Production and characterization of recombinant human anti-HBs Fab antibodies.

Recombinant human Fab antibodies were generated with different reactivities against the hepatitis B virus surface (HBs) antigen. To isolate the antibodies, a method was used that combined transformation of human B cells by Epstein-Barr virus (EBV) infection with a primer-vector system developed for isolating DNA fragments of human Ig Fab portions. With this method, monoclonal and oligoclonal cell lines producing anti-HBs antibodies were established and three anti-HBs Fab antibodies were isolated from two of these cell lines. From analysis of affinity characteristics, immunohistochemical activity, and cytolysis activity, these three Fab antibodies were classified into three different groups. The first group had high affinity for HBs, the second had the ability to kill HBV-infected cells, and the third was applicable to immunohistochemical staining with HBV-infected cells. The combined effect of these antibodies was also investigated by complement-dependent cytotoxicity assay.

A retrospective study of the fluctuation in serum levels of anti-cyclic citrullinated peptide antibody in patients with rheumatoid arthritis.

OBJECTIVE: To investigate the fluctuation in serum levels of anti-cyclic citrullinated peptide antibody (anti-CCP) retrospectively in patients with rheumatoid arthritis (RA). METHODS: Serum levels of anti-CCP were measured retrospectively in 131 patients with RA and 90 patients with non-RA rheumatic diseases using a commercially available kit. All sera were collected from patients during the 22-year period, 1982-2004. To analyze the fluctuation in anti-CCP levels, 17 RA patients were selected on the basis of showing a significantly higher anti-CCP level in a serum sample taken at the first visit (> 80 U/ml), and availability of preserved serum samples that had been taken from each patient at 10 time points. RESULTS: The test gave a sensitivity of 88% (115/131) and a specificity of 81% (73/90). The longitudinal study of 17 RA patients showed that anti-CCP levels were elevated at the first visit in 12 (71%) patients and then decreased gradually, whereas those in the other five (29%) patients fluctuated substantially. In both cases, anti-CCP levels tended to fluctuate in parallel with the erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) level, reflecting the spontaneous aggravation of arthritis and the efficacy of anti-rheumatic drugs. The courses of three representative RA patients are illustrated in detail along with their therapeutic regimens, and these further confirm the correlation of anti-CCP levels with laboratory parameters (ESR and CRP) as well as the activity of arthritis. CONCLUSION: Measurement of serum anti-CCP levels was found to be useful for not only the diagnosis but also the management of RA.

Molecular structure of ras-related small GTP-binding protein genes of rice plants and GTPase activities of gene products in Escherichia coli.

We isolated two rice cDNA clones (ric1 and ric2) encoding proteins homologous to the ras-related small GTP-binding protein. The amino acid sequences of ric1 and ric2 are conserved in four regions involved in GTP binding and hydrolysis which are characteristic in the ras and ras-related small GTP-binding protein genes. In addition, two consecutive cysteine residues near the carboxyl-terminal end required for membrane anchoring are also present in ric1 and ric2. The ric1 and ric2 proteins synthesized in Escherichia coli possessed GTPase activity (i.e. hydrolysis of GTP to GDP).

Measurement of anti-double-stranded DNA antibodies in major immunoglobulin classes.

A solid-phase radioimmunoassay for quantitating anti-double-stranded deoxyribonucleic acid antibodies (anti-dsDNA) in IgG, IgM and IgA classes has been devised. A distinct feature of the method is an application of polystyrene tubes coated with poly-L-lysine, through which dsDNA could be bound firmly to a solid phase. Studies on patients' sea as well as normal sera revealed that anti-dsDNA was not qualitatively but quantitatively characteristic of systemic lupus erythematosus (SLE) and that IgG anti-dsDNA levels correlated well with the disease activity.

A simple and rapid microenzyme-linked immunosorbent assay for antibodies to poly(ADP-ribose) in systemic lupus erythematosus.

