MMP-13 promoter region acts as a surrogate marker of anti-CCP antibody production in HLA-DRB1*SE allele-negative RA patients, which may reflect RA severity.

Olf1/EBF associated zinc finger protein interfered with antinuclear antibody production in patients with systemic lupus erythematosus

IntroductionThe aim of the study was to determine whether Olf1/EBF associated zinc finger protein (OAZ), a transcription factor encoded by a positional systemic lupus erythematosus (SLE) candidate gene, plays a functional role in the pathogenesis in SLE.MethodsGene expression levels in peripheral blood cells (PBLs) measured using quantitative real-time polymerase chain reaction (qPCR) were assessed for association with disease activity and the presence of specific autoantibodies. Peripheral blood mononuclear cells (PBMCs) were incubated with specific siRNAs for three days, then cells were harvested for measuring mRNA levels using qPCR, and supernatants for levels of total immunoglobulin (Ig)G and IgM as well as secreted cytokines, chemokine and antinuclear antibodies (ANA) using ELISA. Indirect immunofluorescence was also applied for ANA detection.ResultsOAZ gene expressions in PBLs from 40 ANA-positive SLE patients were significantly increased than those from 30 normal controls (P < 0.0001) and 18 patients with rheumatoid arthritis (P < 0.01). In SLE patients, OAZ transcripts were positively correlated with SLE disease activity index (SLEDAI) score (r = 0.72, P < 0.0001) and higher in those positive for anti-dsDNA or anti-Sm antibodies (both P < 0.05). Co-culturing with OAZ siRNAs reduced mRNA levels of OAZ by 74.6 ± 6.4% as compared to those co-cultured with non-targeting siRNA and OAZ silencing resulted in reduced total IgG, ANA, interferon (IFN)-γ, interleukin (IL)-10, IL-12 and IL-21, but elevated CCL2 levels in culture supernatants (P < 0.05). The declined ANA levels correlated with inhibited OAZ expression (r = 0.88, P = 0.05), reduced IL-21 levels (r = 0.99, P < 0.01), and elevated chemokine (C-C motif) ligand 2 levels (r = -0.98, P < 0.01). Expressions of ID1-3 were significantly down-regulated by 68.7%, 70.2% and 67.7% respectively after OAZ silence, while ID3 was also highly expressed in SLE PBLs (P < 0.0001) and associated with disease activity (r = 0.76, P < 0.0001) as well as anti-dsDNA or anti-Sm antibodies (both P < 0.05).ConclusionsElevated expression of OAZ transcripts in SLE PBLs were strongly correlated with disease activity. Suppression of OAZ expression inhibited downstream ID levels, and secretion of ANA and IL-21, implicating a role of OAZ pathway in the pathogenesis of SLE.

Expression and immunogenicity of NY‐ESO‐1 in hepatocellular carcinoma

The present study was designed to investigate the expression of and humoral response against NY-ESO-1 in patients with hepatocellular carcinoma and to analyze the relationship between expression of NY-ESO-1 mRNA and clinicopathological features. NY-ESO-1 mRNA and protein expression in surgically resected hepatocellular carcinoma specimens, adjacent non-cancerous liver and non-tumor bearing liver were examined by reverse transcription-polymerase chain reaction and immunohistochemical staining using a monoclonal antibody against NY-ESO-1 (ES121), respectively. The antibody response to NY-ESO-1 was examined by enzyme-linked immunosorbent assay using recombinant NY-ESO-1 protein. NY-ESO-1 mRNA was detected in 18 of 41 (43.9%) hepatocellular carcinomas. No NY-ESO-1 mRNA was expressed in 41 paired non-cancerous specimens and 18 specimens histologically diagnosed as liver cirrhosis or chronic hepatitis. Immunohistochemistry revealed heterogeneous expression of NY-ESO-1 protein in three of 18 NY-ESO-1 mRNA-positive hepatocellular carcinomas. None of 23 NY-ESO-1 mRNA-negative hepatocellular carcinomas expressed NY-ESO-1 protein. Antibody against NY-ESO-1 protein was detected in two of 92 patients with hepatocellular carcinoma. Both of these patients had tumors invading main branches of the portal vein. The present study has demonstrated the expression of NY-ESO-1 mRNA in hepatocellular carcinoma and NY-ESO-1 antibody production in patients with advanced hepatocellular carcinoma. Although the enhancement of NY-ESO-1 protein expression and the activation of immune response of the patients with hepatocellular carcinoma are necessary, NY-ESO-1 has the potential to be a good target molecule for immunotherapy against advanced hepatocellular carcinoma.