A simple and rapid microenzyme-linked immunosorbent assay has been developed for determination of anti-poly(ADP-ribose) antibodies in humans using a combination of protein A-alkaline phosphatase conjugates and poly(ADP-ribose)-coated polyvinyl microplates. After a 1-h treatment of the plates with 100 microliters of poly L-lysine (PLL) solution (50 micrograms/ml), an aliquot of the solution containing 100 ng poly(ADP-ribose) (50 microliters) was added to the PLL-treated plates and evaporated at 37 degrees C overnight to facilitate the adherence of poly(ADP-ribose) to the plates. Nonspecific binding of diluted test sera from patients with systemic lupus erythematosus (SLE) or from normal individuals to the PLL-coated plates was minimized by exposure of the plates for 1 h to Tris-buffered saline (pH 7.4) containing 0.01% bovine serum albumin (BSA). This method was also applicable to the determination of anti-double-stranded DNA antibodies in humans. The present assay is advantageous over those reported so far as it saves time and antigen.

Anti-double strand (ds) DNA antibody formation by NZB/W (F1) spleen cells in a microculture system detected by solid phase radioimmunoassay.

A solid-phase radioimmunoassay method was devised to detect mouse anti-double strand (ds) DNA antibody. This method could easily detect the anti-dsDNA antibody in 1 : 10,000 dilutions (1 unit) of pooled 9-10-month-old female NZB/W F1 sera. The sensitivity was about 10(3)- and 10(2)-fold higher than that of the modified Farr method and of the double antibody technique respectively. NZB/W mice developed high titer anti-dsDNA antibody as they grew older. Spleen cells brought to a microculture system using flat-bottomed polystyrene plates produced anti-dsDNA antibody clearly detectable by solid-phase radioimmunoassay. Anti-dsDNA antibody produced in vitro (y units) was in close correlation with the anti-dsDNA antibody titer of the spleen donor (x units) (y = 4.8 X 10(-2) x -65, gamma = 0.94, P less than 0.001). A combination of the microculture system and solid-phase radioimmunoassay was recommended for the characterization of anti-dsDNA antibody-forming cells.

C1q solid-phase radioimmunoassay: binding properties of solid-phase C1q and evidence that C1q-binding IgG complexes in systemic lupus erythematosus are not bound to endogenous C1q.

The binding properties of C1q solid-phase radioimmunoassay (C1q SPRIA) were examined, using heat-aggregated IgG (HAG) as the model of immune complexes (IC). The free, liquid-phase C1q, which was added to the C1q-coated tubes prior to the addition of HAG, had little inhibitory effect on binding of HAG to the solid-phase C1q, suggesting that the solid-phase C1q has a higher affinity for HAG than the liquid-phase C1q. On the other hand, more than 60% inhibition was seen when HAG was preincubated with the liquid-phase C1q. These binding properties of HAG to the solid-phase C1q in the presence of the liquid-phase C1q were not essentially altered by the heat inactivation or the addition of EDTA, suggesting that these pretreatments are not essential in C1q SPRIA. Next, in similar kinds of experiments, the binding properties of C1q-binding IgG complexes in SLE sera were investigated. In contrast to HAG, the binding capacity of IgG complexes in SLE sera to the solid-phase C1q was not inhibited by the preincubation with excess liquid-phase C1q. These findings suggest that C1q-binding IgG complexes in SLE sera detected by C1q SPRIA may not be bound to endogenous C1q in the circulation.

Age-related changes in auto- and natural antibody in the Werner syndrome.

To assess the immunologic disturbance in WErner syndrome, antibodies to "intrinsic" (auto)antigens (anti-DNA antibodies and rheumatoid factors) and "natural" antibodies to "extrinsic" antigens (hemagglutinins for sheep red cells and antibodies against ABO blood type antigens) were measured in serum samples from 16 patients with Werner syndrome and compared with those from 150 healthy persons ranging in age from less than a year to 98. Employing a sensitive solid-phase radioimmunoassay, we found that the levels of both anti-double-stranded and anti-single-stranded DNA antibodies in the IgG class gradually increased with age in normal donors; a more abrupt increase with age was observed in those with Werner syndrome, although they lacked any complication of renal disease and hypocomplementemia. The titers of rheumatoid factor detected by sensitized sheep cell agglutination also gradually rose in normal persons and patients with Werner syndrome. In contrast, the titers of natural antibodies declined with age in both groups. These disturbances in antibody production suggested that Werner syndrome expresses an accelerated form of aging in immunologic aspect.

Cloning and expression of human anti-tumor necrosis factor-alpha monoclonal antibodies from Epstein-Barr virus transformed oligoclonal libraries.