Monoclonal antibody-based genetic immunotherapy.

The clinical application potential of monoclonal antibodies concerns a wide range of diseases including, among others, viral infections, cancer and autoimmune diseases. Intravenous injection is a simple and obvious mode of administration of purified therapeutic antibodies to patients but may not always be appropriate for long-term treatments for a variety of reasons. One limitation concerns the elevated costs of recombinant proteins certified for human use. Moreover, hour-long infusions require a hospital environment and are often associated with mild to very severe side effects. This makes large-scale clinical applications of a number of monoclonal antibodies with demonstrated therapeutic activity impossible or, at least, severely compromised. In vivo production of therapeutic antibodies in patients, through either genetic modification of their tissues or implantation of antibody-producing cells, might represent an attractive alternative to overcome these drawbacks. Moreover, this method should also provide other benefits. Continuous and sustained delivery of antibodies at a low, but therapeutic level should prevent, or at least delay, induction of neutralizing anti-idiotypic immune responses, which sometimes develop when massive doses of purified immunoglobulins are repeatedly injected into patients. Additionally, it should also limit variations in the bioavailability of therapeutic antibodies that are often detrimental to the efficacy of treatments. The present review reports on the recent developments of gene/cell therapies aiming at the in vivo production and systemic delivery of monoclonal antibodies with the final goal of treating severe chronic diseases.

Anti-myeloperoxidase antibodies in sera from patients with propylthiouracil-induced vasculitis might recognize restricted epitopes on myeloperoxidase molecule.

Myeloperoxidase (MPO) is one of the major target antigens of antineutrophil cytoplasmic antibodies (ANCA) in primary systemic vasculitis. It is known that propylthiouracil (PTU) could induce MPO-ANCA-positive vasculitis. The production of anti-MPO antibodies in patients with PTU-induced vasculitis may be different from that in patients with primary microscopic polyangiitis (MPA). One possible reason for this may be differences in epitope recognition. The aim of this study is to compare the epitopes of antibodies to MPO in sera from patients with PTU-induced vasculitis (n = 10) and MPA (n = 10). The sera were collected and used to inhibit monoclonal antibodies against human MPO (3D8 and 6B9) and affinity purified, horseradish peroxidase conjugated human anti-MPO antibodies (Pab1-HRP, Pab2-HRP) in a competitive inhibition enzyme-linked immunosorbent assay (ELISA) system using soluble human MPO as solid phase ligand. The Pab1-HRP and Pab2-HRP were affinity purified from plasma exchanges of a patient with PTU-induced vasculitis and a patient with MPA, respectively. The inhibition rates were evaluated and compared between the PTU and primary MPA groups. In the PTU group all 10 sera could inhibit 3D8: the average inhibition rate was 44.7% +/- 5.0%; 9/10 sera could inhibit 6B9: the average inhibition rate was 35.6% +/- 6.0%. However, in the MPA group all 10 sera could inhibit 3D8 and 6B9; the average inhibition rates were 68.4% +/- 16.1% (P < 0.01) and 62.2% +/- 17.2% (P < 0.01), respectively. Sera in both the PTU and MPA groups could inhibit Pab1-HRP and the inhibition rates were 81.4% +/- 9.4%versus 86.6% +/- 17.2% (P > 0.05). However, the average inhibition rate for Pab2-HRP in the MPA group was significantly higher than that in the PTU group (76.3% +/- 7.8%versus 58.9% +/- 15.5%, P < 0.01). We conclude that anti-MPO antibodies from patients with PTU-induced vasculitis and from patients with primary MPA could recognize more than one epitope on the native MPO molecule. Although the epitopes overlapped between the two groups, the epitopes of anti-MPO antibodies from patients with PTU-induced vasculitis might be more restricted.

Restricted T-cell receptor β-chain usage by T cells autoreactive to β2–glycoprotein I in patients with antiphospholipid syndrome

We recently identified CD4(+) T cells that are autoreactive to beta(2)-glycoprotein I (beta(2)GPI) and that promote antiphospholipid antibody production in patients with antiphospholipid syndrome (APS). In this study, T-cell receptor (TCR) beta chains of beta(2)GPI-reactive T cells were examined in 8 beta(2)GPI-responders, including 5 patients with APS and 3 healthy subjects, using polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) analysis combined with in vitro stimulation of peripheral blood T cells with recombinant beta(2)GPI. The TCR Vbeta segments that expanded oligoclonally after stimulation with beta(2)GPI varied among responders, but the Vbeta7 and Vbeta8 segments were commonly detected in 6 and 4 beta(2)GPI-responders, respectively. Analysis of the complementarity-determining region 3 sequence of beta(2)GPI-reactive T cells revealed limited diversity, and all Vbeta7(+) TCRs had an amino acid motif of TGxxN/Q or minor variations. The Vbeta8(+) TCRs had another motif, PxAxxD/E. Surprisingly, an identical Vbeta7(+) TCRbeta chain was used by beta(2)GPI-reactive T cells in 3 patients with APS. There was no apparent difference in the TCRbeta usage between APS patients and healthy responders. Some of the Vbeta7(+) TCRs with the TGxxN/Q motif detected by PCR-SSCP analysis were also used by beta(2)GPI-specific CD4(+) T-cell clones responsive to an immunodominant epitope containing the major phospholipid-binding site. Depletion of Vbeta7(+) or Vbeta8(+) T cells from the peripheral blood mononuclear cell cultures significantly inhibited in vitro anti-beta(2)GPI antibody production in response to beta(2)GPI. Our results indicate preferential usage of TCRbeta chains by beta(2)GPI-reactive T cells. These TCRbeta chains can be reasonable targets for TCR-based immunotherapy for patients with APS.

Epitope analysis of HLA class I donor specific antibodies in sensitized renal transplant recipients.

The goal of this study was to evaluate the epitope specificity of donor-specific HLA class I antibodies detected in the serum of alloimmunized from a previous renal graft patients. A total of 410 serum samples from 87 patients who had lost a previous graft, were collected every 4 months during a 2-year follow-up period. All recipients and donors were typed for class I HLA-antigens by a standard lymphocytotoxicity technique. To define the specificities of the HLA class I antibodies, two techniques were used in parallel: the antihuman globulin augmented CDC (AHG-CDC) technique and an ELISA technique. The mismatched HLA-antigens and the detected HLA class I antibodies were categorized as intra-cross-reactive group mismatches (intra-CREGs-MMs) and other-CREG-MMs. For each sensitized patient actual and at risk epitope specificities were defined. Thirty-eight patients (43.7%) had developed IgG HLA class I-specific antibodies with stable specificities against mismatched alloantigens from the previous graft. A total of 60 antibody reactivity patterns and 82 specificities against private and public epitope were recognized. Patients with only intra-CREGs-MMs produced HLA class I-specific antibodies less frequently than patients with only other-CREG-MMs, although the difference was nearly statistically significant (P=0.053). All HLA class I donor-specific antibodies were considered to have specificities against the private epitopes of the mismatched graft HLA-antigens. In the cases where HLA class I alloreactivity was spreading to more than one donor antigens, we considered that the detected antibodies had specificities against the private and the shared between the alloantigens epitope(s). No epitope-specific antibodies were detected against shared epitopes between the mismatched alloantigens and the HLA-antigens of the patients. In 11/38 cases (28.9%) HLA class I alloreactivity spreading to non-graft antigens was detected. These antibodies were directed against HLA-antigens that share epitope(s) and have strong serological reactivity with the immunogenic alloantigens. Our data show that a small number of private and public alloepitopes seem to be responsible for antibody production in patients sensitized from a previous graft. A detailed description of these HLA-epitopes, in the context of clinical graft complications, may lead to an improved organ allocation strategy.