Peripheral blood was obtained from a healthy human volunteer and transformed with Epstein-Barr virus (EBV). This produced an oligoclonal cell library in culture medium that was screened by ELISA for anti-human tumor necrosis factor-alpha (TNFalpha) activity. RNA from two positive clones was applied to RT-PCR using antibody-specific primers, and the light (kappa and lambda) and heavy chain genes (gamma and mu) were cloned into the plasmid vector pFab1-His2. The antibodies produced in Escherichia coli as Fab fragments were assayed for anti-TNFalpha activity utilizing ELISA. Two IgG1/kappa anti-TNFalpha antibodies and two IgM/kappa anti-TNFalpha antibodies were isolated. DNA sequence analysis showed that the VL and VH gene families of IgM and IgG were the same. Both the antibodies showed almost the same activity on ELISA-testing. Ten clones randomly selected from light (kappa and lambda) and heavy (gamma and mu) chain genes in the oligoclonal cell library 1D5 were sequenced, and each gene (kappa, lambda, gamma, and mu) was found to be composed of one to three different genes. These data support the conclusion that the cell clone is oligoclonal at the molecular level.

Suppression of lipoprotein lipase in 3T3-L1 cells by a mediator produced by SEKI melanoma, a cachexia-inducing human melanoma cell line.

Production of a cachexia-inducing factor(s) by the SEKI melanoma cell line, established from a human melanoma, has been well documented. Conditioned medium from cultures of this melanoma cell line contains a factor(s) that inhibits the activity of lipoprotein lipase (LPL) in fully differentiated 3T3-L1 adipocytes. The mode of inhibition of this enzyme by the factor, i.e. its dose-dependency and time course, is very similar to that of LPL-inhibition by a macrophage-derived cachexia-inducing factor, cachectin/tumor necrosis factor (cachectin/TNF). However, the conditioned medium of SEKI melanoma cells does not contain any immuno-reactive substances reactive in enzyme-linked immunosorbent assay (ELISA) with anti-cachectin/TNF antibody, or with anti-interleukin 1 alpha or beta antibodies. This LPL-suppression factor present in the conditioned medium seems to be a peptide because of its heat-lability and apparent molecular weight of more than 25,000. The conditioned media from cultures of four other different cell lines were found to show no significant suppression of LPL activity. These results imply that SEKI melanoma cells produce a cachexia-inducing factor(s) similar to cachectin/TNF but that the molecule involved is different.

Bacterial expression of a neutralizing mouse monoclonal antibody Fab fragment to a 150-kilodalton surface antigen of Entamoeba histolytica.

A mouse monoclonal antibody (MAb) (EH3015, IgG1 with a K light chain) prepared by hybridoma technology recognizes a 150-kD surface antigen of Entamoeba histolytica and inhibits adherence and cytotoxicity of the ameba to mammalian cells. The genes encoding the light chain and the Fd region of the heavy chain of the MAb were cloned and expressed in Escherichia coli. The plasmid used was designed for the expression of Fab with a hexa-histidine tag in the periplasmic space. Recombinant Fab fragments were purified and analyzed by an indirect immunofluorescence antibody test and Western immunoblot. The specificity of the recombinant Fab fragment was comparable with the parent whole IgG. In addition, the Fab fragments significantly inhibited the adherence of E. histolytica to erythrocytes. These results suggest that the production of a neutralizing MAb in Escherichia coli is practical and efficient with this expression system.

Clinical significance of serum thrombomodulin levels in patients with systemic rheumatic diseases.