T cells that are autoreactive to beta2-glycoprotein I in patients with antiphospholipid syndrome and healthy individuals.

To identify the T cells responsive to beta2-glycoprotein I (beta2GPI) that mediate antiphospholipid antibody production in patients with antiphospholipid syndrome (APS). In vitro proliferative responses and anti-beta2GPI antibody production induced by beta2GPI were examined in peripheral blood mononuclear cell (PBMC) cultures from 12 APS patients, 13 systemic lupus erythematosus patients without APS, and 12 healthy donors. Peripheral blood T cells from all subjects failed to respond to beta2GPI in its native form. In contrast, reduced beta2GPI was able to stimulate T cells not only from all 12 patients with anti-beta2GPI antibodies, but also from 10 of 25 individuals without anti-beta2GPI antibodies. The specificity of the responses to beta2GPI was confirmed by activation of the reduced beta2GPI-primed T cells by recombinant beta2GPI in secondary cultures. Characterization of the T cell response induced by beta2GPI revealed that the response was associated with the presence of the DR53-associated alleles, the responding T cells were CD4+ and restricted by HLA class II, and antigenic peptides were located in domains IV and/or V. Anti-beta2GPI antibody production was induced specifically in anti-beta2GPI antibody-positive patients, in PBMC cultures with reduced beta2GPI. Anti-beta2GPI antibodies produced in vitro recognized beta2GPI immobilized with cardiolipin or beta2GPI coated on "high-binding" polystyrene plates. These results strongly suggest that CD4+ and HLA class II-restricted T cells responsive to beta2GPI are involved in the production of antiphospholipid antibodies in APS patients.

Correlation between beta2-glycoprotein I valine/leucine247 polymorphism and anti-beta2-glycoprotein I antibodies in patients with primary antiphospholipid syndrome.

Beta2-Glycoprotein I (beta2GPI) exon 7 polymorphism leads to a valine leucine amino acid exchange at position 247 in domain 5 of beta2GPI, between the phospholipid binding site and the cryptic site of the epitopes for anti-beta2GPI antibodies. Therefore, position 247 polymorphism may affect the conformational change of beta2GPI and the exposure of the epitopes for anticardiolipin antibodies (aCL) (= anti-beta2GPI antibodies). In this study we analysed the genetic polymorphism of beta2GPI in a British cohort of well-defined antiphospholipid syndrome (APS) patients. This study comprised 88 Caucasoid patients with APS [57 with primary APS and 31 with APS secondary to systemic lupus erythematosus (SLE)]. Polymorphism assignment was determined by polymerase chain reaction followed by allele-specific restriction enzyme digestion (PCR-RFLP). The presence of anti-beta2GPI antibodies was detected by ELISA utilizing irradiated ELISA plates. Anti-beta2GPI antibodies were present in 28 of 57 primary APS patients (49%) and in 19 of 31 secondary APS patients (61%). The allele containing valine247 was significantly more frequent in primary APS patients with anti-beta2GPI antibodies than in controls (OR = 2.51, 95%, CI 1.03-6.13, P = 0.0396) or in primary APS patients without anti-beta2GPI antibodies (OR = 2.92, 95% CI 1.16-7.39, P = 0.0204). This tendency was not found in the secondary APS group. In conclusion, the beta2GPI polymorphism, valine/leucine247, is correlated with anti-beta2GPI antibody production in patients with primary APS, and valine247 may be important in the formation of beta2GPI antigenicity.

Defective antibody production in patients with rheumatoid arthritis and bronchiectasis.

Bronchiectasis (BR) occurs in about 3% of patients with rheumatoid arthritis (RA). Defective antibody production is a rare but well-recognised cause of both BR and inflammatory arthritis. We examined the hypothesis that subtle specific antibody defects might play a role in the pathogenesis of BR associated with RA. Identification of defects in antibody production is important because substantial benefits may be gained from immunoglobulin replacement. Specific antibody production was assessed in 20 patients with RA and BR, 20 with BR alone, 20 with RA alone and 20 healthy controls (all groups matched for age and sex). All had normal total IgG. IgA and IgM and IgG subclass levels. Specific antibody production was assessed by assay of antibodies to representative polysaccharide and protein antigens. Subjects with subprotective titres were challenged with the appropriate vaccine. Defective antibody production was defined as a subprotective level despite immunisation. Three out of 20 patients with RA and BR had a defective IgG2 response to the polysaccharide antigen, but normal responses to the protein antigen. All of the subjects in the BR alone or healthy control group had normal antibody production. Two out of 20 patients with RA alone had defective production of antibodies against both protein and polysaccharide antigens; both were receiving gold therapy, a recognised cause of functional antibody defects. It was concluded that some patients with RA and BR have functional antibody defects and may benefit from antibody replacement. An unexpectedly high proportion of patients with RA alone also have functional antibody defects, possibly secondary to gold therapy.

Estrogen enhancement of anti-double-stranded DNA antibody and immunoglobulin G production in peripheral blood mononuclear cells from patients with systemic lupus erythematosus.

To study the in vitro effect of estrogen on IgG anti-double-stranded DNA (anti-dsDNA) antibody and total IgG production in peripheral blood mononuclear cells (PBMC) from patients with systemic lupus erythematosus (SLE), in order to elucidate its regulatory role in SLE. PBMC from SLE patients and normal donors were cultured with 17beta-estradiol (E2). IgG anti-dsDNA antibodies, total IgG, and cytokine activity in the culture supernatants were measured by enzyme-linked immunosorbent assay. E2 enhanced production of IgG anti-dsDNA antibodies as well as total IgG in PBMC from SLE patients. Anti-dsDNA production in patients with inactive disease was less responsive to E2 than that in patients with active disease. E2 also enhanced total IgG, but not anti-dsDNA, production in the PBMC of normal donors. Antibody production was increased by E2 to a lesser extent in patients' B cells than in their PBMC. Anti-interleukin-10 (anti-IL-10) antibodies partially blocked the E2-induced increase in antibody production in patients' PBMC, but anti-IL-10 had no effect on B cells. E2 increased IL-10 production by patients' monocytes. Exogenous IL-10 acted additively with E2 in increasing antibody production in patients' B cells. These results suggest that E2 may polyclonally increase the production of IgG, including IgG anti-dsDNA, in SLE patients' PBMC by enhancing B cell activity and by promoting IL-10 production in monocytes. These findings support the involvement of E2 in the pathogenesis of SLE.

Impaired IgG antibody production to pneumococcal polysaccharides in patients with ataxia-telangiectasia.