OBJECTIVE: To determine the soluble thrombomodulin (TM) level in the sera of patients with systemic rheumatic diseases and to analyze its relationship with clinical and laboratory parameters in patients with systemic lupus erythematosus (SLE). METHODS: Serum levels of TM were measured by an enzyme-linked immunosorbent assay in 124 patients with SLE and in 237 patients with systemic rheumatic diseases other than SLE. RESULTS: The frequency of patients with high TM levels (> or = 4.5 FU/ml) was 21% in SLE, 16% in rheumatoid arthritis (RA), 12% in Sjögren's syndrome (SS), and 4% in systemic sclerosis (SSc). The TM levels were significantly higher in SLE patients with the following abnormalities in their past history or present illness: persistent proteinuria, cellular casts, positive anti-dsDNA antibodies (p < 0.001), hypoalbuminemia, decreased creatinine clearance (Ccr), positive anti-Sm antibody, positive immune complex, leukopenia (p < 0.01), pericarditis, oral ulcer, fluorescent anti-nuclear antibody (FANA), decreased C3 and arthralgia (p < 0.05). The serum TM levels in SLE showed a direct correlation with the urinary protein level (p < 0.01) and serum creatinine level (p < 0.01), and an inverse correlation with the serum albumin level (p < 0.01) and complement C3 level (p < 0.05). In SLE patients with active disease, the median TM level at the time of admission (5.9 FU/ml) was significantly higher than at 6 months before admission (3.8 FU/ml, p < 0.01) and at 6 months after admission (4.2 FU/ml, p < 0.001). Moreover, elevation and reduction of the TM level in lupus patients with active disease parallelled that of the SLE disease activity index (SLEDAI). In two representative SLE cases, one with lupus nephritis (LN) and the other with thrombocytopenia without LN, elevated TM levels at disease flare were reduced along with the amelioration of the disease by administration of corticosteroids. CONCLUSIONS: The serum TM level is closely associated with SLE activity and appears to be useful as a new disease activity parameter of SLE.

Comparable histological appearance of synovitis in seropositive and seronegative rheumatoid arthritis.

OBJECTIVE AND METHODS: To compare the histological characteristics of the synovium between seropositive and seronegative rheumatoid arthritis (RA), synovial tissue was obtained from 19 patients with rheumatoid factor-positive (RF+) RA, 11 with rheumatoid factor-negative (RF-)RA and 11 non-RA controls. RESULTS: There were no differences in the frequency of each histological feature or histological scores between RF+ and RF-RA. However, significant differences in the frequency of histological findings such as lining cell proliferation and inflammatory cell infiltration were found both between RF+ RA and non-RA controls, and between RF-RA and non-RA controls. Analysis of clinical parameters and histology in all RA patients revealed that the level of serum C-reactive protein and the erythrocyte sedimentation rate were directly correlated with the inflammatory cellular infiltration score. Immunohistological staining using a monoclonal antibody against IgM rheumatoid factor (RF) was positive in some plasma cells in the synovium of both RF+ and RF-RA patients, while no IgM-RF-positive cells were observed in the synovium of non-RA controls. CONCLUSION: RF serostatus does not necessarily reflect the histology of synovial inflammation.

Down-regulation by eel calcitonin of interleukin-1 release and production by peripheral blood monocytes from patients with rheumatoid arthritis.

To clarify the reason for the therapeutic efficacy of Elcatonin (eCT), an eel calcitonin derivative, in rheumatoid arthritis (RA), we studied the effect of eCT on the extracellular (EC) release and intracellular (IC) production of interleukin-1 (IL-1) by peripheral blood monocytes from patients with RA. In vitro treatment of RA monocytes with eCT reduced predominantly the EC release of both IL-1 alpha and IL-1 beta. Moreover, EC release and IC production of IL-1 alpha and IL-1 beta by monocytes from RA patients who received non-steroidal anti-inflammatory drugs (NSAIDs) plus eCT (CT group) were significantly lower than in those who received NSAIDs only (NSAID group). The anti-rheumatic effect of eCT may be mediated via the inhibition of EC release and the IC production of IL-1 from RA patients.

Progressive multifocal leukoencephalopathy resembling central nervous system systemic lupus erythematosus.

A 21-year-old woman with a 6-year history of SLE presented with a speech disturbance and right hand clumsiness along with manifestations of active disease suggesting central nervous system SLE. Despite aggressive treatment for SLE, her neurological condition worsened. MRI demonstrated low intensity in T1-weighted images and high intensity in T2-weighted images in the white matter of the bilateral cerebrum and cerebellum, compatible with progressive multifocal leukoencephalopathy (PML). Intraspinal administration of interferon-beta seemed to slow the deterioration of her MRI and neurological findings. However, she eventually developed decerebrate rigidity and died due to candidemia. DNA of the JC virus was detected in the autopsied brain by the polymerase chain reaction technique. PML should always be borne in mind when examining patients with SLE showing neurological abnormalities.