Various factors seem to be etiologic in the susceptibility to sinopulmonary infections in ataxia-telangiectasia (A-T) patients, i.e., low serum and salivary IgA, low serum IgG2, and even aspiration of saliva. S. pneumoniae is a common pathogen responsible from pulmonary infections and impaired antibody response to polysaccharide antigens is seen in patients with IgG2 and IgA deficiency as well as patients with CVID and WAS. We studied IgG-type antibody production to six pneumococcal serotypes in 29 A-T patients by ELISA before and 3-4 weeks after pneumococcal vaccine. The response was considered positive when the antibody titer was >10 U/ml but weak when the titer was 10-20 U/ml. Twenty-two of 29 (76%) patients did not respond to any of the serotypes, 5 (17%) showed a positive response to one serotype, 1 (3.4%) to two serotypes, and 1 (3.4%) to four serotypes. With conversion to gravimetric units (ng IgG/ml) and >1800 ng/ml (300 ng Ab N/ml) considered a positive response, 5 of 29 (17.2%) patients showed a positive response (300 ng ab N/ml) to two or fewer serotypes. All patients tested produced IgG antibody to tetanus toxoid. Sixteen of 27 (59.3%) patients had low IgG2 and four (14.8%) had low IgG3 levels, while 18 (62.1%) of 29 patients had low serum IgA. No correlation was found either between serum Ig isotype levels and antipolysaccharide antibody response or between susceptibility to infection and antibody production. The mechanism responsible for disturbed antipolysaccharide (TI-2 antigen) antibody production in patients with A-T needs to be investigated. It may provide additional information on the function of the ATM gene product and be helpful in clarifying the role of B cells and contribution of T cells in TI-2 responses.

T and B cell collaboration is essential for the autoantibody response to DNA topoisomerase I in systemic sclerosis.

To elucidate the mechanisms controlling anti-DNA topoisomerase I (topo I) antibody production in patients with systemic sclerosis (SSc), in particular the role of interactions between topo I-specific Th cells and B cells, we established an in vitro system for the analysis of anti-topo I antibody production. In vitro anti-topo I antibody synthesis in PBMC cultures was induced by recombinant topo I and PWM, and was measured by a topo I-specific ELISA. Anti-topo I antibody was detected in PBMC culture supernatants from 11 (61%) of 18 anti-topo I-positive SSc patients. In contrast, anti-topo I antibody was not detected in the PBMC culture supernatants from 4 anti-topo I-negative SSc patients or 10 healthy donors. Characterization of in vitro anti-topo I antibody production showed that 1) the anti-topo I antibody isotype produced was IgG; 2) the anti-topo I antibody levels in culture supernatants correlated with those in patients' sera; 3) CD4+ T cells were necessary for antibody synthesis; and 4) antibody synthesis was restricted by HLA-DR, but not by HLA-DQ or DP. In addition, separation of cultured T and B cells by a semipermeable membrane or culture with anti-CD40 ligand mAb blocked in vitro anti-topo I antibody production. These results indicate that a contact-mediated and HLA-DR-restricted collaboration between topo I-specific T and B cells is essential for in vitro anti-topo I antibody production in a subset of SSc patients.

Hydralazine induces Z-DNA conformation in a polynucleotide and elicits anti(Z-DNA) antibodies in treated patients

We studied the effect of hydralazine, an antihypertensive drug with lupus-inducing side effects, on the conformation of poly(dG-m5dC).poly(dG-m5dC) and a plasmid with a 23 bp insert of (dG-dC)n.(dG-dC)n sequences. Using an e.l.i.s.a. with a monoclonal anti-(Z-DNA) antibody Z22, we found that hydralazine provoked the Z-DNA conformation in poly(dG-m5dC).poly(dG-m5dC) at 250-500 microM concentration. The supercoiled form of hydralazine-treated plasmid bound to Z22 in a gel-retardation assay. To examine further whether Z-DNA could act as an inciting agent in anti-nuclear antibody production in patients, we analysed 65 sera from 25 hypertensive patients taking hydralazine and found anti-(Z-DNA) antibodies in 82% of these sera. Sera from age-matched normal controls showed no binding to Z-DNA. Data on sera drawn sequentially from four hypertensive patients showed that antibodies were present after the drug treatment. These data demonstrate the presence of a high incidence of anti-(Z-DNA) antibodies in patients treated with hydralazine and suggest that a possible mechanism for the production of autoantibodies in drug-related lupus might involve the induction and stabilization of Z-DNA by drugs.