Increase of cytokine production by pulmonary artery endothelial cells induced by supernatants from monocytes stimulated with autoantibodies against U1-ribonucleoprotein.

OBJECTIVE: To clarify the pathogenetic role of autoantibodies against U1-RNP (ribonucleoprotein) (anti-U1-RNP) in mixed connective tissue disease (MCTD), we examined whether supernatants of monocytes which were stimulated with anti-U1-RNP could induce the production of proinflammatory cytokines by human pulmonary artery endothelial cells (HPAECs). METHODS: Monocytes from MCTD patients (n = 11) and normal volunteers (n = 11) were stimulated with purified antibodies against U1-RNP or double-stranded DNA and their supernatants were added to cultures of HPAECs. Cell-associated cytokines were assayed by an enzyme-linked immunosorbent assay. RESULTS: The supernatants of anti-U1-RNP-stimulated MCTD monocytes significantly up-regulated the cell-associated production of IL-1 alpha (p < 0.01) and IL-6 (p < 0.01) by HPAECs compared with their production by normal IgG-stimulated MCTD monocytes, whereas the cell-associated production of IL-1 beta and TNF-alpha by HPAECs was not up-regulated. The supernatants of anti-U1-RNP-stimulated monocytes from normal volunteers similarly up-regulated the cell-associated production by HPAECs of IL-1 alpha (p < 0.01) and IL-6 (p < 0.01), but not of IL-1 beta and TNF-alpha. Supernatants of monocytes stimulated with the F(ab')2 preparation of anti-U1-RNP antibodies enhanced the amounts of both Il-1 alpha and IL-6 associated with HPAECs almost as effectively as those stimulated with intact autoantibody molecules. Inhibition experiments employing specific anti-cytokine antibodies of anti-U1-RNP-stimulated monocyte supernatants suggested that soluble factors, including cytokines, in monocyte supernatants could enhance the cytokine association with HPAECs. CONCLUSION: Up-regulation by anti-U1-RNP autoantibodies of proinflammatory cytokines associated with vascular endothelial cells may play a role in the immunopathological processes leading to proliferative vasculopathy, a characteristic of MCTD.

Clinical significance of the serum lipoprotein(a) level in patients with systemic lupus erythematosus: its elevation during disease flare.

OBJECTIVE: To determine the clinical significance of serum lipoprotein(a) [Lp(a)] levels in patients with systemic lupus erythematosus (SLE). METHODS: Serum Lp(a) levels in 77 patients with SLE were measured by turbidimetric immunoassay. RESULTS: The median serum Lp(a) levels in all the SLE patients (14.4 mg/dl) and in those with active disease (Group A; 19.6 mg/dl, n = 39) at admission were significantly higher than those in healthy subjects (11.9 mg/dl, p < 0.05 and p < 0.001, respectively). The serum Lp(a) levels in SLE patients correlated directly with the serum cholesterol (p < 0.001) and urinary protein (p < 0.001) levels and inversely with the serum albumin levels (p < 0.02). Analysis limited to Group A patients with renal disease (Group A + RD, n = 28) revealed that the median serum Lp(a) level at the time of admission (OM) was significantly higher than those at 6 months before (-6M, p < 0.01) and at 6 months after admission (+6M, p < 0.01). Moreover, the serum Lp(a) level decrease from 0M to +6M in Group A+RD correlated significantly with the serum albumin level increase (p < 0.05). Multiple regression analysis demonstrated that the serum albumin level increment, the SLEDAI score decrement, the cholesterol level at 0M and the total dose of oral corticosteroids administered during the 0M to +6M period contributed independently and significantly to the serum Lp(a) level decrement from 0M to +6M in Group A + RD. CONCLUSION: Our study is the first to reveal that hypoalbuminemia appearing during disease flare plays an important role in increasing the serum Lp(a) levels in lupus patients with renal disease and shows that corticosteroid treatment reduced the elevated serum Lp(a) levels almost to original levels.

Increased levels of matrix metalloproteinase-3 in sera from patients with active lupus nephritis.

OBJECTIVE: To determine the matrix metalloproteinase-3 (MMP-3) levels in sera from patients with systemic lupus erythematosus (SLE) and to analyse the relationships between MMP-3 and clinical and laboratory features. METHODS: Serum MMP-3 levels were measured by an enzyme immunoassay in 124 patients with SLE and 237 patients with other systemic rheumatic diseases. RESULTS: The frequencies of patients with high MMP-3 levels were 76% in SLE and 82% in rheumatoid arthritis (RA). The level of MMP-3 in the SLE patients was 193.0 +/- 171.5 ng/ml (mean +/- SD) and was almost equal to the level in the RA patients (259.5 +/- 255.6 ng/ml). The MMP-3 levels were significantly higher in SLE patients who had a history of the following abnormalities: persistent proteinuria, cellular casts, anti-double stranded DNA antibodies, decreased C3, decreased creatinine clearance (p < 0.001), circulating immune complex (p < 0.01), malar rash, hypoalbuminemia, or decreased C4 (p < 0.05). The serum MMP-3 level in patients with SLE at admission showed direct correlations with serum uric acid, total cholesterol (p < 0.001), triglyceride, the white blood cell count, and the neutrophil count (p < 0.05), as well as inverse correlations with the total protein (p < 0.01), and IgG (p < 0.05). In SLE patients with active renal disease, the median MMP-3 level at admission and that at 6 months after admission were significantly higher than that at 6 months before admission. CONCLUSIONS: The increased level of serum MMP-3 in SLE is closely associated with clinical features relevant to lupus nephritis, suggesting that it plays a role in the pathogenesis of this condition.

Clinical and laboratory features of lupus patients with complicating pulmonary disease.

The objective of this study was to determine the incidence of pulmonary involvement in patients with systemic lupus erythematosus (SLE) and to clarify the clinical and laboratory characteristics in SLE patients with various pulmonary involvements. A retrospective study (n = 137) revealed that the types of pulmonary involvement found in SLE patients were: pleuritis (9%), interstitial pneumonia (8%), pulmonary infarction (7%), pulmonary infection (4%), pulmonary hypertension (2%), restrictive dysfunction (28%) and decreased diffusion capacity (43%). The incidences of pericarditis (P < 0.01), arthralgia (P < 0.05) and hypoalbuminemia (P < 0.05) were significantly greater in patients with pleuritis than in those without, while in patients with interstitial pneumonia, the incidence of anti-SS-A antibody (P < 0.05) and sicca syndrome (P < 0.05) were significantly greater than in those without. A longitudinal follow-up study of patient groups with various pulmonary involvements revealed: 1. significant changes of erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), lactate dehydrogenase (LDH) and thrombomodulin (TM) in patients with pleuritis, and 2. significant changes of WBC and LDH in patients with interstitial pneumonia. The increased ESR, CRP and TM levels during disease episodes suggest that the involvement of inflammatory processes is related to vasculitic events in the pathogenesis of lupus pleuritis. A higher incidence of anti-SS-A antibody in lupus patients with interstitial pneumonia suggests a potential role for this autoantibody in the pathogenesis of this complication.

[Autoantibodies associated with vasculitis].

A variety of autoantibodies have been associated with vasculitis, including that to neutrophils or to endothelial cells. Anti-neutrophil cytoplasmic antibodies (ANCA) have been described as sensitive and specific markers for active Wegener's granulomatosis (WG). ANCA in WG produces a characteristic cytoplasmic staining pattern (cANCA) and are directed against proteinase 3 (PR3-ANCA). PR3-ANCA occur in more than 90% of patients with extended WG, in 75% of patients with limited WG without renal involvement and in some 40% to 50% of patients with vasculitic overlap syndrome such as microscopic polyarteritis. Change in levels of cANCA precede disease activity and may be used as guidelines for treatment. Antibodies producing a perinuclear staining of ethanol-fixed neutrophils (pANCA) occur in a wide range of disease. They are directed against different cytoplasmic constituents of neutrophils. Among these, antibodies to myeloperoxidase (MPO-ANCA) are found in patients with idiopathic crescentic glomerulonephritis, the Churg-Strauss syndrome, polyarteritis nodosa and vasculitic overlap syndromes. Anti-endothelial cell antibodies (AECA) have been described in various autoimmune (systemic lupus erythematosus, scleroderma, rheumatoid arthritis) and vasculitic disorders (WG, polyarteritis nodosa, Kawasaki syndrome). They have been implicated in the pathogenesis of vascular injury common to these disorders.