Heterogeneity of Hanganutziu-Deicher antigen glycoproteins in different species animal sera.

A heterophilic Hanganutziu-Deicher (HD) antigen is present in many animal sera except human and chicken sera. To visualize the antigenic molecules, nine species animal and human sera were analyzed by SDS-PAGE, followed by Western blotting and immunostaining with avain anti-N-glycolylneuraminyl-lactosyl ceramide antibody which recognizes the terminal N-glycolylneuraminic acid moiety of glycoconjugates as an epitope of the HD antigen. Several HD antigen-active glycoprotein bands were detected in the sera of fetal calf, calf, horse, goat, monkey, rabbit, guinea pig, rat and mouse, except for human serum. The HD antigenic proteins showed heterogeneities in their molecular weights and were not identical with any major band visualized with silver-staining, indicating that they are minor components of serum proteins in each animal. Neuraminidase treatment destroyed the antigenicity of all proteins, confirming that N-glycolylneuraminic acid (NeuGc) at the non-reducing terminal of carbohydrate chains is the antigenic epitope in serum glycoprotein molecules as already confirmed in glycosphingolipid (GSL) antigens. The finding of HD-antigenic glycoproteins in animal sera suggests that they also stimulate HD antibody production in patients who received animal antiserum for therapeutic aim.

The specific IgM response to Mycoplasma pneumoniae infection: interpretation and application to early diagnosis.

Specific IgM antibody production in patients with serologically proven Mycoplasma pneumoniae infection by the complement fixation (CF) test was studied using a mu-capture ELISA. Sera from 79% of patients were found to be IgM positive. Patients could be divided into two groups relating to the amount of specific antibody produced. High levels of specific IgM (greater than or equal to 10 units) were more commonly found in younger patients. Seventy-six per cent of patients under the age of 20 produced relatively high levels of IgM compared to 35% of patients over the age of 20. In contrast, the number of patients who produced low or undetectable levels of IgM (less than 10 units) was found to increase with age. This trend was found to be significant which suggests that low or undetectable levels of IgM may be due to reinfection with M. pneumoniae. Specific IgM was found to appear in the serum at approximately 7 days after the onset of symptoms, peaking at between 10 and 30 days, and then falling to undetectable levels at an estimated 12-26 weeks post onset of symptoms. Twenty-eight per cent of acute-phase sera (CF titres less than 256) from patients whose sera subsequently showed a fourfold or greater rise in M. pneumoniae CF antibody titre were IgM positive. Thus using mu-capture ELISA a diagnosis of M. pneumoniae infection may often be made more rapidly than by the complement fixation test.

DNA-daunorubicin complexes specifically suppress in vitro spontaneous anti-DNA antibody production in lymphocytes of patients with systemic lupus erythematosus.

Elevated production of anti-DNA antibody in patients with systemic lupus erythematosus (SLE) is a central problem in the pathogenesis of tissue injury. In the present study, we attempted to manipulate anti-DNA antibody production through the antigen-cytotoxic drug conjugates, DNA-daunorubicin complexes. The effect of DNA-daunorubicin complexes was determined by examining SLE lymphocytes for spontaneous in vitro production of anti-DNA antibody. These complexes, at 2 micrograms/ml, suppressed anti-DNA antibody production, but not total IgG production, which suggests that specific suppression of anti-DNA antibody production was achieved at this concentration. We believe that the DNA-daunorubicin complexes affected mainly B cells, since such suppression was obtained by treating B cells, as well as B plus T cells. Furthermore, the complexes had no effect on the proliferative responses of SLE T cells to DNA, phytohemagglutinin, or concanavalin A. These results indicate that DNA-daunorubicin complexes may have the potential for selectively suppressing anti-DNA antibody production in patients with SLE.